Rapid and Systematic Exploration of Chemical Space Relevant to Artemisinins: Anti-malarial Activities of Skeletally Diversified Tetracyclic Peroxides and 6-Aza-artemisinins

Article abstract of DOI:10.1021/acs.joc.0c01017

To achieve both structural changes and rapid synthesis of the tetracyclic scaffold relevant to artemisinins, we explored two kinds of de novo synthetic approaches that generate both skeletally diversified tetracyclic peroxides and 6-aza-artemisinins. The anti-malarial activities of the tetracyclic peroxides with distinct skeletal arrays, however, were moderate and far inferior to artemisinins. Given the privileged scaffold of artemisinins, we next envisioned element implantation at the C6 position with a nitrogen without the trimmings of substituents and functional groups. This molecular design allowed the deep-seated structural modification of the hitherto unexplored cyclohexane moiety (C-ring) while keeping the three-dimensional structure of artemisinins. Notably, this approach induced dramatic changes of retrosynthetic transforms that allow an expeditious catalytic asymmetric synthesis with generation of substitutional variations at three sites (N6, C9, and C3) of the 6-aza-artemisinins. These de novo synthetic approaches led to the lead discovery with substantial intensification of the in vivo activities, which undermine the prevailing notion that the C-ring of artemisinins appears to be merely a structural unit but to be a functional area as the anti-malarial pharmacophore. Furthermore, we unexpectedly found that racemic 6-aza-artemisinin (33) exerted exceedingly potent in vivo efficacies superior to the chiral one and the first-line drug, artesunate.

Full text of DOI:10.1021/acs.joc.0c01017

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Article
Rapid and Systematic Exploration of Chemical Space
Relevant to Artemisinins: Anti-malarial Activities of Skeletally
Diversified Tetracyclic Peroxides and 6-Aza-artemisinins
Karunakar Reddy Bonepally, Norihito Takahashi, Naoya Matsuoka, Hikari Koi, Haruki MIZOGUCHI,
Takahisa Hiruma, Kyohei Ochiai, Shun Suzuki, Yutaka Yamagishi, Hideaki Oikawa, Aki Ishiyama,
Rei Hokari, Masato Iwatsuki, Kazuhiko Otoguro, Satoshi Omura, Nobutaka Kato, and Hiroki Oguri
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01017 • Publication Date (Web): 02 Jul 2020
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Page 1 of 20
The Journal of Organic Chemistry
Rapid and Systematic Exploration of Chemical Space Relevant to Artemisinins:
Anti-malarial Activities of Skeletally Diversified Tetracyclic Peroxides and 6-Aza-
artemisinins
1
2
3
4
5
Karunakar Reddy Bonepally, Norihito Takahashi, Naoya Matsuoka, Hikari Koi, Haruki Mizoguchi,
Takahisa Hiruma, Kyohei Ochiai, Shun Suzuki, Yutaka Yamagishi, Hideaki Oikawa, Aki Ishiyama, Rei
Hokari, Masato Iwatsuki,* Kazuhiko Otoguro, Satoshi Ōmura, Nobutaka Kato, and Hiroki Oguri*
6
7
8
9
10
11
12
13
14
15
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42
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45
46
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48
49
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51
52
53
54
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57
58
59
60
R2
Me
H
O
O
H
O
O
CO2R
SiMe3
O
O
H
O
O
4 steps
R2
N
NH
H
O
O
N
O
R3
R1
N
O
de novo
synthesis
H
Me
H
O
R1
H
R3
In vivo activities
racemic > chiral
6-aza-artemisinins
X
ABSTRACT: To achieve both structural changes and rapid synthesis of the tetracyclic scaffold relevant to artemisinins, we explored
two kinds of de novo synthetic approaches that generate both skeletally diversified tetracyclic peroxides and 6-aza-artemisinins. The
anti-malarial activities of the tetracyclic peroxides with distinct skeletal arrays, however, were moderate and far inferior to artemis-
inins. Given the privileged scaffold of artemisinins, we next envisioned element implantation at the C6 position with a nitrogen
without the trimmings of substituents and functional groups. This molecular design allowed the deep-seated structural modification
of the hitherto unexplored cyclohexane moiety (C-ring), while keeping the three-dimensional structure of artemisinins. Notably, this
approach induced dramatic changes of retrosynthetic transforms that allow an expeditious catalytic asymmetric synthesis with gen-
eration of substitutional variations at three sites (N6, C9 and C3) of the 6-aza-artemisinins. These de novo synthetic approaches led
to the lead discovery with substantial intensification of the in vivo activities, which undermine the prevailing notion that the C-ring
of artemisinins appears to be merely a structural unit but to be a functional area as the anti-malarial pharmacophore. Furthermore,
we unexpectedly found that racemic 6-aza-artemisinin (33) exerted exceedingly potent in vivo efficacies superior to the chiral one
and the first-line drug, artesunate.
semi-synthesis
O
Me
H
OMe
10 O
Me
H
O
■ INTRODUCTION
10 O
O
Malaria poses a deadly threat particularly to young children and
expectant mothers. The World Health Organization (WHO) recom-
mends artemisinin-based combination therapy (ACT) as the first-
line treatment for uncomplicated Plasmodium falciparum malaria,
including chloroquine-resistant strains.1 Artemisinin-based chemo-
therapies have made significant contribution toward reducing the
malaria burden throughout the world. Artemisinin (1), a sesquiter-
pene peroxide, is found in Artemisia annua (sweet wormwood), a
plant that contains highly variable amounts of artemisinin in its
leaves (Figure 1).2 Artemisinin was originally identified as an anti-
malarial agent by its ability to rapidly kill blood-stage malaria par-
asites.3 Among the various semi-synthetic derivatives 2–5 of 1, ar-
tesunate (2) with improved aqueous solubility is widely used as part
of fixed-dose ACT.
OH
O
H
O
H
O
O
O
O
H
H
Me
Me
Me
Me
artesunate (2)
artemether (3)
Me
H
O
10
9
O 11
H
D
C
O
1
O
6
B
A
O
Me
H
3
Me
artemisinin (1)
O
O
S
Until recently, the supply of 1 has been completely reliant on
plant extraction. This necessitated a lead time of approximately 18
months to grow the plants, resulting in frequent shortages and price
fluctuations. A notable achievement of synthetic biology is the ef-
ficient microbial production of the biosynthetic precursor, artemis-
inic acid (8), by genetically engineered strains of Saccharomyces
cerevisiae (Figure 2).4 This precursor 8 can be chemically con-
verted to 1 in four-step semisynthetic manipulations.4a,5,6 The recent
development of continuous-flow processes substantially streamline
these conversions.7
Me
H
O
Me
H
N
11
N
O
10 O
H
O
H
O
O
O
O
O
H
Me
Me
H
Me
Me
artemisone (4)
11-aza-artemisinin (5)
Figure 1. Artemisinin (1) and semi-synthetic derivatives (2-5).
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Page 2 of 20
engineered biosynthesis
Compared to the semi-synthetic approach, the chemical synthesis
Me
1
2
3
4
5
6
7
8
of 1 and its analogs would be more flexibly applicable for explora-
tion of chemical space relevant to artemisinins. Extensive efforts
have been made to achieve total synthesis of artemisinin (1).8 Avery
developed an efficient synthetic route to 1 in 10 steps starting from
(R)-(+)-pulegone and conducted structure-activity relationship
studies (Figure 3a).9 In 2012, Cook’s group reported an elegant
and highly concise asymmetric synthetic route to 1 from cyclohex-
enone.10 As an alternative approach to access the anti-malarial
agents by structural simplification of 1, fully synthetic peroxides
represented by OZ439 (9)11,12 have been developed (Figure 3b).
amorphadiene
synthase
(ADS)
Me
O
OPP
Me
Me
SCoA
Me
acetyl CoA
farnesyl diphosphate (6)
Me
Me
Me
Me
Me
H
1,3-hydride
shift
Me
H
9
H
Me
In efforts to develop a chemical assembly line that enables both
the expeditious synthesis and unprecedented levels of structural di-
versification of the tetracyclic sp3-rich scaffold of artemisinins, we
reported two synthetic approaches. The first one is divergent syn-
thesis of skeletally diversified tetracyclic peroxides reminiscent of
artemisinins (Figure 4a).13 Whilst generation of anti-trypanosomal
lead candidates were reported in the previous paper,13b herein we
report relationship between structure and anti-malarial activity of
the fully synthetic peroxides having diverse arrays of skeletal, func-
tional group properties with an identical molecular weight.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Me
H
Me
Me
Me
OPP
Me
H
I
II
III
H
H
CYP71AV1
CPR1
Me
H
H
H
H
Me
H
H
H
CYB5
OH
H
H
H
Me
H
Me
H
Me
Me
Me
Me
H
Our approaches
IV
skeletally diversified tetracyclic peroxides
amorphadiene (7)
(a)
O
Me
O
O
Br
O
O
A
Me
O
semi-synthesis
R
O
Me
9
OH
SiMe3
H
H
C
H
1. H2, (Ph3P)3RhCl
2. (COCl)2, MeOH
H
H
H
CO2H
CN
CN
Me
11
D
O
6 steps
XMg
I
i. ADH1
ii. ALDH1
H
H
O
H
CN
O
3. Li2MoO4, H2O2
4. Cu(OTs)2
O
1
6
O
B
A
H
O
O
Me
O
racemic
Me
RB
NC
H
O
Me
3
CN
Me
H
Dowex resin
O
Me
H
artemisinin (1)
artemisinic acid (8)
(b)
Figure 2. Microbial production of artemisinic acid (8) from farne-
syl diphosphate via intermediates I-IV and amorphadiene (7), fol-
lowed by the semi-synthesis of artemisinin (1).
R2
R2
CO2R
O
O
H
O
H
SiMe3
O
9
O
H
O
4 steps
de novo
R2
NH
10
O
O
11
R3
R3
1O
N
6
N
R1
O
H
O
(a)
R3
O
O
H
3
H
synthesis
R1
R1
Avery
Me
H
Me
O
OH
SiMe3
H
6-aza-artemisinins
H
12
X
Me
Me
SiMe3
O
catalytic
asymmetric
synthesis
1
installation of peroxide
acetal formations
modular
assembly
H
Me
Me
O
Me
Me
O
O
O
Me
R2
R2
R2
CO2R
[M]
(R)-(+)-pulegone
Me
Me
CO2R
SiMe3
Me
Me
H
CO2R
H
H
8
5
stereocontrolled
cyclization
Cook
TIPSO
H+
TIPSO Me
N
H
SiMe3
R3
N
SiMe3
R3
N
Me
Br
Me
OMe
H
R1
O
R1
O
R1
H
R3
H
ZnMe2
Me
X
O
X
H
X
V
13
14
1
Figure 4. (a) Divergent synthesis of skeletally diverse tetracyclic
peroxides (b) Catalytic asymmetric modular synthesis of 6-aza-ar-
temisinins generating substitutional variations at N6, C3 and C9
positions.
Me
Me
Me
Me
(b)
Vennerstrom
OMe
N
O
The second approach features modular de novo synthesis of
6-aza-artemisinins (Figure 4b). Based on the molecular design-
ing concept of 6-aza-artemisinins, replacing a sp3 stereogenic
carbon center at C6 position with a nitrogen revealed in the pre-
vious preliminary communication,14 we report herein the devel-
opment of the concise modular de novo synthetic approach gen-
erating substitutional variations at three positions (N6, C9 and
C3). Structure-activity relationship (SAR) studies allowed se-
lection of optimum substituents and led to generation of lead
candidates exhibiting promising in vivo anti-malarial activities
comparable to the natural product, artemisinin (1) as well as the
H
O
O
N
O
O
Cl
N
Cl
O
O
OZ439 (9)
OR
Figure 3. (a) Total syntheses of artemisinin (1). (b) Concise syn-
thesis of anti-malarial drug candidate OZ439 (9).
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The Journal of Organic Chemistry
semi-synthetic drug, artesunate (2). Intriguing configurational (ADS) would be next to impossible. Isomerization of allylic di-
1
2
3
4
5
6
7
8
effects on the in vivo therapeutic efficacies are also described.
phosphate in farnesyl diphosphate (6) and subsequent cycliza-
tion via nucleophilic attack of a trisubstituted double bond gen-
erates a tertiary cation (I →II). 1,3-Hydride shift followed by
the second cyclization (II→III→IV) and subsequent deproto-
nation lead to amorphadiene (7). Since the installation of nitro-
gen at C6 position would preclude the cationic cyclization cas-
cade (I →II→III), the de novo synthetic approach generating
6-aza-artemisinins is expected to provide an alternative means
to access the tetracyclic scaffold related to artemisinins.
■ RESULTS AND DISCUSSION
Anti-malarial activities of of tetracyclic peroxides The de-
sign and development of the highly divergent synthetic process
generating skeletally distinct tetracyclic peroxides were re-
ported previously.13 In vitro anti-malarial activities of the tetra-
cyclic peroxides were evaluated by employing the chloroquine-
resistant P. falciparum K1 strain.15 As shown in Figure 5, the
endoperoxide 15 exhibited modest anti-malarial efficacies (IC50
= 10 µM). The other three kinds of tetracyclic artemisinin ana-
logs 16–19 with skeletal diversification of the endo-peroxide
moiety resulted in almost negligible activities. Although the tet-
racyclic skeletal array of 16 is almost identical to that of 1, the
structural modifications involving incorporation of double bond,
nitrile, and trimming of the lactone carbonyl group in 1 caused
a substantial decrease in the anti-malarial activities. These find-
ings indicated the importance of both the arrangement of func-
tional groups and the three-dimensional structure of the perox-
ide bridge installed in the tetracyclic skeleton of 1. In theory,
the synthetic approach generating an assortment of skeletally
distinct natural product-relevant sp3-rich scaffolds bearing an
identical molecular weight (15–19: M.W. 261) would offer a
relatively unexplored means for SAR studies identifying 3D
pharmacophore despite weak activities of the tested compounds
in this study.13b
9
Unlike conventional medicinal chemistry approaches, the
molecular design replacing the stereogenic carbon at the C6 po-
sition of artemisinin with a nitrogen opens new opportunities
for the deep-seated structural modification of the tetracyclic
skeleton specifically for the cyclohexane moiety (C-ring).17
This attempt featuring the element implantation rather than
mere structural simplification could inspire an innovative retro-
synthetic disconnection into three simple building blocks:
amine, aldehyde, and commercially available alkyne (Figure
4b). Harnessing the versatile reactivity of nitrogen, we con-
ceived catalytic asymmetric synthesis of the 6-aza-artemisinins
in just four steps starting from the modular assembly of rela-
tively simple building blocks. This approach featuring “point
mutation” of the natural product allowed concise de novo syn-
thesis of the unprecedented analogs of artemisinins without
simplifying the complex molecular architecture.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of 6-aza-artemisinins The previously communicated
modular synthetic process14 was refined and substantially expanded
based on the synthetic plan (Figure 4b), which allowed not only the
installation of a series of nitrogen substituent but also diversifica-
tion of the C3 and C9 substituents. Catalytic asymmetric three-com-
ponent condensation, commonly called the A3-coupling, could al-
low direct assembly of the chiral ene-yne 13, bearing all of the
framework carbon and nitrogen atoms of the 6-aza-artemisinins.
