Lead Optimization of Second-Generation Acridones as Broad-Spectrum Antimalarials

Article abstract of DOI:10.1021/acs.jmedchem.0c00539

The global impact of malaria remains staggering despite extensive efforts to eradicate the disease. With increasing drug resistance and the absence of a clinically available vaccine, there is an urgent need for novel, affordable, and safe drugs for prevention and treatment of malaria. Previously, we described a novel antimalarial acridone chemotype that is potent against both blood-stage and liver-stage malaria parasites. Here, we describe an optimization process that has produced a second-generation acridone series with significant improvements in efficacy, metabolic stability, pharmacokinetics, and safety profiles. These findings highlight the therapeutic potential of dual-stage targeting acridones as novel drug candidates for further preclinical development.

Full text of DOI:10.1021/acs.jmedchem.0c00539

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Article
Lead Optimization of Second-Generation
Acridones as Broad-Spectrum Antimalarials
Papireddy Kancharla, Rozalia A. Dodean, Yuexin Li, Sovitj Pou, Brandon Pybus, Victor Melendez,
Lisa Read, Charles E. Bane, Brian Vesely, Mara Kreishman-Deitrick, Chad Black, Qigui Li, Richard
J. Sciotti, Raul Olmeda, Thu-Lan Luong, Heather Gaona, Brittney Potter, Jason Sousa, sean
marcsisin, Diana Caridha, Lisa Xie, Chau Vuong, Qiang Zeng, Jing Zhang, Ping Zhang, Hsiuling
Lin, Kirk Butler, Norma Roncal, Lacy Gaynor-Ohnstad, Susan E. Leed, Christina Nolan, Frida G.
Ceja, Stephanie A. Rasmussen, Patrick K. Tumwebaze, Philip J. Rosenthal, Jianbing Mu, Brett R.
Bayles, Roland Cooper, Kevin A. Reynolds, Martin J. Smilkstein, Michael K. Riscoe, and Jane X. Kelly
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00539 • Publication Date (Web): 11 May 2020
Downloaded from pubs.acs.org on May 12, 2020
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Zhang, Ping ; Walter Reed Army Institute of Research, Division of
Experimental Therapeutics
Lin, Hsiuling ; Walter Reed Army Institute of Research, Division of
Experimental Therapeutics
Butler, Kirk; Walter Reed Army Institute of Research, Division of
Experimental Therapeutics
Roncal, Norma ; Walter Reed Army Institute of Research, Division of
Experimental Therapeutics
Gaynor-Ohnstad, Lacy ; Walter Reed Army Institute of Research,
Division of Experimental Therapeutics
Leed, Susan; Walter Reed Army Institute of Research, Division of
Experimental Therapeutics
Nolan, Christina ; Walter Reed Army Institute of Research, Division of
Experimental Therapeutics
Ceja, Frida ; Dominican University of California, Department of Natural
Sciences and Mathematics
Rasmussen, Stephanie ; Dominican University of California, Department
of Natural Sciences and Mathematics
Tumwebaze, Patrick ; Infectious Diseases Research Collaboration
Rosenthal, Philip; University of California San Francisco, Department of
Medicine
Mu, Jianbing; National Institutes of Health,
Bayles, Brett ; Dominican University of California, Department of Natural
Sciences and Mathematics
Cooper, Roland; Dominican University of California, Department of
Natural Sciences & Mathematics
Reynolds, Kevin; Portland State University, Department of Chemistry
Smilkstein, Martin; Portland Veterans Affairs Medical Center,
Riscoe, Michael; VA Medical Center, Portland State Univ, Experimental
Chemotherapy Lab
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Kelly, Jane; Oregon Health and Science University,
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Lead Optimization of Second-Generation Acridones
as Broad-Spectrum Antimalarials
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Papireddy Kancharla,*,†,#Rozalia A. Dodean, ‡,# Yuexin Li, ‡,# Sovitj
‡
§
Brandon Pybus,§ Victor Melendez, Lisa Read, Charles E.