Metal-mediated intramolecular cyclization of the ene-yne 13 would
provide the intermediate 14, composed of a piperidine ring with a
(Z)-vinyl silane moiety and two contiguous stereogenic centers, via
the formation of metallacyclic intermediate V, followed by proto-
nation. The tetracyclic framework of 6-aza-artemisinins could be
assembled via deprotection and oxidative installation of silyl per-
oxide and aldehyde, followed by tandem cyclic acetal formations.
Exploiting the modular nature of the catalytic asymmetric synthetic
process, substitutional variations at the N6, C9 and C3 positions
could be systematically generated through judicious choice of the
building blocks, in which each of the substituents (R1, R2 and R3)
orients to three distinct directions of the globular-shaped tetracyclic
scaffold (Figure 4b).
O
Me
Me
O
O
O
H
O
O
O
NC
Me
O
H
H
H
O
H
CN
CN
15: IC50 = 10 µM
16: IC50 = > 45 µM
17: IC50 = 32 µM
H
O
Me
H
O
O
H
O
O
H
O
NC
CN
Me
H
18: IC50 = > 45 µM
19: IC50 = > 45 µM
Figure 5. Structures of tetracyclic peroxides 15–19 and in vitro
anti-malarial activities against P. falciparum K1 strain.
Design of 6-aza-artemisinins Taking into accounts for the
SAR studies with skeletally diverse artemisinin analogs 15–19,
the second-generation molecular design featuring element im-
plantation on the sp3-rich tetracyclic scaffold of 1 was then de-
veloped as reported in the previous communication.14 We de-
signed 6-aza-artemisinins by replacing a stereogenic carbon
center at the C6 position of 1 with a nitrogen (Figure 4b). Im-
plantation of the nitrogen on the tetracyclic scaffold of 1 could
allow facile structural diversification of the hitherto unexplored
region of artemisinins, especially the cyclohexane ring (C-ring)
while minimizing changes of the three-dimensional cyclic ar-
rays especially for the trioxane core responsible for the potent
anti-malarial activity.13
First of all, we prepared building blocks bearing either an amino
or an aldehyde group based on the reported procedure (Figure 6).14
The amine building blocks 10a/10b were synthesized in two steps
from the known sulfonamide 2018 with improved yield of 10a. The
aldehyde building block 12a was synthesized from commercially
available ethyl levulinate (21) in two steps: Brønsted acid-mediated
formation of cyclic ketal, and subsequent reduction of the ethyl es-
ter with DIBAL-H. Likewise, the aldehyde 12b bearing a t-butyl
group was synthesized using 2,2-dimethylhept-6-en-3-one (22)19
in two steps: acid-catalyzed ketalization, followed by ozonolysis.
Given the biosynthetic mechanism of 1 via 7 (Figure 2),4,16
enzymatic assembly of the tetracyclic scaffold of 6-aza-artemis-
inins exploiting the key terpene cyclase amorphadiene synthase
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two cyclic acetals to construct the trioxane core (Figure 7).14 First
of all, treatment of 14a with trifluoroacetic acid allowed deprotec-
R2
O
1) acrolein, NaOAc, n-Bu4NCl
toluene/THF (1/1)
NHNs
1
2
3
4
5
6
7
8
Ot-Bu
then Ph3P=CR2CO2t-Bu
tions of a ketone and a carboxylic acid and simultaneous formation
of an ammonium salt (VI). To prevent oxidation of the tertiary
amine in the following ozonolysis, temporary protection of the
amine was conceived to be critical. The resultant ammonium salt
VI was thus directly subjected ozonolysis using conditions modi-
fied from Avery’s protocol.9c Since two substituents at C5a and C8a
adopt pseudo-axial conformation (VI) by minimizing the allylic
strain of the vinyl silane 14a, [3+2] dipolar cycloaddition of ozone
predominantly underwent at the less-hindered α-face to form
molozonide (VII). Spontaneous migration of the silyl group ena-
bled the simultaneous installation of aldehyde and trimethylsilyl
peroxide (VIII). Subsequent treatment with trifluoroacetic acid in
one-pot affected sequential formation of two cyclic acetals giving
rise to the trioxane 24 in 46% overall yield from 14a. The overall
yield and reproducibility were substantially improved by azeotropic
distillation with toluene of the resulting mixture containing the am-
monium salt VI prior to ozonolysis and tandem acetalizations.14 In
this manner, we have achieved streamlined asymmetric synthesis of
the tetracyclic array of 6-aza-artemisinins in just four steps starting
from modular assembly of the three simple building blocks.
NH
2) PhSH, Cs2CO3, CH3CN
MeO
PMB
20
10a: R2 = Me, 40% (2 steps)
10b: R2 = H, 69% (2 steps)
1) 2,2-dimethyl-1,3-propanediol
p-TsOH·H2O, HC(OEt)3
CH2Cl2, 98%
O
Me
O
O
9
H
Me
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EtO
2) DIBAL-H, –78 °C
CH2Cl2, 83%
O
12a
Me
Me
21
Me
1) 2,2-dimethyl-1,3-propanediol
p-TsOH·H2O, HC(OEt)3
CH2Cl2, 40 °C, 39%
O
H
Me
Me
Me
Me
O
O
Me
2) O3, NaHCO3, then PPh3
MeOH/CH2Cl2
–78 °C, 84%
O
Me
Me
22
12b
Not only to improve Ti(II)-mediated cyclization but also to ex-
plore SAR studies involving modifications at the C9 position,21,22
we next conceived an intramolecular cyclization employing a less
sterically demanding substrate 13b without a methyl substituent in
place of 13a and subsequent manipulation of the C9 position to gen-
erate derivatives (25–26, 28, 30, 32 35, 42 and 44) (Figure 7, 9–10).
Cu(I)-catalyzed three-component condensation of the amine build-
ing block 10b, aldehyde 12a, and trimethylsilylacetylene 11 gave
13b in 85% yield with high enantioselectivity (94% ee). Subsequent
Ti(II)-mediated cyclization of the C9-desmethylated ene-yne 13b
smoothly underwent as the temperature was gradually increased (–
78 °C → –40 °C) to afford the desired syn-14b in 61% yield accom-
panied with the formation of a small amount of the C8a epimer 14c
(7% yield). Thus, the cyclization process leading to 14b was sub-
stantially improved, relative to the corresponding conversion of the
C9-methylated 13a into 14a (40% yield). Meanwhile, Ni(0)-medi-
ated cyclizations of 13b resulted in almost exclusive formation of
the undesired diastereomer 14c having an anti-stereochemical rela-
tionship between 5a and 8a positions (Figure 8).23 These results im-
ply that the chelation of oxophilic titanium and magnesium species
with oxygen-containing functional groups in 13b is likely to play
pivotal roles in the predominant formation of syn-isomer 14b.
Figure 6. Synthesis of the building blocks amines 10a/10b and al-
dehydes 12a/12b.
The four-step access to the tetracyclic scaffold of 6-aza-artemis-
inins, began with Cu-catalyzed modular assembly of the three
building blocks: amine 10a, aldehyde 12a, and commercially avail-
able trimethylsilylacetylene 11 (Figure 7).14 To achieve a catalytic
asymmetric de novo synthesis, Carreira’s chiral catalyst CuBr/(R,
M)-PINAP was employed for Cu(I)-mediated assembly.20 In the
presence of only 1 mol% of the chiral catalyst and 4Å molecular
sieves in dimethyl carbonate, the three-component assembly under-
went smoothly at room temperature to afford the ene-yne 13a in
92% yield with excellent enantioselectivity (93% ee).
The second step was the intramolecular cyclization of the ene-
yne 13a to forge the densely-functionalized piperidine 14a with ste-
reo-controls both for a syn-relationship between C5a and C8a and
for a 9R-methyl group. The stereochemical controls of the vicinal
stereogenic centers were achieved by adopting low-valent titanium
(II)-mediated cyclization.21 Upon treatment of 13a with “Ti(Oi-
Pr)2”, generated in situ at low temperature in diisopropyl ether, the
intended cyclization occurred, presumably via metallacyclic inter-
mediate V (Figure 4b) as the temperature was gradually increased
(–78 °C → –30 °C). Subsequent protonation of the intermediate
provided the desired 14a in 40% yield as the major product. The
careful control of reaction temperature keeping below –30 °C was
turned to be important for improving the yield for this conver-
sion.14a The three-dimensional structure including the relative con-
figurations of the three stereogenic centers as well as Z-geometry of
the tri-substituted double bond were confirmed by X-ray analysis
of the crystalline 14a. Despite the modest yield of 14a, this ap-
proach enabled direct and highly diastereo-controlled installation of
consecutive stereogenic centers (C8a and C9).
Alkylation of 14b upon treatment with lithium diisopropylamide,
followed by methyl iodide, furnished a 1:1 mixture of diastereomers
14a and 23 in excellent yield (94%), which were easily separable
by silica-gel chromatography (Figure 7). C9 epimerization of 23 by
treatment with lithium diethylamide followed by protonation effi-
ciently afforded 14a in 81% yield with the recovery of 23 (17%).
Collectively, this approach provides a high-yielding alternative
route to the precursor 14a and also enables stereochemical diversi-
fication at the C9 position (Figure 7). The one-pot conversions of
the resulting precursors 14b and 23 afforded two kinds of 6-aza-
artemisinins: 25 without the C9 methyl group and 26 having the 9S-
methyl group, respectively. The change of stereochemistry of the
C9 methyl group caused negative impacts on the one-pot synthesis
of the trioxane core. The corresponding conversion of 23 to provide
26 resulted in 7% overall yield.
To construct the tetracyclic scaffold 24 from the vinyl silane 14a,
we sought to perform a one-pot procedure involving (1) removal of
the protecting groups, (2) temporary protection of the amine, (3)
oxidative conversion of the vinyl silane for installations of an alde-
hyde and a silylperoxide, and (4) acid-mediated formation of the
ACS Paragon Plus Environment

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The Journal of Organic Chemistry
step 1
step 2
step 3, 4
Me
O
X
Me
H
Me
H
CO2H
H
1
2
3
4
5
6
7
Y
9
(R, M)-PINAP
(1 mol%)
CO2t-Bu
SiMe3
CO2t-Bu
H
Ot-Bu
Ti(Oi-Pr)4
i-PrMgCl
8a
5a
i) CF3CO2H
CH2Cl2-H2O
PMB
CF3CO2
CuBr (0.8 mol%)
HN
H
NH
N
H
10a
N
SiMe3
4 Å MS, r.t.
(MeO)2CO
(i-Pr)2O
−78 to −30 °C
SiMe3
PMB
H
PMB
H
VI
PMB
SiMe3
11
O
Me
O
Me
O
92%, 93% ee
then
1 M HCl (aq)
40%
O
O
ii) O3, CH2Cl2
−78 °C
Me
Me
13a
Me
(R, M)-PINAP:
Me
O
Me
Me
HN
Ph
O
14a
N
N
H
12a
H
8
Me
PMB
HN
Me
SiMe3
PPh2
H
O
9
CF3CO2
O
X
O
VII
Y
H
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
CO2t-Bu
CO2t-Bu
SiMe3
Ti(Oi-Pr)4
i-PrMgCl
8a
5a
O
H
(R, M)-PINAP
(1 mol%)
OH
O
O
(i-Pr)2O
N
SiMe3
N
Ot-Bu
H
CuBr (0.8 mol%)
−78 to −40 °C
H
H
PMB
PMB
X
Y
O
Me
O
O
O
Me
NH
then
1 M HCl (aq)
61%
4 Å MS, r.t.
(MeO)2CO
86%, 94% ee
CF3CO2
PMB
O
SiMe3
10b
+ 11 and 12a
O
O
NH
PMB
Me
Me
13b
Me
H
Me
Me
14b: X = H, Y = H
14a: X = Me, Y = H
23: X = H, Y = Me
LDA, MeI, THF
−78 °C to rt
94% (14a:23 = 1 : 1)
VIII
LiNEt2, THF
−78 to −10 °C
81%
iii) CF3CO2H
X
O
Y
H
Me
H
R
H
O
O
H
O
O
O
H
O
R
H
O
O
H
O
O
H
9
O
H
O
’R
DDQ, CH2Cl2
6
N
O
pH 11 buffer
O
O
O
O
O
6
HN
N
N
O
O
O
MeO
reductive
amination
H
27 (48% from 24)
N
O
H
H
Me
H
Me
Me
(84% BRSM)
Me
24: X = Me, Y = H (46% from 14a)
25: X, Y = H (49% from 14b)
33
31: R = Me, 32: R = H
27: R = Me, 28: R = H
26: X = H, Y = Me (7% from 23)
O
HCHO, AcOH
NaBH(OAc)3
Br
Cl
ClCH2CH2Cl
29 (99% from 27)
acylation
R
H
O
O
R
O
O
H
O
9
H
H
O
O
N
6
O
O
N
Br
H
O
O
Me
H
Me
Me
X-ray (35)
34: R = Me, 35: R = H
X-ray (29)
artemisinin (1)
29: R = Me, 30: R = H
Me
H
O
O
H
O
Me
H
Me
H
O
O
H
O
O
O
H
O
Me
H
O
Me
H
O
O
H
O
Me
H
O
O
H
O
O
H
O
F
F
O
O
O
O
N
O
O
O
N
N
N
N
N
O
O
N
O
N
N
MeO
O
O
N
H
N
F
H
H
H
H
H
Me
HN
Me
Me
O
Me
Me
Me
37
41
38
39
36
40
Figure 7. Modular de novo synthesis of 6-aza-artemisinins. Four-step synthesis of the tetracyclic scaffold of 6-aza-artemisinin (24–40)
and three-dimensional structures of 6-aza-artemisinins (29 and 35) and artemisinin (1) based on X-ray analyses.
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Given the frequent appearance of the cyclopropyl ring systems in
drug molecules,24 we also conceived the installation of a cyclopro-
pane ring at C9 position (Figure 9). Stepwise dialkylation of 14b
with 1-bromo-2-chloroethane was achieved in good yields to afford
14d, which was then converted into 42 in modest yield (overall
11%).
H
CO2t-Bu
H
H
CO2t-Bu
H
CO2t-Bu
SiMe3
8a
5a
8a
5a
Ni(COD)2
DBU
N
N
SiMe3
SiMe3
N
H
H
H
PMB
PMB
PMB
O
Me
O
O
Me
O
O
Me
THF
125 °C (M.W.)
47%
O
Me
Me
Me
Next, we installed several substituents on the nitrogen to generate
a series of compounds for SAR studies (Figure 7). Removal of the
p-methoxy benzyl group in 24/25 with DDQ liberated the second-
ary amines 27/28. Reductive amination of 27 and 28 with formal-
dehyde gave rise to the targeted 6-aza-artemisinin 29 and its C9-
desmethyl analog 30, respectively, in good yields.
Me
14c
Me
14b: not observed
Me
13b
Figure 8. Attempted cyclizaiton of ene-yne 13b.
ACS Paragon Plus Environment

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P. falciparum K1 strain to conduct SAR studies (Table 1).26 The
6-aza-artemisinin 29, which bears N6, C9 and C3 methyl groups,
9
9
H
CO2t-Bu
H
H
CO2t-Bu
H
1
2
3
4
5
6
7
8
8a
8a
5a
1. LDA, BrCH2CH2Cl
THF, –78 °C to r.t., 69%
exhibited potent activity (entry 3, IC50 = 30 nM) comparable to
those of artemisinin (1) and artesunate (2) (entries 1 and 2). The
C9-desmethyled analog 30, however, showed substantially re-
duced activity (entry 4: IC50 = 1,043 nM). Installation of an ar-
ylalkyl group at the nitrogen in place of the methyl substituent
exerted profound effects on improving the anti-malarial activity
of the C9-desmethylated series (entries 4–6). In contrast to the
N-methylated 30, N-p-methoxybenzylated 25 and N-benzylated
32 showed potent activities (entries 5 and 6), which led to im-
proved IC50 values of around 11 and 16 times, respectively,
compared to that of 30. These results suggested that the instal-
lation of a substituent on the N6 position could exert a favorable
influence on the anti-malarial activities.
The N-p-bromobenzoylated 35, however, showed weak activ-
ity (entry 7), suggesting the superiority of the methylene linkage
between the nitrogen and aryl groups in 25/32 over the amide
bond in 35. Since the installation of the amide linkage caused
substantial conformational changes of the piperidine substruc-
ture (C-ring) of 6-aza-artemisinins as shown in the X-ray anal-
yses for 29 and 35 (Figure 7), the changes both the C-ring con-
formation and the configuration of the N6 substituents might be
associated with the distinct in vivo anti-malarial activities. The
installation of C9 methyl group allowed substantial intensifica-
tion of the activities for the N-benzoylated 34 (entries 7 and 8).
The 6-aza-artemisinins (24, 31, 33 and 36–40), bearing a C9 me-
thyl group and the N-arylalkyl group on the nitrogen, exhibited high
potencies (entries 9–15). Briefly, the exchange of the benzene ring
in 31 into the heteroaromatic ring as well as the incorporation of the
three fluorine substituents on the benzene ring resulted in the nearly
unchanged efficacies (entries 10 and 11–13). Appendages of the
medicinally relevant and larger substituents are found to be well
tolerated (entries 14–16). Among the three derivatives, 6-aza-arte-
misinin 33 bearing a 2-morpholinoethoxy substituent on the ben-
zene ring exhibited an optimum activity (entry 16). The secondary
amine 27 without a substituent at N6 showed a diminished activity
(entry 17). The different configuration of the C9 methyl group of
9R–24 and 9S–26 had little effect on in vitro anti-malarial efficacy
(entries 9 and 18). Incorporation of the cyclopropane ring at C9 in
42 was tolerated to exhibit potent activity (entry 19). Installation of
the t-butyl group at the C3 position resulted in decreased potencies
(entries 20 and 21), despite our expectation that the sterically de-
manding substituent in the vicinity of the labile trioxane moiety
would exert kinetic stabilization effects.
5a
N
SiMe3
N
SiMe3
H
PMB
H
2. LiHMDS, THF/DMPU
–78 °C to r.t., 86%
PMB
O
Me
O
O
Me
O
Me
Me
Me
Me
14b
14d
i) CF3CO2H
CH2Cl2, H2O
ii) O3, CH2Cl2
–78 °C
O
H
9
9
O
H
O
6
N
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
iii) amberlyst 15
11%
O
MeO
3
Me
42
Figure 9. Synthesis of the C9-cyclopropyl-6-aza-artemisinin 42.
The relative configuration of the tetracyclic structure of the 6-
aza-artemisinins was unambiguously confirmed based on the X-ray
analysis of crystalline 29 (Figure 7). It is worth noting that the N6
methyl group of 29 adopts a pseudo-equatorial conformation and
thereby the overall three-dimensional architecture of 29 was turned
to be essentially identical to that of 1. Thus, this approach featuring
nitrogen implantation allowed precise imitation of the methyl sub-
stituent at the stereogenic C6 position of 1. A series of 6-aza-arte-
misinins (31–33, and 36–40) with the installation of a medicinally
relevant substituent at N6 were systematically synthesized via sim-
ilar reductive amination of 27/28 with benzaldehyde or various al-
dehydes. Acylation of 27/28 with p-bromobenzoyl chloride pro-
duced 34/35. X-ray analysis of crystalline 35. confirmed the abso-
lute configuration of the 6-aza-artemisinins synthesized in this
study (Figure 7).
Exploiting the modular nature of this synthetic process, we fur-
ther synthesized 6-aza-artemisinins 43 and 44 bearing the tert-butyl
group as an unnatural substituent (R3) at the C3 position (Figure
10).25 Employing the building block 12b in place of 12a, the three-
component assembly proceeded in good yields with high enantiose-
lectivities (97 and 88% ee with 10a and 10b, respectively). The 6-
aza-artemisinins 43 and 44 having a distinct R3 substituent were
synthesized by applying essentially the same four-step protocol de-
spite modest yields. Occasionally, desoxy byproduct such as 45
having cyclic ketal moiety was also formed.
R2
O
R2
CO2t-Bu
SiMe3
Ot-Bu
Ti(Oi-Pr)4
i-PrMgCl
(i-Pr)2O
(R, M)-PINAP
(1–2.2 mol%)
CuBr (0.8–2 mol%)
SiMe3
NH
PMB
N
–78 to –30 °C
H
PMB
4 Å MS, r.t.
(MeO)2CO
then
1 M HCl (aq)
O
t-Bu
10a/10b
11
As a whole, the low cytotoxicity of N-alkylated 6-aza-artemis-
inins with a 9R-methyl substituent (24, 29, 31, 33, 36, 37, 39, and
40) in a mammalian cell line established a selectivity index [SI: IC50
for MRC-5 cells / IC50 for P. falciparum K1 strain] of greater than
approximately 1,000. The almost negligible activity of the deoxy-
analog 41 (Figure 7), occasionally obtained as a byproduct from the
one-pot sequence from 14a to 24, is consistent with the fact that the
1,2,4-trioxane moiety is responsible for the potent anti-malarial ac-
tivity of these compounds. These preliminary SAR results illustrate
the considerable yet previously unexplored anti-malarial efficacies
of 6-aza-analogs through chemical modification of the C6, C9 and
C3 positions of artemisinin (1).
O
Me
O
H
Me
Me
Me
Me
O
O
13c: R2 = Me, 98%, 97% ee
13d: R2 = H, 97%, 88% ee
Me
Me
12b
R2
H
9
CO2t-Bu
i) CF3CO2H
CH2Cl2, H2O
ii) O3, CH2Cl2
–78 °C
R2
O
O
H
O
8a
H
H
9
Me
H
O
O
5a
N
SiMe3
6
H
O
PMB
N
O
t-Bu iii) CF3CO2H
H
O
MeO
H
O
Me
O
N
3
O
Me
H
PMB
Me
Me
Me
t-Bu
43: R2 = Me (6% from 14e)
44: R2 = H (14% from 14f)
45
Whilst the installation of the arylalkyl substituent slightly in-
creases the lipophilicity of the compound, the pre-installation of the
nitrogen on the scaffold would counterbalance the lipophilic prop-
erties. Indeed, the ClogP values of 6-aza artemisinins possessing the
hydrophobic substituents are in the same range as that of artemis-
inin (1), as shown in Table 1.
14e: R2 = Me, 30%
14f: R2 = H, 38%
Figure 10. Synthesis of 6-aza-artemisinins 43 and 44 bearing a t-
butyl group at C3.
In Vitro anti-malarial activities Having established the syn-
thetic process, in vitro anti-malarial activities of the 6-aza-arte-
misinins were evaluated by employing the chloroquine-resistant
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Table 1. In vitro anti-malarial activity and cytotoxicity of 6-aza-artemisinins.
1
2
3
4
anti-malarial
cytotoxicityb)
IC50 (nM)
Selectivity
Indexc)
activitiesa)
ClopPd)
com-
pound
R1
R2
R3
IC50 (nM)
entry
5
6
7
8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
artemisinin (1)
artesunate (2)
29
30
25
32
21
6.8
30
1,043
91
159,987
37,001
37,378
27,330
89,233
49,451
7,618
3,700
1,168
26
981
738
2.52
2.35
Me
Me
PMB
Bn
Me
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
t-Bu
t-Bu
1.14
0.68
2.11
2.13
2.62
3.00
2.45
2.54
1.86
1.76
3.19
2.54
1.56
2.32
0.56
2.45
2.46
3.54
3.16
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
67
35
34
24
31
36
37
38
39
40
33
27
26
42
43
44
p-Br-Bz
p-Br-Bz
PMB
11,522
130
69
86
26
94
77
55
43
33
267
51
80
788
434
117,575
91,245
10
702
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Mee)
CH2CH2
Me
H
203,288
160,480
139,589
117,144
23,053
>250,346
88,286
35,572
136,726
117,679
>124,546
>231,734
>239,520
2,946
1,866
5,369
1,246
299
>4,551
2,053
1,078
512
2,307
>1,557
>294
>552
Bn
furan-CH2
pyridine-CH2
C6H2F3-CH2
see Fig. 7
see Fig. 7
see Fig. 7
H
PMB
PMB
PMB
PMB
a) Anti-malarial activity against P. falciparum K1 strain, b) Cytotoxicity against MRC-5 cell line, c) Selectivity index [cyto-
toxicity/anti-malarial activity], d) ClogP was calculated using ALGOPS 2.1 software (http://www.vcclab.org/lab/alogps/), e)
Compound 26 has an epimeric methyl substituent at C9.
Substituent effects on In Vivo anti-malarial activities
Evaluation of in vivo anti-malarial activities of 6-aza-artemisinins
revealed discernible SAR relationships indicating the importance of
the substituents at N6 and C9 positions. Four kinds of chiral 6-aza
artemisinins (24, 29, 31 and 33) bearing C9 methyl groups were
compared based on Peter’s 4 day suppressive test using a mouse
model infected with rodent malaria P. berghei N strain (Figure 11).
Each compound was administrated intraperitoneally (dosage 15
mg/kg, four times every 24 h). Despite the high in vitro activity, N-
methylated 29 resulted in the lowest efficacy among the four com-
pounds. In contrast, three compounds bearing the N6 arylalkyl sub-
stituents, exerted potent in vivo activity 33 (69.1%), 24 (82.9%),
and 31 (98.6%) superior to artemisinin (1) (59.1%). These experi-
mental results clearly indicated that the installation of the N6 ar-
ylalkyl substituents at the C6 position of 1 substantially intensifies
the in vivo therapeutic efficacy, while structure modifications at C6
position have largely remained unexplored through semi-synthetic
and total synthetic approaches. Notably, the chiral N-benzylated 31
exhibited almost identical or even superior in vivo efficacy (98.6%)
to that of the first-line drug artesunate (2, 93.5%).
Figure 11. In vivo efficacy data obtained using the P. berghei rodent
malaria model. Activities resulting in average parasitemia reduction
were evaluated [I.P.; q.d. 15 mg/kg x 4] of 6-aza-artemisinins (24,
29, 31, 32, and 33), artemisinin (1), and artesunate (2). The inhibi-
tory activities of 6-aza-artemisinins were assessed by three distinct
experiments using 1 and 2 as the positive controls for each run [first
run: 33, second run: 29, 31 and 32, and third run: 24]. Regarding 1
and 2 the experimental data obtained from the first run was pro-
vided.
The effects of the C9 methyl group of the chiral N-benzylated 31
was then verified. While removal of the C9 methyl group led to di-
minished in vitro efficacy in all cases (Table 1), the C9-desmethyl-
ated 32 bearing the N-benzyl group still exerted meaningful in vivo
therapeutic activity (51.5%) comparable to artemisinin (1).
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Survival assays of chiral 6-aza-artemisinin 31 The survival Configurational effects on the in vivo anti-malarial activi-
1
2
3
4
5
6
7
8
assays were conducted employing five mice for each compound.
As shown in Figure 12, intraperitoneal administration [dosage
15 mg/kg, four times every 24 h] of the chiral 31 exhibited su-
perior therapeutic efficacy than artemisinin (1) and artesunate
(2). Upon administration of 1 and 2, all of the five mice infected
with the rodent malaria were dead within either seven or eight
days, respectively. In contrast, treatment of chiral 31 allowed the
survival of three mice until nine days and one for thirteen days.
Thus, this de novo catalytic asymmetric synthetic process paved
a new avenue to rapid access to fully synthetic analogs of arte-
misinins with promising therapeutic potentials.
ties Influence of absolute configuration of the 6-aza-artemis-
inins were then evaluated. The racemic 6-aza-artemisinins (25,
30, 32, and 33) were synthesized from racemic ene-yne 13b
(Figure 14). Whilst the three component condensation reaction
employing N,N,N',N'-tetramethylethylenediamine (TMEDA) as
ligand to cuprous catalyst in place of chiral (R, M)-PINAP (Fig-
ure 14) was sluggish at room temperature, micro-wave assisted
heating in dioxane for 25 min allowed rapid and gram-scale syn-
thesis of 13b in good yield (86%). The essentially identical syn-
thetic transformations described in Figure 7 provided racemic
aza-artemisinins (25, 30, 32, and 33).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CO2t-Bu
SiMe3
Me
Me
Me
Me
SiMe3
O
N
N
11
Ot-Bu
(10 mol%)
N
O
H
CuBr (10 mol%)
PMB
Me
O
NH
O
Me
H
dioxane
O
O
PMB
(µ-wave)
150 °C
Me
10b
86%
Me
Me
Me
12a
racemic 13b
O
O
H
H
9
9
see Figure 7
O
H
O
O
H
O
6
6
O
O
N
N
O
O
MeO
H
Me
H
Me
Me
racemic 30
racemic 25
Me
H
O
O
O
H
9
9
O
O
H
H
O
O
O
N
O
N
O
N
O
O
Figure 12. Survival curves upon intraperitoneal administration of
chiral 6-aza-artemisinin (31: q.d. 15 mg/kg x 4), artesunate (2: q.d.