§
§
Pou,
§
§
§
Bane, Brian Vesely, Mara Kreishman-Deitrick, Chad Black, Qigui
Li,§ Richard J. Sciotti,§ Raul Olmeda,§ Thu-Lan Luong,§ Heather
Gaona,§ Brittney Potter,§ Jason Sousa,§ Sean Marcsisin,§ Diana
Caridha,§ Lisa Xie,§ Chau Vuong,§ Qiang Zeng,§ Jing Zhang,§ Ping
Zhang,§ Hsiuling Lin,§ Kirk Butler,§ Norma Roncal,§ Lacy Gaynor-
Ohnstad,§ Susan E. Leed,§ Christina Nolan,§ Frida G. Ceja,
Stephanie A. Rasmussen, Patrick K. Tumwebaze, ‖
Philip J.
Rosenthal, Jianbing Mu, Brett R. Bayles,, Roland A. Cooper,


Kevin A. Reynolds,† Martin J. Smilkstein, Michael K. Riscoe,
‡
†, ‡
and Jane X. Kelly*,†, ‡
†Department of Chemistry, Portland State University, Portland,
Oregon 97201, United States
‡Department of Veterans Affairs Medical Center, Portland, Oregon
97239, United States
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§Division of Experimental Therapeutics, Walter Reed Army
Institute of Research, Silver Spring, Maryland 20910, United
States
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Department of Natural Sciences and Mathematics, Dominican
University of California, San Rafael, CA 94901, United States
‖Infectious Diseases Research Collaboration, Kampala, Uganda

Department of Medicine, University of California, San Francisco,
CA 94143, United States

Laboratory of Malaria and Vector Research, National Institute
of Allergy and Infectious Diseases, National Institutes of
Health, Rockville MD 20852, USA.

Global Public Health Program, Dominican University of
California, San Rafael CA 94901
KEYWORDS.
Malaria,
lead
optimization,
antimalarials,
antiplasmodial activity, second-generation, acridones, Plasmodium
falciparum, dual-stage activity, broad-spectrum.
ABSTRACT
The global impact of malaria remains staggering despite extensive
efforts to eradicate the disease. With increasing drug resistance
and the absence of a clinically available vaccine, there is an
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urgent need for novel, affordable, and safe drugs for prevention
and treatment of malaria. Previously we described
a
novel
antimalarial acridone chemotype that is potent against both blood-
stage and liver-stage malaria parasites. Here we describe an
0
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0
optimization process that has produced
a
second-generation
acridone series with significant improvements in efficacy,
metabolic stability, pharmacokinetics, and safety profiles. These
findings highlight the therapeutic potential of dual-stage
targeting acridones as novel drug candidates for further
preclinical development.
INTRODUCTION
Malaria has been a scourge on humankind down through the centuries
of recorded time and even now it accounts for over 200 million
clinical cases worldwide each year along with roughly half a
million deaths.1, 2 The global eradication effort in the past decade
has propelled a surge in antimalarial drug development.3-5 However,
many challenges remain for a sustainable elimination campaign,
including the failing effectiveness of front-line artemisinin-
combination therapy (ACT) due to emerging resistance,6-8 and safety
concerns associated with limited radical cure options for
relapsing Plasmodium vivax.9,
10
Without any clinically proven
vaccine in sight, there is a continuous need to develop new drugs
that overcome artemisinin resistance and ideally such agents would
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be active in both treatment of active bloodstream infections as
well as preventing the disease at the liver-stage.3, 4, 11-13
We recently described acridones with dual-stage activity against
both liver- and blood-stage malaria parasites.14 However, these
acridones only displayed moderate metabolic stability along with
an unsatisfactory risk of cardiotoxicity and evidence of cross-
resistance to atovaquone (ATV) in P. falciparum. With the aim of
improving metabolic stability, safety profile and other desired
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“drug-like” properties, we created
a
second-generation of
acridones by varying the halogen substitution pattern on ring-A
and placement of aryl groups on ring-B (Figure 1). A rigorous lead
optimization process produced candidates with enhanced potency
against blood- and liver-stage malaria parasites, improved
metabolic stability and PK profile, and with a diminished risk of
cardiotoxicity and cross-resistance with ATV, while maintaining a
high selectivity index toward killing the parasites.
O
O
alkyl
N
1
A
4
8
O-Ar
NH-Ar
Ar
F
Cl
OMe
F
Cl
O
alkyl
B
n
N
H
N
H
5
O-CH -Ar
2
First-generation
Second-generation
Figure 1. Transition from first-generation acridones to the
second-generation acridones.