30 mg/kg x 4) and artemisinin (1: q.d. 15 mg/kg x 4).
H
H
Me
Me
racemic 32
racemic 33
Figure 14. Synthesis of racemic 6-aza-artemisinins (25, 30, 32,
and 33).
Oral treatment of the optimum 6-aza-artemisinin 31 was then
conducted (Figure 13). Chiral 31 [dosage 30 mg/kg, four times
every 24 h] exhibited a potent therapeutic efficacy (95.6%),
which is superior to artemisinin (1, 74.5%) and comparable to
artesunate (2, 97.7%).
To our surprise, the racemic C9-desmethylated 32 with the N-
benzyl group exhibited apparently greater in vivo therapeutic ac-
tivities than that of chiral 32 (Figure 15). Despite the difference
of the dosage chiral 32 (15 mg x 4) and racemic 32 (30 mg x 4),
the content of the chiral 32 having corresponding absolute con-
figuration to that of artemisinin 1, are identical in each experi-
ment (15 mg x 4).
Figure 13. In vivo anti-malarial activity of 6-aza-artemisinin (31)
on P. berghei N strain-infected mice through oral administration
[q.d. 30 mg/kg x 4].
Figure 15. In vivo anti-malarial therapeutic effects of 6-aza-arte-
misinins chiral 32 [q.d. 15 mg/kg x 4] and racemic 32 [q.d. 30
mg/kg x 4].
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The Journal of Organic Chemistry
1
2
3
4
5
6
O
O
O
H
O
O
O
O
H
O
O
O
H
O
O
O
H
O
Me
H
O
H
H
H
H
9
9
9
9
10 O
H
O
OH
6
N
6
N
6
N
6
N
O
O
O
O
O
O
O
O
O
O
MeO
MeO
H
H
Me
H
H
Me
Me
Me
Me
H
Me
Me
control
(vehicle)
2: artesunate
32 (racemic)
25 (racemic)
30 (racemic)
25 (chiral)
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 16. In vivo blood-stage efficacy study of artesunate (2) and 9-desmetnyl 6-aza-artemisinins [32, 25, 30 as racemate and chiral 25].
Exploiting the in vivo imaging system (IVIS) for the mice
model, in vivo efficacy of racemic and chiral C9-desmethylated
25 with N-p-methoxyl benzyl group were then evaluated against
blood-stage model with the rodent malaria parasite P. berghei
that expresses firefly luciferase.26 Each compound (100 mg/ kg)
involving C9-desmethylated racemic 6-aza-artemisinins, (32,
25, and 30), chiral 25 and artesunate (2) was administrated
against four mice and monitored for 48 h (Figure 16 and 17).
Bioluminescence intensity was utilized to measure parasite
growth. Consistent with Peter’s 4 day suppressive test (Figure
11) for the C9-methylated 31 and 24, N-benzylated 32 exhibited
greater efficacy than N-p-methoxybenzylated 25, while N-meth-
ylated 30 resulted in the weakest efficacy among tested. More
importantly, the racemic C9-desmethylated 25 with the N-p-
methoxyl benzyl group was demonstrated to exert superior in
vivo therapeutic activity compared to the chiral one.
Asimilar phenomenon, intensification of the in vivo therapeu-
tic activities employing the racemic sample in place of chiral
one, was also observed for the other member of 6-aza-artemis-
Figure 17. In vivo efficacy study: CD-1 mice were inoculated with
inins having the C9 methyl group. Taking into accounts for the
unexpected findings with chiral and racemic 25, the configura-
tional effects were then verified employing an identical amount
of chiral and racemic 33 having a 2-morpholinoethoxy substit-
uent on the N-arylalkyl substituent (Figure 18). While the chiral
33 already exhibited superior activity (69.1%) than that of 1 as
P. berghei (ANKA luc-GFP) at 24 h before treatment. Each sample,
artesunate (2) or 6-aza-artemisins [32, 25, 30 as a racemate and chi-
ral 25], was administered as a single dose (100 mg/kg) at 0 h (n =
4) for each compound. Infections were monitored using the in vivo
imaging system (IVIS). Bioluminescent intensity was quantified
from each mouse and plotted against time.
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mentioned above (Figure 11), racemic 33 exerted the uppermost
Page 10 of 20
1
therapeutic activity (~100% inhibition) among the trials (Figure
2
18).
3
4
5
6
7
8
9
The survival assay of racemic 33 employing five mice was
also conducted (Figure 19). Intraperitoneal treatment of racemic
33 (dosage 15 mg/kg, four times every 24 h) allowed survival
of four mice until eight days, three for 10 days, two for 11 days
and one for 15 days (Figure 19b). In contrast, all of the five mice
were dead until eight days upon the intraperitoneal treatment of
chiral 33 (dosage 15 mg/kg, four times every 24 h) as shown in
Figure 19a.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 18. In vivo anti-malarial therapeutic effects of 6-aza-arte-
misinins [I.P.; q.d. 15 mg/kg x 4], chiral 33 and racemic 33.
The in vitro anti-malarial activities of the racemic 6-aza-arte-
misinins (32 and 25 and 33) were evaluated against the P. falci-
parum K1 and FCR3 strains and compared with the chiral ones
(Table 2). Whilst racemic 25 exhibited slightly greater activities
than the racemic one, the other two 6-aza-artemisinins (32 and
33) resulted in the virtually identical efficacies against both
strains between the racemic and chiral samples. These results
imply that the several factors of the pharmacokinetics and phar-
macodynamics nature of the racemic 6-aza-artemisinins might
play important roles on the substantial intensification of the in
vivo efficacies.
Figure 19. Survival curves upon intraperitoneal administration of
6-aza-artemisinin (33: 15 mg/kg x 4), artesunate (2: 30 mg/kg x 4)
and artemisinin (1: 15 mg/kg x 4). (a) chiral 33, (b) racemic 33.
SAR relationships focused on the scaffold variations Since
the fully synthetic tetracyclic peroxides 15–19 with skeletal di-
versification of 1 resulted in the significant reduction of the in
vitro anti-malarial activities (Figure 5), the cyclic arrays as well
as the functional groups and substituents of artemisinins were
demonstrated to be important elements for the anti-malarial
pharmacophore. These experimental findings were then sub-
stantiated through the generation of 6-aza-artemisinins, exhibit-
ing potent anti-malarial activities, that minimize the structural
simplifications and modifications except for the element im-
plantation at C6 position. The X-ray analyses suggested that 6-
aza-artemisinins adopt almost identical three-dimensional struc-
ture to that of 1, especially for the derivatives having N-methyl
and N-arylalkyl substituents (Figure 7). In contrast, the installa-
tion of the N-acyl group caused both substantial changes in the
conformation of the C-ring of 35 (Figure 7) and a decrease in
the anti-malarial activities. These results suggested that the
nearly complete imitation of the skeletal and functional group
properties of 1 is critical for designing the fully synthetic chem-
ical entities with promising anti-malarial activities comparable
to that of artemisinins.
Table 2. In vitro anti-malarial activities of chiral and racemic 6-
aza-artemisinins (32, 25 and 33).
anti-malarial activitiesa) IC50 (nM)
compound
K1
67
55
91
35
33
64
FCR3
52
43
56
26
chiral 32
racemic 32
chiral 25
racemic 25
chiral 33
17
29
racemic 33
a) anti-malarial activities against P. falciparum K1 (chloroquine-re-
sistant) and FCR3(chloroquine-sensitive) strains.
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The Journal of Organic Chemistry
human target proteins involving gephyrin,42 the intrinsically dis-
SAR relationships focused on the stereochemical proper-
ordered Bcl-2-antagonist of cell death promoter (BAD).43 As a
consequence, this concise modular catalytic asymmetric syn-
thetic approach featuring systematic structural diversification of
the nitrogen-containing sp3-rich scaffold could be beneficial for
the further the chemical genetic and medicinal investigations re-
lated to cancer biology and chemotherapy. The next challenges
would involve the generation of either novel or hybrid mole-
cules exploiting the 6-aza-artemisinin scaffold capable of com-
pensating the increased lipophilicity.
1
ties According to the recent report by Giannis and co-workers,
in vitro anti-malarial activities of the fully synthetic ent-1, (–)-
artemisinin, resulted in nearly identical to 1, clarifying in vitro
anti-malarial activity of 1 against P. falciparum NF54 strain has
almost no dependence on the absolute configuration of the tet-
racyclic scaffold.27 O’Neill and Posner previously reported
comparable results by generating enantiomeric analogs pos-
sessing the 1,2,4-trioxane moiety.28 Racemates and individual
enantiomers of the structurally simplified analogs showed sta-
tistically the same levels of in vitro anti-malarial activities
against P. falciparum 3D7, Dd2, K1, HB3 and NF54 strains. In
sharp contrast to these reports, the experimental findings dis-
closed herein indicate that chiral and racemic 6-aza-artemisinins
(25, 32 and 33) exhibited marked differences in the in vivo effi-
cacy against rodent malaria P. berghei N strain. Whilst admin-
istration of the chiral and racemic molecules sometimes causes
distinct outcomes due to distinct pharmacological properties be-
tween the enantiomeric samples and racemates, the apparently
superior in vivo therapeutic efficacy of racemic 33 is noteworthy
(Figure 18-19). Taking into accounts the unexpected but con-
sistent results found in the other two 9-desmethyl analogs 32
and 25 (Figures 15-17), the installation of the nitrogen on the
tetracyclic scaffolds as well as the appendage of N-arylaklyl
group are assumed to cause substantial changes on the pharma-
cological effects against P. berghei.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
■ CONCLUSIONS
In conclusion, we designed both tetracyclic peroxides and 6-
aza-artemisinins and developed concise and divergent synthetic
processes. The fully-synthetic tetracyclic peroxides 15–19 with
modification of skeletal arrays, functional groups, and peroxide
bridge exhibited moderate or almost negligible anti-malarial ac-
tivities, indicating the importance of the privileged tetracyclic
scaffold of artemisinins. These experimental findings led to the
design of 6-aza-artemisinin without substantial structural sim-
plifications of the artemisinins. The installation of the nitrogen
offers an entirely different retrosynthetic disconnection and
paved the way for a modular catalytic asymmetric synthesis.
This molecular design featuring “point mutation” of natural
product allowed both the development of the most concise fully
synthetic process to access the chemical space relevant to arte-
misinins and the diversification of the three substituents ori-
ented towards three distinct directions of the nearly spherical-
shaped scaffold. Our synthetic efforts resulted in the discovery
of a novel scaffold bearing the N-arylalkyl group generated by
the element implantation at the hitherto unexplored substructure
(C-ring) of artemisinins. Notably, this de novo synthetic cam-
paign is capable of overturning the prevailing belief: the C-ring
of artemisinins has been demonstrated not to be a merely struc-
tural unit, but to be a functional area for modulation of anti-
malarial activities. The small structural modifications at the C6,
C9 and C3 positions of 1 were demonstrated to have substantial
impacts on in vitro/in vivo anti-malarial activities. While the in-
corporation of a bulky substituent at C3 position resulted in di-
minished activities, the installation of the methyl, as well as cy-
clopropyl groups at C9 position, increased the activities. The
lead candidate 31 exerts the potent in vivo anti-malarial efficacy
comparable to the first-line semi-synthetic drug, artesunate (2).
Furthermore, racemic 6-aza-artemisinin 33 exhibited potent in
vivo therapeutic efficacy superior to chiral 33 as well as ar-
tesunate (2). Thus, we have successfully demonstrated the
promising but still untapped therapeutic potential of 6-aza-arte-
misinins bearing customizable structural, physical and pharma-
cological properties.44 This fully synthetic anti-malarial scaffold
could provide a basis for the next challenges to cope with
emerging artemisinin-resistant parasites33,45 and the develop-
ment of lead candidates for the chemotherapy of cancers and
other infectious diseases.
The mode of action of artemisinins remains under debate.29
Regarding their anti-malarial activities, the widely accepted
mechanism involves a heam-dependent cleavage of the peroxide
and subsequent alkylation of proteins with the resultant radical
species within the parasites.30 The heam-dependent activation of
peroxide moiety is likely to be triggered only inside the parasite
but not in the uninfected red blood cells. Several target proteins
including a parasitic Ca-ATPase (PfATP6),31 a membrane glu-
tathione S-transferase (PfEXP1)32 and phosphatidylinositol-3-
kinase (PfPI3K)33 have been identified. Recent chemical prote-
omics studies showed that a derivative of artesunate is capable
of alkylating at least 124 parasite proteins in a promiscuous
manner.34 Given the chiral molecular recognition with the tar-
geted proteins of parasites might not to be indispensable to the
iron-dependent pathways,35 these experimental findings with
the 6-aza-artemisinins as well as the precedented reports11,12 im-
ply that the use of racemate of artemisinins and their analogs
could be an alternative and convenient option for the develop-
ment of anti-malarial agents without the need for the asymmet-
ric synthesis, despite the concern of the adverse effects of the
racemic agents.
It is generally accepted that artemisinins are almost non-toxic
to normal cells but display polypharmacology and intriguing
anti-cancer activities based on mechanisms related to the one
proposed for the anti-malarial activities.36 It has been suggested
that artemisinins exert anti-cancer activities through ferropto-
sis,37 as well as inhibitions of angiogenesis38 and cancer-related
signaling pathways.39 Apart from the promiscuous mode of ac-
tions for the antimalarial activities against the parasite-infected
red blood cells, it holds the possibility of the specific molecular
recognition between artemisinins and human proteins in either
an iron-independent or dependent manner.40 In fact, Woerden-
bag reported stereochemistry-dependent cytotoxicity against
EN2 tumor cells,41 suggesting the involvement of chiral molec-
ular recognition between artemisinins with cellular biomacro-
molecules in cancer cells. Consistently, recent studies identified
■ EXPERIMENTAL SESSON
General Methods All reactions were performed under nitro-
gen atmosphere unless otherwise specified. Upon warming, the
reaction vessel was placed on a metal heating block. Microwave
reactions were performed using a Biotage Initiator. NMR spec-
tra were recorded on Bruker VSP 500, JEOL ECA 500 (1H/500
MHz, 13C/125 MHz), JEOL ECX 400 (1H/400 MHz, 13C/100
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Page 12 of 20
MHz), and JEOL AL 300 (1H/300 MHz, 13C/75 MHz) spectrom-
extracted with CH2Cl2 (300 mL x 2). The combined organic lay-
1
2
3
4
5
6
7
8
eters. Chemical shifts are reported in δ (ppm) using chloroform
as an internal standard of δ 7.26 and 77.16 for 1H and 13C-NMR,
respectively. Data for 1H NMR are reported as follows: chemi-
cal shift (number of hydrogens, multiplicity, coupling constant).
Multiplicity is abbreviated as follows: s (singlet), d (doublet), t
(triplet), q (quartet), quin (quintet), m (multiplet), br (broad).
ESI-Mass spectra were recorded on JEOL AccuTOF LC-Plus
JMS-T100 and Bruker micrOTOF control 3.0 systems. Optical
rotations were recorded on JASCO P-2200 and JASCO DIP-
1000 digital polarimeters. The medium pressure liquid chroma-
tography (MPLC) purifications were performed on YAMAZEN
YFLC-AI-580 and Biotage Isolera. Where necessary, solvents
were distilled from appropriate drying agents prior to use. Re-
actions were monitored by thin layer chromatography using
Merck Millipore TLC Silica gel F254 plates (0.25 mm) which
were visualized using UV light, p-anisaldehyde stain, ninhydrin
stain and PMS stain. Flash column chromatography was per-
formed using Kanto Silica Gel 60N.
Materials Commercial solvents and reagents were used as re-
ceived with the following exceptions. The (R, M)-PINAP cata-
lyst was prepared according to the reported protocol by Carreira
and co-workers.20 The CuBr was prepared according to the re-
ported protocol.46 The wet CH2Cl2 was prepared as follows, the
mixture of CH2Cl2 (20 mL) and water (1 mL) in a vial (50 mL)
was shaken vigorously, and a portion of CH2Cl2 layer was used
for the hydrolysis of cyclic ketal and t-butyl ester in 14a-f.
Synthesis of skeletally diversified tetracyclic peroxides All
synthetic procedures and analytical data for compounds (15–
19) shown in Figures 5 were reported in the supporting infor-
mation of the previous papers.13a,b
ers were washed with brine. After filtration and concentration
in vacuo, the residue was purified by silica-gel column chroma-
tography (CH2Cl2/EtOAc) to afford amine 10a (3.80 g, 12.4
mmol, 97%) as a brown oil. 10a: 1H-NMR (500 MHz, CDCl3):
δ 7.21 (2H, d, J = 8.6 Hz), 6.84 (2H, d, J = 8.6 Hz,), 6.62 (1H,
td, J = 7.3, 1.3 Hz), 3.76 (3H, s), 3.71 (2H, s), 2.71 (2H, t, J =
7.3 Hz), 2.34 (2H, q, J = 7.3 Hz), 1.78 (3H, d, J = 1.1 Hz), 1.46
(9H, s); 13C{1H}-NMR (125 MHz, CDCl3): δ 167.4, 158.7,
138.5, 132.3, 130.6, 129.3, 113.8, 80.1, 55.2, 53.3, 47.9, 29.5,
28.1, 12.6; HRMS (ESI, m/z): calcd. for C18H28NO3, [M+H]+
306.2064; found, 306.2080.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-(2-(tert-butyl)-5,5-dimethyl-1,3-dioxan-2-yl)propanal
12b To a solution of ketone 22 (1.00 g, 7.15 mmol) and 2,2-
dimethyl-1,3-propanediol (1.86 g, 17.9 mmol) in CH2Cl2 (15
mL) was added triethylorthoformate (2.97 mL, 18.0 mmol) and
p-toluenesulfonic acid monohydrate (142 mg, 0.739 mmol). Af-
ter being stirred for 14 h at 40 °C, additional portion of 2,2-
dimethyl-1,3-propanediol (1.86 g, 17.9 mmol), triethylorthofor-
mate (4.47 mL, 27.1 mmol) and p-toluenesulfonic acid mono-
hydrate (160 mg, 0.841 mmol) were added. After being stirred
for 48 h at 40 °C, the reaction mixture was cooled to room tem-
perature and quenched with Na2CO3 (1.14 g). The mixture was
stirred for 1 h, and the resulting suspension was filtrated through
a cotton plug. After concentration of the filtrate in vacuo, the
residue was purified by silica-gel column chromatography
(Hexane/EtOAc) to afford the corresponding ketal (637 mg,
2.81 mmol, 39%) as a colorless oil.
To a solution of the ketal (2.35g, 10.4 mmol) in MeOH (8.0
mL) and CH2Cl2 (43 mL) was added NaHCO3 (638 mg, 7.59
mmol). A stream of O3 (1.0 L/min) was bubbled into the solu-
tion at −78 °C until a persistent blue color was observed. The
solution was then purged with O2 until the blue color disap-
peared. This process, treatment with O3 followed by the O2
purge, was repeated for three times. The resulting mixture was
diluted with MeOH (43 mL) and treated with tri-
phenylphosphine (4.09 g, 15.6 mmol) at −78 °C. The mixture
was then warmed up to room temperature. After being stirred
for 2 h, the mixture was cooled to 0 °C and filtrated through a
cotton plug. The filtrate was concentrated in vacuo, and the res-
idue was purified by silica-gel column chromatography (Hex-
ane/EtOAc) to afford aldehyde 12b (1.98 g, 8.67 mmol, 84%)
as a colorless oil. 12b: 1H-NMR (300 MHz, CDCl3): δ 9.81 (1H,
t, J = 0.9 Hz), 3.52 (2H, d, J = 11.4 Hz), 3.37–3.28 (2H, m),
2.65–2.56 (2H, m), 2.26–2.16 (2H, m), 1.16 (3H, s), 0.99 (9H,
s), 0.71 (3H, s); 13C{1H}-NMR (75 MHz, CDCl3) δ 201.9, 101.8,
70.5, 40.9, 40.5, 29.7, 25.7, 23.2, 22.6, 16.6; HRMS (ESI, m/z):
calcd. for C13H25O3, [M+H]+ 229.1798; found, 229.1784.
Synthesis of racemic ene-yne 13b To a solution of amine
10b (2.91g, 10.0 mmol) and aldehyde 12a (2.24 g, 12.0 mmol)
in dioxane (8.0 mL) was added CuBr (143 mg, 1.00 mmol),
N,N,N',N'-tetramethylethylenediamine (151 µL, 1.00 mmol),
and dioxane (7.0 mL). The resulting mixture was divided into
two halves, and the micro-wave assisted reaction was con-
ducted with two glass vials (Biotage microwave vial: 10–20
mL) sealed with Teflon septum lined cap. Each vial was initially
sonicated at room temperature for 15 min under nitrogen atmos-
phere. The mixture was then treated with trimethylsilylacety-
lene 11 (885 µL, 6.25 mmol: the half quantity for each vial, total
1.77 mL, 12.5 mmol) at room temperature and then heated at
150 °C with microwave irradiation. The resultant mixture was
filtered twice through cotton plugs, and the residues were
washed with hexane and Et2O. The filtrate was concentrated in
Synthesis of 6-aza-artemisinins Synthetic protocols and ana-
lytical data for compounds (10a–10b, 12a, 13a–13b, 14a–14c,
24–35, 41) were provided in the supporting information of the
previous communication.14 All synthetic procedures and analyt-
ical data for new compounds (12b, 13c–13d, 14d–f, 36–40, 42–
45) were described in this supporting information. Improved
synthetic protocols for compounds (10a, 14a), nickel-mediated
cyclization of ene-yne 13b as well as three-component conden-
sation leading to the racemic ene-yne 13b were also provided
below.
Modified synthetic procedure of tert-butyl (E)-5-((4-meth-
oxybenzyl)amino)-2-methylpent-2-enoate 10a To a solution
of sulfonamide 20 (3.00 g, 9.31 mmol) in a 1:1 mixture tolu-
ene/THF (9.4 mL) was added n-Bu4NCl (518 mg, 1.86 mmol).
The reaction mixture was treated with NaOAc (764 mg, 9.32
mmol) and acrolein (1.86 mL, 27.9 mmol) at 0 °C, then the re-
sulting mixture was warmed up to room temperature and stirred
for 1.5 h. After evaporation of solvents in vacuo keeping tem-
perature at around 10 °C, the residue was then dissolved in dry
CH2Cl2 (60 mL). The solution was treated with
Ph3P=C(CH3)CO2t-Bu (7.28 g, 18.6 mmol) and stirred for 12 h
at room temperature. The resulting mixture was filtered through
a short pad of Celite and washed with CH2Cl2 (30 mL x 2). After
concentration of the filtrate in vacuo, the residue was purified
by silica-gel column chromatography (Hexane/EtOAc) to af-
ford the corresponding trisubstituted olefin (1.87 g, 3.81 mmol,
41%) as a colorless oil.
To a solution of the trisubstituted olefin (6.28 g, 12.8 mmol)
in CH3CN (51 mL) was added cesium carbonate (5.01 g, 15.4
mmol) and benzenethiol (1.42 mL, 13.8 mmol) at 0 °C. After
being stirred for 18 h at room temperature, water (100 mL) was
added. After separation of the organic layer, aqueous layer was
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The Journal of Organic Chemistry
vacuo, and the residue was purified by silica-gel column chro- mixture was poured into a solution of saturated aqueous Na-
1
2
3
4
5
6
7
8
matography (Hexane/EtOAc) to afford racemic ene-yne 13b
(4.81 g, 8.62 mmol, 86%) as a pale yellow oil. The analytical
data was provided the previous communication.14
HCO3 (65 mL) and EtOAc (80 mL) at 0 °C, and then stirred for
20 min. Organic layer was separated, and the aqueous layer was
extracted with EtOAc (100 mL x 2). The combined organic ex-
tracts were washed with brine, dried over Na2SO4. After filtra-
tion and concentration in vacuo, the residue was purified by sil-
ica-gel column chromatography (Hexane/EtOAc) to afford 14a
(377 mg, 0.657 mmol, 40%) as a colorless oil. 14a: [α]25D −12°
(c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3): δ 7.25 (2H, d, J =
8.5 Hz), 6.87–6.78 (2H, d, J = 8.5 Hz), 5.33 (1H, s), 3.79 (3H,
s), 3.71 (1H, d, J = 13.2 Hz), 3.59 (1H, d, J = 13.2 Hz), 3.53
(2H, d, J = 11.0 Hz), 3.47 (2H, d, J = 11.0 Hz), 3.14 (1H, dd, J
= 11.3, 3.2 Hz), 3.05 (1H, td, J = 13.9, 2.1 Hz), 2.74–2.61 (1H,
m), 2.49–2.38 (2H, m), 2.38–2.23 (1H, m), 2.16–2.05 (1H, m),
1.98–1.81 (1H, m), 1.46–1.32 (14H, m), 1.24–1.14 (1H, m),
1.01 (3H, d, J = 6.9 Hz), 0.98 (3H, s), 0.94 (3H, s), 0.05 (9H, s);
13C{1H}-NMR (125 MHz, CDCl3): δ 176.2, 158.7, 154.3, 132.2,
130.9, 129.8, 113.6, 99.0, 80.1, 70.4, 70.3, 64.1, 57.5, 55.3, 49.0,
42.9, 39.7, 34.3, 29.9, 28.2, 27.3, 24.8, 22.8, 22.6, 22.1, 16.8,
0.6; HRMS (ESI, m/z): calcd. for C33H56NO5Si, [M+H]+
574.3922; found, 574.3926.
Catalytic asymmetric synthesis of chiral ene-yne 13c A
mixture of 4Å molecular sieves (8.32 g), CuBr (9.50 mg, 0.0662
mmol) and (R, M)-PINAP (46.6 mg, 0.0833 mmol) in dimethyl
carbonate (20 mL) was stirred for 2 h at room temperature. To
the reaction mixture was added a solution of amine 10a (2.79 g,
9.15 mmol) in dimethyl carbonate (9.0 mL), a solution of alde-
hyde 12b (1.90 g, 8.32 mmol) in dimethyl carbonate (9.0 mL)
and then trimethylsilylacetylene 11 (2.42 mL, 17.5 mmol) at
room temperature. After being stirred for 23 h, the reaction mix-
ture was filtered through a pad of Celite. After concentration of
the filtrate in vacuo, the residue was purified by silica-gel col-
umn chromatography (Hexane/EtOAc) to afford the ene-yne
9
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13
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13c (5.02 g, 8.18 mmol, 98%, 97% ee) as a colorless oil. 13c:
1
[α]22 +81° (c 1.0, CHCl3); H-NMR (500 MHz, CDCl3): δ
D
7.29–7.23 (2H, m), 6.84 (2H, dt, J = 9.3, 2.4 Hz), 6.67 (1H, td,
J = 7.2, 1.3 Hz), 3.82–3.71 (4H, m), 3.53 (2H, d, J = 11.5 Hz),
3.40–3.23 (4H, m), 2.70–2.57 (1H, m), 2.56–2.46 (1H, m),
2.39–2.28 (2H, m), 2.01–1.81 (2H, m), 1.77 (3H, s), 1.74–1.63
(2H, m), 1.49 (9H, s), 1.14 (3H, s), 0.98 (9H, s), 0.65 (3H, s),
0.20 (9H, s); 13C{1H}-NMR (75 MHz, CDCl3): δ 167.5, 158.7,
139.1, 131.7, 130.0, 129.9, 113.8, 104.7, 102.2, 89.4, 80.0, 70.5,
70.4, 55.3, 54.8, 54.2, 49.3, 40.7, 29.6, 29.3, 28.3, 27.7, 25.9,
23.3, 23.2, 22.6, 12.6, 0.5; HRMS (ESI, m/z): calcd. for
C36H60NO5Si, [M+H]+ 614.4236; found, 614.426.
Nickel-mediated cyclization of ene-yne 13b leading to
piperizine 14c A Biotage microwave vial was charged with
Ni(COD)2 (235 mg, 0.854 mmol) and DBU (255 µL, 1.71
mmol) and THF (10 mL) in the globe box. The vial was sealed
with Teflon septum lined cap. After being stirred for 3 min, a
solution of 13b (318 mg, 0.569 mmol) was added. The reaction
mixture was heated at 125 °C for 5 h under microwave irradia-
tion. After cooling to room temperature, the mixture was treated
with water, stirred for 15 min, and then extracted with EtOAc.