RESULTS AND DISCUSSION
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Chemistry. Our previous structural optimizations of the first-
generation acridones demonstrated that halogen substitutions on
ring-A and (dialkylamino)alkoxy moieties at the 6 position on ring-
B are required for optimal broad-spectrum antimalarial activity.14
Subsequently, we have designed and synthesized a large library of
the second-generation acridones with various mono- and di-
substitutions on ring-A and aryloxy, arylamino, aryl or benzyloxy
moieties at either the 6 or 7 position of ring-B, via simple and
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efficient synthetic routes from commercially available and
inexpensive starting materials. The synthetic routes for the
second-generation acridones are outlined in Schemes 1−3.
Synthesis of 6-aryloxy-, 6-arylamino- and 6-aryl-acridones
(18−58). Synthesis of the first series of acridones 18−58 with
various mono and di-substitutions on ring-A and aryloxy, arylamino
and aryl moieties at the 6 position of ring-B started from
commercially available methyl 4-iodo-2-methoxybenzoate (1) and
methyl 4-bromo-2-methoxybenzoate (2). Initially the copper-
catalyzed arylation15 of
1
with phenols 3a−e delivered the
corresponding diaryl ether intermediates 4a−e, which were then
converted into 2-hydroxy-diaryl ethers 5a−e, by selective
deprotection of the methoxy (OMe) group with boron trichloride
16
(BCl ).
Intermediates 5a−e were treated with triflic anhydride
3
(
Tf O) in the presence of pyridine to give the triflates 6a−e,
2
which were further subjected to Buchwald-Hartwig cross coupling
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with various substituted anilines 7a−l to give the key anthranilate
intermediates 8a−q (Scheme 1).14,
17
Conversely, synthesis of
anthranilate intermediates 12a−j and 17a−f, key synthons in the
synthesis of acridones 43−52 and 53−58, commenced with the coupling
of 2 with corresponding anilines 7m−q and boronic acids 13a−d under
standard Buchwald-Hartwig and Suzuki cross-coupling reaction
conditions to derive intermediates 9a−e and 14a−d, respectively.
Intermediates 9a−e and 14a−d then delivered 12a−j and 17a−f in
good yields, via the appropriate intermediates 10a−e, 11a−e and
1
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0
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15a−d, 16a−d, respectively. Finally, our recently developed method
18
(boron trifluoride etherate (BF ·Et O)/microwave irradiation)
3
2
efficiently delivered the desired acridones 18−42 and 53−58 in
good to excellent yields in short reaction time (1.0 min), via an
intramolecular cyclization of the anthranilate intermediates 8a−q
and 17a−f, respectively (Scheme 1). Similarly, Eaton’s acid-
mediated cyclization of 12a−j provided the corresponding acridones
4
3−52 in good yields.14 Positional isomers were separated either
by recrystallization or flash column chromatography.
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O
O
O
MeO
MeO
MeO
MeO
HO
+
R1
i
R1
ii
R1
I
HO
MeO
X
X
1
1
1
4a-e: X = O
9a-e: X = NH
3
a: R = 4-OCF ; 3b: R = 4-Cl
5a-e: X = O
10a-e: X = NH
3
1
1
3
c: R = 3-OCF ; 3d: R = 2-OCF
3
3
1
3e: R = 4-
N
O
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iii
O
7m-q
iv
O
MeO
MeO
O
MeO
R3
Br
R2
R1
MeO
TfO
R1
7a-l
2
N
X
X
H
iv
8
a-q: X = O
12a-j: X = NH
B(OH)2
6a-e: X = O
1a-e: X = NH
1
1
v or vi
13a: R = Cl
vii
1
1
3b: R = OCF
3
1
1
3c: R = H
O
O
R1 13d: R =
1
R3
R2
R3
N
O
R1
R2
O
N
X
N
H
H
MeO
R1
1
2
3
53-58
1
8-42: X = O; R , R , R = Various substitutions
MeO
O
1
2
3
R1, R2, R3 = Various substitutions
4
3-52: X = NH; R , R , R = Various substitutions
1
4a-d
R1
v
ii
O
O
R3
MeO
MeO
MeO
TfO
iii
7d/7h/7k
R2
HO
N
H
iv
1
5a-d
R1
R1
R1
16a-d
17a-f
NH2
NH2
NH2
NH2
NH2
NH2
Cl
F
F
Cl
Cl
Cl
Cl
F
Cl
F
7
a
7d
NH2
7e
7f
Cl
7b
7c
NH2
NH2
NH2
NH2
NH2
Cl
OMe
F
OMe
OMe
OMe
OMe
7l
7
g
7h
OMe
7j
F
7i
Cl
7k
H2N
H2N
H2N
OCF3
H2N
H2N
O
N
OCF3
CF3O
Cl
7q
7n
7
o
7p
7m
a
Reagents and conditions: (i) K2CO3, CuCl, N,N-dimethylglycine, 1-methyl-2-pyrrolidone, MS 4A , 150 oC, 4 h; (ii) BCl3, DCM, -78 oC-
o
rt, 1 h; (iii) Tf2O, pyridine, DCM, 0 oC-rt, 5 h; (iv) Pd(OAc)2, XPhos, Cs2CO3, toluene, 110 oC, 5 h; (v) BF3.OEt2, mw, 150 oC, 1.0 min;
o
(
vi) Eaton's acid, 90 oC, 5 h; (vii) Pd(PPh3)4, Na2CO3, dioxane/H2O (9:1), 100 C, 5 h.