Organic extract was washed with saturated aqueous NaHCO3
and brine, dried over Na2SO4. After filtration and concentration
in vacuo, the residue was purified by silica-gel column chroma-
Catalytic asymmetric synthesis of chiral ene-yne 13d A
mixture of 4Å molecular sieves (1.71 g), CuBr (4.80 mg, 0.0335
mmol) and (R, M)-PINAP (20.7 mg, 0.0370 mmol) in dimethyl
carbonate (3.5 mL) was stirred for 2 h at room temperature. To
the reaction mixture was added a solution of amine 10b (547
mg, 1.88 mmol) in dimethyl carbonate (2.4 mL), a solution of
aldehyde 12b (390 mg, 1.71 mmol) in dimethyl carbonate (2.4
mL) and then trimethylsilylacetylene 11 (497 μL, 3.59 mmol)
at room temperature. After being stirred for 16 h, additional so-
lution of amine 10b (99.7 mg, 0.342 mmol) in dimethyl car-
bonate (0.25 mL) was added to the solution. After being stirred
for 4 h, the reaction mixture was filtered through a pad of Celite
and washed with EtOAc. After concentration of the filtrate in
vacuo, the residue was purified by silica-gel column chroma-
tography (Hexane/EtOAc) to afford ene-yne 13d (1.00 g, 1.67
mmol, 97%, 88% ee) as a colorless oil. 13d: [α]20D +69° (c 1.0,
tography (Hexane/EtOAc) to afford 14c (150 mg, 0.268 mmol,
1
47%). 14c: [α]25 −22° (c 1.0, CHCl3); H-NMR (500 MHz,
D
CDCl3): δ 7.24 (2H, d, J = 8.5 Hz), 6.83 (2H, d, J = 8.5 Hz),
5.15 (1H, s), 3.79 (3H, s), 3.67 (1H, d, J = 13.6 Hz), 3.60–3.51
(3H, m), 3.47 (2H, dd, J = 11.5, 6.0 Hz), 3.39–3.32 (1H, m),
2.96 (1H, td, J = 12.8, 3.6 Hz), 2.86–2.77 (1H, m), 2.58–2.47
(2H, m), 2.15 (1H, dd, J = 15.3, 7.3 Hz), 2.02–1.83 (2H, m),
1.69–1.57 (2H, m), 1.56–1.45 (2H, m), 1.43 (9H, s), 1.38 (3H,
s), 1.00 (3H, s), 0.94 (3H, s), 0.04 (9H, s); 13C{1H}-NMR (125
MHz, CDCl3): δ 172.3, 158.7, 158.3, 132.2, 129.8, 120.1, 113.7,
99.3, 80.2, 70.5, 70.4, 65.2, 57.7, 55.4, 44.1, 39.4, 36.6, 32.9,
31.2, 30.1, 28.2, 23.0, 22.7, 22.5, 22.4, 0.9; HRMS (ESI, m/z):
calcd. for C32H54NO5Si, [M+H]+ 560.3766; found, 560.3795.
Piperizine 14d A flame-dried 50 mL two-necked round-bot-
tom flask was charged with diisopropylamine (0.34 ml, 2.4
mmol) in dry THF (10.5 mL) and then cooled to −78 °C. A so-
lution of n-BuLi (1.50 mL, 2.33 mmol, 1.55 M in n-hexane) was
slowly added dropwise. The mixture was warmed up to 0 °C
and stirred for 40 min. After being cooled to −78 °C, a solution
of substrate 14b (420 mg, 0.750 mmol) in dry THF (10.5 mL)
was added dropwise. The mixture was stirred at −78 °C for 5
min, and then warmed up to room temperature and stirred for 1
h. After being cooled to −78 °C, BrCH2CH2Cl (0.40 mL, 4.8
mmol) was added dropwise. The mixture was warmed to room
temperature and stirred for 2 h. The resulting mixture was
treated with Et2O (20 mL) and H2O (5 mL) at 0 °C. After sepa-
ration of organic layer, aqueous layer was extracted with Et2O
(50 mL x 2). The combined organic layers were washed with
brine, dried over Na2SO4. After filtration and concentration in
1
CHCl3); H-NMR (300 MHz, CDCl3): δ 7.25 (2H, d, J = 8.5
Hz), 6.88–6.78 (3H, m), 5.74 (1H, d, J = 15.8 Hz), 3.79 (3H, s),
3.75 (1H, d, J = 13.9 Hz), 3.53 (2H, d, J = 11.7 Hz), 3.36–3.25
(4H, m), 2.73–2.59 (1H, m), 2.58–2.44 (1H, m), 2.44–2.23 (2H,
m), 2.00–1.76 (2H, m), 1.74–1.60 (2H, m), 1.48 (9H, s), 1.14
(3H, s), 0.97 (9H, s), 0.65 (3H, s), 0.19 (9H, s); 13C{1H}-NMR
(75 MHz, CDCl3): δ 166.0, 158.8, 146.1, 131.6, 130.1, 124.0,
113.8, 104.7, 102.2, 89.5, 80.2, 70.5, 70.4, 55.4, 54.9, 54.3, 49.4,
40.7, 31.2, 29.7, 29.3, 28.3, 26.0, 23.3, 22.6, 0.5; HRMS (ESI,
m/z): calcd. for C35H58NO5Si, [M+H]+ 600.4079; found,
600.4085.
Improved synthetic procedure for diastereoselective cy-
clization leading to piperizine 14a To a solution of 13a (940
mg, 1.64 mmol) in i-Pr2O (31 mL) was added Ti(Oi-Pr)4 (0.997
mL, 3.37 mmol) and then treated dropwise with i-PrMgCl (6.73
mL, 6.73 mmol, 1.0 M solution in Et2O) at −78 °C. The reaction
mixture was gradually warmed up to −30 °C over 100 min and
then stirred at −30 °C for 17 h. The reaction mixture was then
treated with 1 M HCl (25 mL) and EtOAc (70 mL) at −30 °C.
After being stirred at room temperature for 1 h, the resultant
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vacuo, the residue was purified by silica-gel column chroma- (200 mL). The combined organic extracts were washed with
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tography (Hexane/EtOAc) to give a 50:50 mixture of diastere-
omers (322 mg, 0.517 mmol, 69%) as a colorless oil.
brine, dried over Na2SO4. After filtration and concentration in
vacuo, the residue was purified by silica-gel column chroma-
tography (Hexane/CH2Cl2/MeCN) to afford 14f (383 mg, 0.636
mmol, 38%) as a colorless oil. 14f: [α]21D −21° (c 1.0, CHCl3);
1H-NMR (500 MHz, CDCl3): δ 7.28 (2H, d, J = 8.4 Hz), 6.83
(2H, d, J = 8.4 Hz), 5.41 (1H, s), 3.79 (3H, s), 3.78–3.71 (3H,
m), 3.57 (1H, d, J = 13.4), 3.36–3.27 (2H, m), 3.11–2.98 (2H,
m), 2.89 (1H, br. q, J = 6.9 Hz), 2.60 (1H, t, J = 12.9 Hz), 2.49–
2.38 (3H, m), 2.24–2.12 (1H, m), 1.91 (1H, br. q, J = 11.7 Hz),
1.64–1.53 (1H, m), 1.49–1.33 (10H, m), 1.20–1.14 (4H,
m),1.03 (9H, s), 0.73 (3H, s), 0.08 (9H, s); 13C{1H}-NMR (100
MHz, CDCl3): δ 172.0, 158.8, 157.8, 132.3, 130.0, 128.1, 113.7,
102.5, 80.4, 70.5, 69.8, 65.0, 57.7, 55.4, 42.7, 42.0, 40.7, 39.0,
30.8, 29.8, 28.4, 26.1, 25.2, 24.6, 23.5, 22.6, 0.7; HRMS (ESI,
m/z): calcd. for C35H60NO5Si, [M+H]+ 602.4239; found,
602.4242.
Improved synthesis of 6-aza-artemisinin 24 To a solution
of 14a (509 mg, 0.887 mmol) in wet CH2Cl2 (8.87 mL) was
added trifluoroacetic acid (4.44 mL) at 0 °C. The reaction mix-
ture was warmed up to room temperature and then stirred for
1.5 h. After concentration in vacuo, the residue containing the
corresponding ammonium salt was subjected to azeotropic de-
hydration with dry toluene for three times. The crude ammo-
nium salt was dissolved in CH2Cl2 (120 mL) and transferred
into a 300 mL three-necked round bottom flask. A stream of O3
(1.0 L/min) was bubbled into the solution for 5 min at −78 °C.
The solution was then purged with nitrogen gas until the blue
color disappeared. This process, treatment with O3 followed by
the nitrogen purge, was repeated twice. The resulting mixture
was then treated with trifluoroacetic acid (135 µL, 1.77 mmol)
at −78 °C, then warmed up to room temperature and stirred for
12 h. The reaction mixture was quenched with saturated aque-
ous solution of NaHCO3 (70 mL) at 0 °C. After separation of
organic layer, the aqueous layer was extracted with CH2Cl2 (150
mL x 2). The combined organic layers were dried over Na2SO4.
After filtration and concentration in vacuo, the residue was pu-
rified by silica-gel column chromatography (Hexane/AcOEt) to
To a mixture of the resultant diastereomers (782 mg, 1.25
mmol) in THF/DMPU (1/1) (12.6 mL) was treated dropwise
with lithium bis(trimethylsilyl)amide (2.80 mL, 2.80 mmol, 1.0
M solution in THF) at −78 °C. The mixture was warmed up to
room temperature and stirred for 4.5 h. The resulting mixture
was added with Et2O (180 mL) and H2O (65 mL) at 0 °C. After
separation of organic layer, aqueous layer was extracted with
Et2O (200 mL x 2). The combined organic layers were washed
with brine, dried over Na2SO4. After filtration and concentration
in vacuo, the residue was purified by silica-gel column chroma-
tography (Hexane/EtOAc) to afford 14d (635 mg, 1.08 mmol,
86%) as a yellow oil. 14d: [α]21D −13° (c 1.0, CHCl3); 1H-NMR
(500 MHz, CDCl3): δ 7.22 (2H, d, J = 8.0 Hz), 6.83 (2H, d, J =
8.0 Hz), 5.53 (1H, m), 3.79 (3H, s), 3.69 (1H, d, J = 13.2 Hz),
3.63 (1H, d, J = 13.2 Hz), 3.53 (2H, d, J = 12.6 Hz), 3.47 (2H,
d, J = 11.5 Hz), 3.31 (1H, dd, J = 11.5, 3.4 Hz), 3.09–2.99 (1H,
m), 2.63–2.50 (1H, m), 2.34–2.17 (2H, m), 2.08–1.95 (1H, m),
1.95–1.84 (1H, m), 1.79–1.66 (2H, m), 1.58 (1H, td, J = 12.9,
4.2 Hz), 1.46–1.35 (13H, m), 1.09–1.03 (1H, m), 0.99 (3H, s),
0.94 (3H, s), 0.82–0.72 (1H, m), 0.71–0.61 (1H, m), 0.05 (9H,
s); 13C{1H}-NMR (125 MHz, CDCl3): δ 173.9, 158.8, 158.6,
132.8, 129.7, 123.0, 113.7, 99.3, 80.3, 70.5, 70.4, 63.2, 55.4,
45.8, 44.9, 33.9, 30.4, 30.1, 28.3, 26.8, 24.9, 22.93, 22.87, 22.5,
16.7, 13.9, 0.7; HRMS (ESI, m/z): calcd. for C34H56NO5Si,
[M+H]+ 586.3928; found, 586.3939.
9
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24
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Piperizine 14e To a solution of 13c (3.90 g, 6.35 mmol) in
(i-Pr)2O (129 mL) was added Ti(Oi-Pr)4 (3.76 mL, 12.4 mmol)
and then treated dropwise with i-PrMgCl (12.7 mL, 25.4 mmol,
2.0 M solution in Et2O) at −78 °C. The reaction mixture was
gradually warmed up to −30 °C over 100 min. After being
o
stirred for 18 h at −30 C, 1 M HCl (64 mL) and EtOAc (180
mL) was added. The reaction mixture was warmed up to room
temperature and then poured into a solution of saturated aque-
ous NaHCO3 (80 mL) and EtOAc (150 mL) at 0 °C. Organic
layer was separated, and the aqueous layer was extracted with
EtOAc (200 mL). The combined organic extracts were washed
with brine, dried over Na2SO4. After filtration and concentration
in vacuo, the residue was purified by silica-gel column chroma-
tography (Hexane/CH2Cl2/MeCN) to afford 14e (1.19 g, 1.93
afford 24 (160 mg, 0.411 mmol, 46% from 14a) as a colorless
1
foamy solid. 24: [α]25 +1.4° (c 1.0, CHCl3); H-NMR (500
D
MHz, CDCl3): δ 7.17 (2H, d, J = 8.5 Hz), 6.85 (2H, d, J = 8.5
Hz), 6.28 (1H, s), 4.10 (1H, d, J = 13.2 Hz), 3.80 (3H, s), 3.39–
3.31 (1H, m), 3.03 (1H, d, J = 13.2 Hz), 2.85 (1H, dt, J = 12.0,
3.2 Hz), 2.52–2.43 (1H, m), 2.35 (1H, dd, J = 10.1, 7.3 Hz),
2.27–2.19 (1H, m), 2.10 (1H, ddd, J = 14.8, 4.9, 3.3 Hz), 1.94–
1.80 (2H, m), 1.76 (1H, dt, J = 13.6, 5.0 Hz), 1.71–1.64 (1H,
m), 1.47 (3H, s), 1.24–1.15 (4H, m); 13C{1H}-NMR (125 MHz,
CDCl3): δ 171.7, 158.9, 130.5, 129.8, 113.9, 105.0, 94.0, 77.5,
65.1, 56.2, 55.3, 51.1, 44.1, 34.5, 32.7, 25.7, 25.1, 23.5, 12.4;
HRMS (ESI, m/z): calcd. for C21H28NO6, [M+H]+ 390.1911;
found, 390.1903.
6-aza-artemisinin 42 To a solution of 14d (214 mg, 0.365
mmol) in wet CH2Cl2 (5.4 mL) was added trifluoroaceticacid
(3.4 mL) at 0 °C. The reaction mixture was warmed up to room
temperature and then stirred for 2 h. After concentration in
vacuo, the residue containing the corresponding crude ammo-
nium salt was directly used for the next reaction without further
purification.
mmol, 30%) as a yellow oil. 14e: [α]22 −2.7° (c 1.0, CHCl3);
D
1H-NMR (500 MHz, CDCl3): δ 7.30–7.24 (2H, m), 6.84–6.81
(2H, m), 5.34 (1H, s), 3.79 (3H, s), 3.78–3.67 (3H, m), 3.58 (1H,
d, J = 13.2 Hz), 3.32 (2H, ddd, J = 15.5, 11.2, 2.6 Hz), 3.13–
3.02 (2H, m), 2.69–2.56 (2H, m), 2.49–2.39 (2H, m), 2.19–2.07
(1H, m), 1.84 (1H, q, J = 11.6 Hz), 1.58–1.51 (1H, m), 1.42 (9H,
s), 1.34–1.27 (1H, m), 1.21–1.14 (4H, m), 1.04–0.99 (12H, m),
0.73 (3H, s), 0.10 (9H, s); 13C{1H}-NMR (75 MHz, CDCl3): δ
176.3, 158.7, 154.9, 132.3, 130.7, 130.0, 113.6, 102.5, 80.3,
70.5, 69.8, 65.0, 57.6, 55.4, 49.2, 42.6, 40.6, 39.2, 29.8, 29.4,
28.2, 26.0, 24.3, 23.4, 22.6, 16.8, 0.7; HRMS (ESI, m/z): calcd.
for C36H62NO5Si, [M+H]+ 616.4392; found, 616.4431.
Piperizine 14f To a solution of 13d (1.00 g, 1.67 mmol) in
(i-Pr)2O (33 mL) was added Ti(Oi-Pr)4 (1.18 mL, 4.00 mmol)
and then treated dropwise with i-PrMgCl (3.33 mL, 6.66 mmol,
2.0 M solution in Et2O) at −78 °C. The reaction mixture was
gradually warmed to −40 °C over 90 min. After being stirred
for 3 h at −40 °C, 1 M HCl (17 mL) and EtOAc (50 mL) was
added. The reaction mixture was warmed up to room tempera-
ture and then poured into a solution of saturated aqueous Na-
HCO3 (20 mL) and EtOAc (100 mL) at 0 °C. Organic layer was
separated, and the aqueous layer was extracted with EtOAc
The crude ammonium salt was dissolved in CH2Cl2 (34 mL)
and transferred into a two-necked round bottom flask. A stream
of O3 (1.0 L/min) was bubbled into the solution for 5 min at
−78 °C. The solution was then purged with nitrogen gas until
the blue color disappeared. This process, treatment with O3 fol-
lowed by the nitrogen purge, was repeated for two times. The
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The Journal of Organic Chemistry
resulting mixture was then treated with Amberlyst-15 (149 mg)
12.7; HRMS (ESI, m/z): calcd. for C24H34NO5, [M+H]+
416.2432; found, 416.2431.
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at −78 °C, then warmed up to room temperature and stirred for
23 h. The reaction mixture was quenched with saturated aque-
ous solution of NaHCO3 (20 mL) at 0 °C and stirred for 5 min.
After filtration and separation of organic layer, the aqueous
layer was extracted with CH2Cl2 (150 mL x 2). The combined
organic layers were dried over K2CO3. After filtration and con-
centration in vacuo, the residue was purified by silica-gel col-
umn chromatography (Hexane/EtOAc) to afford 42 (16.1 mg,
6-aza-artemisinin 44 To a solution of 14f (298 mg, 0.495
mmol) in wet CH2Cl2 (5.0 mL) was added trifluoroacetic acid
(2.5 mL) at 0 °C. The reaction mixture was warmed up to room
temperature and then stirred for 1.5 h. After concentration in
vacuo, the residue containing the corresponding crude ammo-
nium salt was directly used for the next reaction without further
purification.
40.1 µmol, 11% from 14d) as a colorless foamy solid. 42: [α]21
D
The crude ammonium salt was dissolved in CH2Cl2 (98 mL)
and transferred into a three-necked round bottom flask. A
stream of O3 (1.0 L/min) was bubbled into the solution for 5
min at −78 °C. The solution was then purged with nitrogen gas
until the blue color disappeared. This process, treatment with
O3 followed by the nitrogen purge, was repeated for three times.
The resulting mixture was then treated with trifluoroacetic acid
(75.8 μL, 0.990 mmol) at −78 °C, then warmed up to room tem-
perature and stirred for 8 h. The reaction mixture was quenched
with saturated aqueous solution of NaHCO3 (45 mL) at 0 °C.
After separation of organic layer, the aqueous layer was ex-
tracted with CH2Cl2 (100 mL x 2). The combined organic layers
were dried over K2CO3. After filtration and concentration in
vacuo, the residue was purified by silica-gel column chroma-
tography (Hexane/CH2Cl2) to afford 44 (29.4 mg, 0.0704 mmol,
14% from 14f) as a white solid. 44: [α]21D +12° (c 1.0, CHCl3);
1H-NMR (500 MHz, CDCl3): δ 7.19 (2H, d, J = 8.0 Hz), 6.85
(2H, d, J = 8.0 Hz), 6.35 (1H, s), 4.11 (1H, d, J = 12.6 Hz), 3.80
(3H, s), 3.16 (1H, dd, J = 18.3, 6.9 Hz), 3.00 (1H, d, J = 13.2
Hz), 2.80 (1H, dt, J = 12.0, 2.9 Hz), 2.38–2.23 (5H, m), 1.96–
1.85 (2H, m), 1.81–1.72 (1H, m), 1.60–1.44 (2H, m), 1.00 (9H,
s); 13C{1H}-NMR (125 MHz, CDCl3): δ 168.8, 158.9, 130.6,
129.9, 113.9, 109.4, 94.1, 76.4, 65.2, 56.3, 55.4, 51.2, 38.6, 38.0,
31.2, 29.2, 28.7, 26.0, 24.2; HRMS (ESI, m/z): calcd. for
C23H32NO6, [M+H]+ 418.2224; found, 418.2227.
6-aza-artemisinin 36 To a solution of amine 27 (37.2 mg,
0.138 mmol) in 1,2-dichloroethane (2.8 mL) was added 3-fur-
aldehyde (26.6 mg, 0.277 mmol), acetic acid (7.9 µL, 0.138
mmol) and NaBH(OAc)3 (117 mg, 0.552 mmol) at room tem-
perature. After being stirred for 11 h at 39 °C, the resulting mix-
ture was cooled to room temperature, diluted with CH2Cl2 (10
mL) and quenched with saturated aqueous solution of NaHCO3
(8 mL) at 0 °C. After separation of organic layer, aqueous layer
was extracted with CH2Cl2 (30 mL x 2). The combined organic
extracts were washed with brine, dried over K2CO3. After fil-
tration and concentration in vacuo, the residue was purified by
silica-gel column chromatography (Hexane/EtOAc) to afford
36 (40.9 mg, 0.117 mmol, 85%) as a light brown solid. 36:
[α]22D +9.3° (c 2.0, CHCl3); 1H-NMR (500 MHz, CDCl3): δ 7.39
(1H, t, J = 1.7 Hz), 7.30 (1H, s), 6.30 (1H, d, J = 1.1 Hz), 6.19
(1H, s), 3.78 (1H, d, J = 14.3 Hz), 3.39 (1H, d, J = 14.3 Hz),
3.35–3.28 (1H, m), 2.95–2.89 (1H, m), 2.49–2.39 (1H, m), 2.27
(1H, dd, J = 10.3, 6.9 Hz), 2.21–2.12 (1H, m), 2.12–2.03 (2H,
m), 1.84–1.67 (3H, m), 1.45 (3H, s), 1.29–1.21 (1H, m), 1.18
(3H, d, J = 7.3 Hz); 13C{1H}-NMR (125 MHz, CDCl3): δ 171.8,
143.3, 140.8, 120.4, 111.3, 105.0, 93.9, 77.4, 63.9, 51.2, 47.0,
44.0, 34.5, 32.7, 25.3, 25.1, 23.4, 12.4; HRMS (ESI, m/z): calcd.
for C18H24NO6, [M+H]+ 350.1598; found, 350.1598.
−18° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3): δ 7.18 (2H,
d, J = 8.6 Hz), 6.86 (2H, d, J = 8.6 Hz), 6.42 (1H, s), 4.10 (1H,
d, J = 13.2 Hz), 3.80 (3H, s), 3.02 (1H, d, J = 13.2 Hz), 2.82
(1H, dt, J = 11.6, 3.3 Hz), 2.52–2.44 (1H, m), 2.34 (1H, dd, J =
10.3, 6.9 Hz), 2.27–2.20 (1H, m), 2.12 (1H, dd, J = 14.9, 2.7
Hz), 1.93–1.81 (2H, m), 1.63–1.46 (7H, m), 1.12 (1H, dd, J =
13.2, 5.2 Hz), 0.98–0.93 (1H, m), 0.73–0.67 (1H, m); 13C{1H}-
NMR (125 MHz, CDCl3): δ 172.3, 158.9, 130.6, 129.9, 113.9,
105.1, 94.0, 78.3, 65.4, 56.3, 55.4, 51.3, 46.4, 34.6, 27.8, 25.8,
25.3, 23.5, 20.7, 12.1; HRMS (ESI, m/z): calcd. for C22H28NO6
[M+H]+ 402.1917; found, 402.1921.
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6-aza-artemisinin 43 To a solution of 14e (476 mg, 0.773
mmol) in wet CH2Cl2 (7.7 mL)8 was added trifluoroacetic acid
(3.9 mL) at 0 °C. The reaction mixture was warmed up to room
temperature and then stirred for 2 h. After concentration in
vacuo, the residue containing the corresponding crude ammo-
nium salt was directly used for the next reaction without further
purification.
The crude ammonium salt was dissolved in CH2Cl2 (106 mL)
and transferred into a three-necked round bottom flask. A
stream of O3 (1.0 L/min) was bubbled into the solution for 5
min at −78 °C. The solution was then purged with nitrogen gas
until the blue color disappeared. This process, treatment with
O3 followed by the nitrogen purge, was repeated for two times.
The resulting mixture was then treated with trifluoroacetic acid
(119 μL, 1.55 mmol) at −78 °C, then warmed up to room tem-
perature and stirred for 11 h. The reaction mixture was
quenched with saturated aqueous solution of NaHCO3 (50 mL)
at 0 °C. After separation of organic layer, the aqueous layer was
extracted with CH2Cl2 (100 mL x 2). The combined organic lay-
ers were dried over K2CO3. After filtration and concentration in
vacuo, the residue was purified by silica-gel column chroma-
tography (Hexane/CH2Cl2/MeCN) to afford 43 (20.3 mg, 47.0
μmol, 6.1% from 14e) as a white solid and 45 (8.9 mg, 21 μmol,
2.8%) as a white solid. 43: [α]22D −1.8° (c 1.0, CHCl3); 1H-NMR
(500 MHz, CDCl3): δ 7.19 (2H, d, J = 8.6 Hz), 6.85 (2H, d, J =
8.6 Hz), 6.30 (1H, s), 4.11 (1H, d, J = 13.2 Hz), 3.80 (3H, s),
3.37–3.31 (1H, m), 3.03 (1H, d, J = 13.2 Hz), 2.86 (1H, dt, J =
11.8, 3.2 Hz), 2.34–2.24 (4H, m), 1.89 (1H, td, J = 12.2, 2.7 Hz),
1.79–1.72 (2H, m), 1.71–1.65 (1H, m), 1.22–1.17 (4H, m), 1.00
(9H, s); 13C{1H}-NMR (125 MHz, CDCl3): δ 172.1, 158.9,
130.6, 129.9, 113.9, 109.3, 94.1, 65.0, 56.3, 55.4, 51.1, 44.1,
38.6, 32.8, 29.9, 29.2, 26.0, 24.2, 23.6, 12.5; HRMS (ESI, m/z):
calcd. for C24H34NO6, [M+H]+ 432.2381; found, 432.2383. 45:
[α]22 −117° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3): δ
D
7.17 (2H, d, J = 8.6 Hz), 6.85 (2H, d, J = 8.6 Hz), 6.07 (1H, s),
4.00 (1H, d, J = 13.2 Hz), 3.80 (3H, s), 3.14–3.06 (1H, m), 3.02
(1H, d, J = 13.2 Hz), 2.86 (1H, dt, J = 11.6, 3.3 Hz), 2.28 (1H,
q, J = 5.5 Hz), 2.20–2.14 (1H, m), 2.03 (1H, dt, J = 14.0, 4.4
Hz), 1.98–1.89 (2H, m), 1.79–1.70 (2H, m), 1.58–1.48 (1H, m),
1.19–1.13 (4H, m), 1.02 (9H, s); 13C{1H}-NMR (125 MHz,
CDCl3): δ 172.0, 158.8, 130.8, 129.8, 114.6, 113.9, 100.4, 81.0,
60.8, 55.4, 55.3, 51.3, 41.5, 36.6, 32.9, 27.2, 25.1, 24.0, 23.2,
6-aza-artemisinin 37 To a solution of amine 27 (30.2 mg,
0.112 mmol) in 1,2-dichloroethane (3 mL) was added 4-pyri-
dinecarboxaldehyde (24.0 mg, 0.224 mmol), acetic acid (12.8
µL, 0.112 mmol) and NaBH(OAc)3 (95.1 mg, 0.449 mmol) at
room temperature. After being stirred for 11 h at 40 °C, the re-
sulting mixture was cooled to room temperature, diluted with
CH2Cl2 (5 mL) and quenched with saturated aqueous solution
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6-aza-artemisinin 40 To a solution of amine 27 (32.9 mg,
of NaHCO3 (10 mL) at 0 °C. After separation of organic layer,
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aqueous layer was extracted with CH2Cl2 (30 mL x 2). The com-
bined organic extracts were washed with brine, dried over
K2CO3. After filtration and concentration in vacuo, the residue
was purified by silica-gel column chromatography
(CH2Cl2/MeOH) to afford 37 (28.6 mg, 0.0794 mmol, 71%) as
a brown oil. 37: [α]22D +1.7° (c 2.0, CHCl3); 1H-NMR (500 MHz,
CDCl3): δ 8.56–8.50 (2H, m), 7.21 (2H, d, J = 4.6 Hz), 6.28 (1H,
d, J = 1.1 Hz), 4.15 (1H, d, J = 14.3 Hz), 3.42–3.30 (1H, m),
3.12 (1H, d, J = 14.3 Hz), 2.80 (1H, d, J = 11.5 Hz), 2.55–2.36
(2H, m), 2.19–2.00 (3H, m), 1.81–1.67 (3H, m), 1.46 (3H, d, J
= 1.7 Hz), 1.29–1.17 (4H, m); 13C{1H}-NMR (125 MHz,
CDCl3): δ 171.6, 150.1, 148.3, 123.4, 105.1, 93.7, 77.3, 65.2,
56.1, 52.0, 44.0, 34.4, 32.7, 25.7, 25.1, 23.5, 12.4; HRMS (ESI,
m/z): calcd. for C19H25N2O5, [M+H]+ 361.1758; found,
361.1777.