Scheme 1. Synthesis of Acridones (18−58) with Aryloxy, Arylamino
and Aryl Groups at the 6 Position of Ring-Ba
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Synthesis of 7-aryloxy- and 7-arylamino-acridones (68−80). The
second series of acridones 68−80 with di-substitutions on ring-A,
and aryloxy and arylamino moieties at the 7 position of ring-B
were synthesized from commercially available methyl 5-iodo-2-
methoxybenzoate (59) accompanying 3a and methyl 5-bromo-2-
hydroxybenzoate (60) with appropriate anilines 7, via the same
synthetic methods that were employed in the Scheme 1. Eaton’s acid
or polyphosphoric acid (PPA)-mediated cyclization of the key
anthranilate intermediates 66a−c and 67a−i provided the desired
acridones 68−71 and 72−80, respectively, in good yields (Scheme
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
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4
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4
4
4
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5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
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5
6
7
8
9
0
2).
O
O
O
I
OCF3
O
Br
MeO
MeO
i
MeO
MeO
+
HO
MeO
ii
OCF3
iii
HO
59
3a
61
60
+
R1
O
O
H2N
iv
X
iii
X
1
7
m: R = 4-OCF
MeO
MeO
HO
3
R1
4: X = O
R1
1
7n
:
R = 3-OCF3
1
TfO
7o: R = 2-OCF3
6
6
62: X = O
3a-c: X = NH
5a-c: X = NH
6
7
O
O
MeO
R3
R3
X
X
R2
R1
R2
R1
v or vi
N
H
N
H
1
2
3
6
6
6a-c: X = O
7a-i: X = NH
68-71: X = O; R , R , R = Various substitutions
1
2
3
72-80: X = NH; R , R , R = Various substitutions
a
o
o
Reagents and conditions: (i) K2CO3, CuCl, N,N-dimethylglycine, 1-methyl-2-pyrrolidone, MS 4A , 150 C, 4 h; (ii) BCl3,
DCM, -78 oC-rt, 1 h; (iii) Pd(OAc)2, XPhos, Cs2CO3, toluene, 110 oC, 5 h; (iv) Tf2O, pyridine, DCM, 0 oC-rt, 5 h; (v)
o
o
Eaton's acid, 90 C, 5 h; (vi) PPA, 110 C, 2 h.
Scheme 2. Synthesis of Acridones (68−80) with Aryloxy and Arylamino
Groups at the 7 Position of Ring-Ba
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesis of 6- and 7-benzyloxy-acridones (86−93, 99−102 and
07−109). The third series of acridones 86−93, 99−102 and 107−109
1
with di-substitutions on ring-A and a variety of benzyloxy moieties
at either the 6 or 7 position of ring-B were synthesized starting
with commercially available methyl 2-hydroxy-4-methoxybenzoate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(81), methyl 2,4-dihydroxybenzoate (94) and methyl 2-bromo-5-
methoxybenzoate (103) (Scheme 3). The key hydroxy-acridone
intermediates 85a−c and 106 were synthesized via the same synthetic
methods that were employed in the previous Schemes 1 and 2. By
treating with various benzyl bromides in the presence of anhydrous
K CO , 85a−c and 106 provided the corresponding acridones 86−93 and
2
3
107−109 in good to excellent yields (top and bottom panels, Scheme
3). Conversely, a different strategy was employed for the synthesis
of acridones 99−102 (Scheme 3, middle panel), in which the
benzyloxy group was installed at the 6 position of ring-B before
the Buchwald-Hartwig cross coupling reaction. Synthesis of 99−102
began with the O-benzylation of 94 in the presence of appropriate
benzyl bromides and K CO to provide the 4-benzyloxy intermediates
2
3
95a and 95b.19 Intermediates 95a and 95b were treated with Tf O in
2
the presence of pyridine to give the triflate intermediate 96a and
9
6b, which were further subjected to Buchwald-Hartwig cross-
coupling with methoxy- anilines to give the anthranilate
intermediates 97a−d. BF ·Et O-mediated cyclization of 97a−d under
7
3
2
microwave irradiation conditions provided the desired acridones
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9
9
9−102 as major products along with the 6-hydroxy-acridones 98a
and 98b as minor products. These two products were separated by
column chromatography using a mixture of ethyl acetate and MeOH as
a mobile phase. Upon treatment with benzyl bromides in the presence
of anhydrous K CO , 98a and 98b efficiently delivered the desired
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
acridones 99−102 in excellent yields.19 It is noteworthy that this
new synthetic strategy (Scheme 3, middle panel) efficiently
generates acridones that contain a OMe group on ring-A and
benzyloxy groups on ring-B.
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
7
i
MeO
HO
MeO
MeO
HN
ii
OMe
TfO
OMe
OMe
83a-c
8
1
82
R2
R1
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R3
iii
R1
O
O
R1
R1
v
iv
N
O
R2
H
8
6-93
1
2
R , R = F, Cl
R2
N
OH
N
OMe
R2
H
H
3
R = H, OCF , CF , SCF , Cl
85a-c
84a-c
3
3
3
O
O
O
MeO
MeO
TfO
MeO
i
v
HO
O
O
HO
OH
R2
R1
R1
94
1
9
5a: R = OCF
96a-b
3
1
95b: R = CF
3
O
7, ii
MeO
N
H
O
R1
iii
9
9-102
+
v
O
MeO
O
R2
R2
MeO
MeO
N
O
H
N
H
OH
97a-d
R1
98a-b
1
2
R = OCF , CF ; R = F, Cl
3
3
F
O
O
O
OMe
OMe
7
OMe
vi
MeO
MeO
ii
F
N
H
Br
HN
1
03
104
105
iv
F
F
F
O
F
O
O
OH
1
1
1
1
07: R = OCF3
v
1
08: R = CF3
F
N
H
1
F
N
09: R = Cl
H
106
R1
a
o
o
Reagents and conditions: (i) Tf O, pyridine, DCM, 0 C-rt, 5 h; (ii) Pd(OAc) , XPhos, Cs CO , toluene, 110 C, 5 h;
2
2
2
3
o
(
iii) BF .OEt , mw, 150 C, 1.0 min; (iv) HI, phenol, reflux, 3 h; (v) appropriate benzyl bromide, K CO , acetone,
3
2
2
3
o
reflux, 3 h; (vi) Eaton's acid, 90 C, 5 h.
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7
8
9
Scheme 3. Synthesis of Acridones (86−93, 99−102 and 107−109) with
Benzyloxy Groups at the 6 Position (Top and Middle Panel) and the
7
Position (Bottom Panel) of the Ring-Ba
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Biological Activity. The library of second-generation acridones
with various mono- and disubstitutions on ring-A and aryloxy,
arylamino, aryl or benzyloxy moieties at either the 6 or 7 position
of ring-B were evaluated for both blood-stage and liver-stage
antimalarial efficacy, safety, and metabolic/PK properties. The
results are summarized and discussed in the following sections.
In
Vitro
Blood-Stage
Activity
and
Structure−Activity
Relationships (SAR) of 6-Aryloxy- Acridones (18−42). In our
previous work, a number of first-generation acridones with di-
halogens on ring-A and (dialkylamino)alkoxy moieties at the 6
position on ring-B showed promising potency and efficacy against
both blood-stage and liver-stage malarial infections.14 Considering
that the alkyl moieties are probably easily metabolized in vivo,
we hypothesized that the replacement of the (dialkylamino)alkoxy
groups on ring-B with aryloxy groups might improve the metabolic
stability and PK profiles. Therefore, we generated an initial
series of acridones 18−42 bearing mono and di-substitutions on
ring-A and aryloxy moieties at the 6 position of ring-B (Table 1).