6-aza-artemisinin 38 To a solution of amine 27 (26.3 mg,
0.0977 mmol) in 1,2-dichloroethane (2.5 mL) was added 3,4,5-
trifluorobenzaldehyde (31.3 mg, 0.196 mmol), acetic acid (5.6
µL, 0.098 mmol) and NaBH(OAc)3 (82.9 mg, 0.391 mmol) at
room temperature. After being stirred for 12 h at 39 °C, the re-
sulting mixture was cooled to room temperature, diluted with
CH2Cl2 (10 mL) and quenched with saturated aqueous solution
of NaHCO3 (9 mL) at 0 °C. After separation of organic layer,
aqueous layer was extracted with CH2Cl2 (40 mL x 2). The com-
bined organic extracts were washed with brine, dried over
K2CO3. After filtration and concentration in vacuo, the residue
was purified by silica-gel column chromatography (Hex-
ane/EtOAc) to afford 38 (22.8 mg, 0.0552 mmol, 56%) as a
white solid. 38: [α]22D +8.2° (c 1.0, CHCl3); 1H-NMR (500 MHz,
CDCl3): δ 6.95–6.86 (2H, m), 6.25 (1H, s), 4.09 (1H, d, J = 14.3
Hz), 3.42–3.31 (1H, m), 3.02 (1H, d, J = 13.7 Hz), 2.81 (1H, dt,
J = 11.9, 3.3 Hz), 2.55–2.43 (1H, m), 2.39 (1H, dd, J = 10.0, 7.2
Hz), 2.22–2.05 (2H, m), 1.98 (1H, td, J = 12.2, 2.5 Hz), 1.84–
1.69 (3H, m), 1.47 (3H, s), 1.28–1.17 (4H, m); 13C{1H}-NMR
(125 MHz, CDCl3): δ 171.5, 152.4, 150.4, 135.4, 112.1, 111.95,
105.1, 93.7, 77.3, 65.1, 56.0, 51.7, 44.1, 34.4, 32.7, 25.7, 25.1,
23.5, 12.4; HRMS (ESI, m/z): calcd. for C20H23F3NO5, [M+H]+
414.1523; found, 414.1541.
0.122 mmol) in 1,2-dichloroethane (2.4 mL) was added 2-(4-
morpholinyl)-5-pyrimidinecarbaldehyde (47.2 mg, 0.244
mmol), acetic acid (7.0 µL, 0.12 mmol) and NaBH(OAc)3 (104
mg, 0.491 mmol) at room temperature. After being stirred for
10 h at 40 °C, the resulting mixture was cooled to room temper-
ature, diluted with CH2Cl2 (15 mL) and quenched with saturated
aqueous solution of NaHCO3 (10 mL) at 0 °C. After separation
of organic layer, aqueous layer was extracted with CH2Cl2 (40
mL x 2). The combined organic extracts were washed with brine,
dried over K2CO3. After filtration and concentration in vacuo,
the residue was purified by silica-gel column chromatography
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(CH2Cl2/MeOH) to afford 40 (37.7 mg, 0.0844 mmol, 69%) as
1
a white solid. 40: [α]22 –3.1° (c 1.0, CHCl3); H-NMR (500
D
MHz, CDCl3): δ 8.20 (2H, s), 6.17 (1H, s), 3.94 (1H, d, J = 13.7
Hz), 3.80–3.72 (8H, m), 3.33 (1H, qd, J = 7.3, 4.9 Hz), 3.03–
2.99 (1H, d, J = 14 Hz), 2.90–2.81 (1H, m), 2.53–2.40 (1H, m),
2.32 (1H, dd, J = 10.3, 6.9 Hz), 2.27–2.17 (1H, m), 2.10 (1H,
dq, J = 14.7, 2.7 Hz), 1.95 (1H, td, J = 11.9, 2.1 Hz), 1.87–1.78
(1H, m), 1.76–1.68 (2H, m), 1.46 (3H, s), 1.23–1.14 (4H, m);
13C{1H}-NMR (125 MHz, CDCl3): δ 171.6, 161.6, 158.4, 119.0,
105.0, 93.7, 77.3, 66.9, 64.8, 51.5, 50.9, 44.4, 44.1, 34.5, 32.6,
25.8, 25.1, 23.3, 12.4; HRMS (ESI, m/z): calcd. for C22H31N4O6,
[M+H]+ 447.2238; found, 447.2227.
In vitro anti-malarial assay against Plasmodium falciparum
In vitro cultivation and in vitro anti-malarial activities against
Plasmodium falciparum K1 and FCR3 strains were measured,
using the method described previously.14,15 Briefly, P. falcipa-
rum Kl (chloroquine resistant strain) and FCR3 (chloroquine
sensitive strain) were cultured in human erythrocytes in RPMI
medium supplemented with 10% human plasma at 37°C, under
93% N2, 4% CO2, and 3% O2. Asynchronous parasites (2%
hematocrit and 0.5 or 1% parasitemia) were seeded in a 96-well
micro-titer plate and serially diluted test compounds were added.
Chloroquine (K1: 30 µM-7.5 µM, FCR3: 1 µM-0.25 µM) and
artesunate (2500 µM-0.052 µM) were added in a similar fashion.
After 72-hours incubation, parasite lactate dehydrogenase (p-
LDH) was assayed using a slight modification of the procedure
reported by Makler et al47 and Vivas et al.48 The 50% inhibitory
concentration (IC50) value was calculated from a dose response
curve.
6-aza-artemisinin 39 To a solution of amine 27 (47.1 mg,
0.175 mmol) in 1,2-dichloroethane (3 mL) was added 7-azain-
dole-3-carboxaldehyde (51.2 mg, 0.350 mmol), acetic acid
(20.0 µL, 0.350 mmol) and NaBH(OAc)3 (148 mg, 0.698
mmol) at room temperature. After being stirred for 12 h at 40 °C,
the resulting mixture was cooled to room temperature, diluted
with CH2Cl2 (10 mL) and quenched with saturated aqueous so-
lution of NaHCO3 (15 mL) at 0 °C. After separation of organic
layer, aqueous layer was extracted with CH2Cl2 (30 mL x 2).
The combined organic extracts were washed with brine, dried
over K2CO3. After filtration and concentration in vacuo, the res-
idue was purified by silica-gel column chromatography
Cytotoxic assay against MRC-5 cells Measurement of cyto-
toxic activity against human fetal lung fibroblast MRC-5 cells
was carried out as described previously.49
In vivo anti-malarial assay against P. berghei N strain in
mouse model. In vivo anti-malarial activities of test com-
pounds were evaluated by a mouse model infected with rodent
malaria Plasmodium berghei N strain, according to “Peter’s 4-
days suppressive test” as described previously.14,15 Male CD-1
(ICR) mice (n = 5, Charles River Japan Inc., Japan) at weight
of ca. 20 g were infected intravenously with a 2 x 106 parasitized
red blood cells of P. berghei N strain (donated by Dr. W Peters,
Northwick Park Institute for Medical Research, UK), and para-
sitemia determined using standard blood smear techniques. All
test compounds were prepared in 10% DMSO/0.5% Tween80
aqueous solution prior to either intraperitoneal (i.p.) or oral
(p.o.) administration. Treatment was started 2 hours after the
mice had been inoculated with the parasite on day 0. Treatment
was then continued daily for 4 days from day 0 to day 3. On day
4, thin blood smears were made from blood samples collected
from tails of each mouse to determine parasitemia and percent-
age inhibition. Percentage inhibition was calculated using the
(CH2Cl2/MeOH) to afford 39 (27.8 mg, 0.0696 mmol, 40%) as
1
a white solid. 39: [α]22 –0.2° (c 2.0, CHCl3); H-NMR (500
D
MHz, CDCl3): δ 10.82 (1H, s), 8.34 (1H, dd, J = 4.8, 1.3 Hz),
8.01 (1H, dd, J = 7.7, 1.4 Hz), 7.26 (1H, br. s), 7.10 (1H, dd, J
= 7.7, 4.9 Hz), 6.29 (1H, s), 4.32 (1H, d, J = 13.2 Hz), 3.38–
3.32 (2H, m), 2.98 (1H, dt, J = 11.6, 3.3 Hz), 2.57–2.47 (1H,
m), 2.38–2.30 (2H, m), 2.15 (1H, dq, J = 14.9, 2.7 Hz), 2.00–
1.89 (2H, m), 1.75 (1H, td, J = 9.2, 4.8 Hz), 1.70–1.63 (1H, m),
1.49 (3H, s), 1.21–1.12 (4H, m); 13C{1H}-NMR (125 MHz,
CDCl3): δ 171.8, 149.3, 143.0, 128.3, 124.1, 120.3, 115.9, 111.0,
105.1, 94.1, 77.6, 65.4, 51.2, 48.5, 44.2, 34.6, 32.7, 25.8, 25.2,
23.5, 12.4; HRMS (ESI, m/z): calcd. for C21H26N3O5, [M+H]+
400.1867; found, 400.1875.
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The Journal of Organic Chemistry
following formula: Percentage inhibition (%) = [(mean para- Shun Suzuki – Division of Chemistry, Graduate School of
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8
Science, Hokkaido University, North 10 West 8, Kita-ku, Sap-
poro 060-0810, Japan
sitemia of negative control-mean parasitemia of treated
group)/mean parasitemia of negative control] x 100.
Yutaka Yamagishiv – Division of Chemistry, Graduate
School of Science, Hokkaido University, North 10 West 8,
Kita-ku, Sapporo 060-0810, Japan
Hideaki Oikawa – Division of Chemistry, Graduate School of
Science, Hokkaido University, North 10 West 8, Kita-ku, Sap-
poro 060-0810, Japan
Aki Ishiyama – Research Center for Tropical Diseases, Kita-
sato Institute for Life Sciences, Kitasato University, 5-9-1
Shirokane, Minato-ku, Tokyo 108-8641, Japan
Rei Hokari – Research Center for Tropical Diseases, Kitasato
Institute for Life Sciences, Kitasato University, 5-9-1 Shiro-
kane, Minato-ku, Tokyo 108-8641, Japan
Kazuhiko Otoguro – Research Center for Tropical Diseases,
Kitasato Institute for Life Sciences, Kitasato University, 5-9-
1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Satoshi Ōmura – Research Center for Tropical Diseases,
Kitasato Institute for Life Sciences, Kitasato University, 5-9-
1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
In addition, each mouse was monitored the survival and par-
asitemia on day 7. The mice surviving beyond day 7 were con-
tinuously monitored to record the survival time. All analyses
were performed by using JMP® 8 (SAS institute Inc., Cary, NC,
USA). Results of parasitemia at day 4 were analyzed using Dun-
nett’s-test. Survival data were analyzed using Kaplan-Meier
method with log-rank significance test. The data was consid-
ered significant at P < 0.05.
This study was approved by “The Kitasato Institute Hospital
Research Ethics Committee (No. 12102)” for the donation of
human blood from volunteers. The approval for the amimal ex-
periment was obtained from the Institutional Animal Care and
Use Committee for Kitasato University.
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In vivo imaging system (IVIS)26a Female CD-1 mice (7–8-
week-old, 23–26 g, n = 4 per experimental group;) were intra-
venously inoculated with approximately 1 × 105 P. berghei
(ANKA GFP-luc) asexual-stage parasites 24 h before treatment
and compounds were administered orally (at 0 h). The biolumi-
nescence signal was measured by the in vivo imaging system
(IVIS SpectrumCT, PerkinElmer) every six hours for the first
24 hours (at 6, 12, 18, and 24 h) and every 12 hours on the sec-
ond day (at 36 and 48 h). Luciferin (150 mg kg−1) was intraper-
itoneally injected approximately 10 min before imaging.
Nobutaka Kato – Broad Institute of Harvard and MIT, 415
Main Street, Cambridge, MA 02142, USA.
The two authors, Karunakar Reddy Bonepally and Norihito
Takahashi contributed equally to this work.
Notes: A patent application for anti-malarial 6-aza-artemisinins
has been filed by Tokyo University of Agriculture & Technol-
ogy and Kitasato University.
■ AUTHOR INFORMATION
Corresponding Authors
Masato Iwatsuki – Research Center for Tropical Diseases,
Kitasato Institute for Life Sciences, Kitasato University, 5-9-
1 Shirokane, Minato-ku, Tokyo 108-8641, Japan; or-
cid.org/0000-0001-8373-4314; Email: iwatuki@lisci.kita-
sato-u.ac.jp
Hiroki Oguri – Department of Chemistry, School of Science,
The University of Tokyo, 7‒3‒1, Hongo, Bunkyo-ku 113‒
0033, Japan; orcid.org/0000-0001-8007-1631; Email:
oguri@chem.s.u-tokyo.ac.jp
■ ACKNOWLEDGMENTS
The authors thank Drs. Timothy N. C. Wells, Jeremy Burrows,
and Didier Leroy of Medicines for Malaria Venture (MMV) as
well as Prof. Stuart L Schreiber (Broad Institute and Harvard
Univ.) for their invaluable advice and continuous support. The
authors thank Mr. Takashi Matsumoto (Rigaku) and Mr. Ryo
Watanabe (Hokkaido Univ.) for X-ray analysis. This work was
supported in part by the GHIT fund HTLP RFP (2014-001),
JSPS KAKENHI Grant Numbers JP23310156, JP26102702,
JP16H01135, and JP18H04388, the Naito Foundation, and the
Uehara Memorial Foundation. This work was inspired by the
international and interdisciplinary environments of the JSPS
Asian CORE Program, “Asian Chemical Biology Initiative”,
and JSPS A3 Foresight Program.
Authors
Karunakar Reddy Bonepally – Division of Applied Chem-
istry, Graduate School of Engineering, Tokyo University of
Agriculture and Technology, 2-24-16 Nakacho, Koganei, To-
kyo 184-8588, Japan
Norihito Takahashi – Division of Applied Chemistry, Grad-
uate School of Engineering, Tokyo University of Agriculture
and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588,
Japan
Naoya Matsuoka – Division of Applied Chemistry, Graduate
School of Engineering, Tokyo University of Agriculture and
Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588,
Japan
■ ASSOCIATED CONTENT
Supporting Information
The Supporting Information providing NMR spectra and chi-
ral HPLC analysis is available free of charge on the ACS Publi-
cations website at DOI:.
■ REFERENCES
1. World Malaria Report 2019; World Health Organization:
Geneva, 2019.
Hikari Koi – Division of Applied Chemistry, Graduate School
of Engineering, Tokyo University of Agriculture and Technol-
ogy, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan
Haruki Mizoguchi – Division of Chemistry, Graduate School
of Science, Hokkaido University, North 10 West 8, Kita-ku,
Sapporo 060-0810, Japan
Takahisa Hiruma – Division of Chemistry, Graduate School
of Science, Hokkaido University, North 10 West 8, Kita-ku,
Sapporo 060-0810, Japan
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