In vitro antimalarial blood-stage activity of acridone derivatives
was tested against
a
panel of multidrug-resistant (MDR) P.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
falciparum (D6, Dd2, 7G8 and TM90-C2B) with different geographic
and genetic backgrounds using a SYBR Green based assay20 and the
results are listed in Table 1. Chloroquine (CQ) and atovaquone
(ATV) were used as reference drugs in all experiments.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Starting from our first-generation lead acridone compound T31,
the (dialkylamino)alkoxy group at the 6 position on ring-B of T31
was first replaced with 4-(trifluoromethoxy)phenyloxy moiety,
leading to acridone 18. Although 18 exhibited respectable in vitro
antiplasmodial potency with low nanomolar IC50 values against all
tested P. falciparum strains (Table 1), it was less potent than
T31 (18 IC50 = 3.2 nM vs. T31 IC50 = 0.022 nM against D6, Table 1).
Notably, compound 18 showed reduced cross-resistance with ATV
against Tm90-C2B (ATV resistant clinical isolate) compared to T31
and ATV (the ratio of Tm90-C2B IC /D6 IC for 18 = 329 vs. T31 =
50
50
10363 and ATV = 82560). These findings demonstrated that the
aryloxy moiety at the 6 position of ring-B is beneficial for
overcoming ATV cross-resistance. Consequently, we retained the
aryloxy moiety at the 6 position on ring-B in further modifications
and introduced various mono- and di-substitutions at different
positions on ring-A. Shifting the chlorine (Cl) of 18 from position
1
to the position 3 as in 19, led to 6-fold reduced antiplasmodial
potency (19 IC50 = 21 nM vs. 18 IC50 = 3.2 nM against D6), whereas
acridone 20, with Cl substitution of 18 shifting from position 2
to the position 3, showed 6-fold enhanced potency (20 IC50 = 0.52
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nM vs. 18 IC50 = 3.2 nM against D6). Removal of Cl substitution at
position 1 of 20, as in acridone compound 21, showed 80% decrease
in antiplasmodial activity (21 IC50
=
2.6 nM vs. 20 IC50 = 0.52 nM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
against D6). Replacement of the dichloro substitutions at
positions 1 and 3 of ring-A from 20 with difluoro substitutions,
as in acridone compound 22, led to significant increase (>100-
fold) in the antiplasmodial potency (22 IC50 = 0.0047 nM vs. 20
IC50 = 0.52 nM against D6) with excellent selectivity. Only a two-
fold antiplasmodial potency loss was observed when the OCF group
3
of 6-aryloxy moiety on ring-B of 22 was replaced with Cl as in 23
(23 IC50 = 0.010 nM vs. 22 IC50 = 0.0047 nM against D6). The greatest
loss of antiplasmodial potency was observed with mono fluoro
substituted
acridones
24
and
25
(51-fold
and
680-fold,
respectively) when compared with the corresponding difluoro
substituted acridones 22. Interestingly, the antiplasmodial
potency was retained against all tested P. falciparum strains even
after introducing a combination of F and Cl substitutions at both
positions 1 and 3 on ring-A with acridones 26 (IC50 = 0.028 nM
against D6) and 27 (IC50 = 0.050 nM against D6). These results
demonstrated that the dihalogen substitutions on ring-A aryloxy
moiety at the 6 position on ring-B are required for optimal
potency. The cross-resistance against blood-stage parasites Tm90-
C2B for mono and di-halogen substituted acridone compounds 18–27
was reduced compared to the first-generation acridones (Table 1).
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To further diminish cross-resistance with ATV, we generated a
series of acridones 28–42, with halogen and methoxy moieties on
ring-A and aryloxy groups at the 6 position of ring-B, then tested
for blood-stage antiplasmodial activity (Table 1). The acridones
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28−30, which contain a halogen at position 1 and OMe group at
position 3 on ring-A and an aryloxy moiety at position 6 on ring-B,
retained the potency against all tested P. falciparum strains as
compared to the corresponding di-halogenated acridones 20, 22, 23,
26 and 27. In contrast, the 6-aryloxy-acridones 31−33 containing
a methoxy group at the 1 position and halogen at the 3 position
on ring-A, showed decreased antiplasmodial activities compared to
the corresponding positional isomers 28−30 (31 IC50 = 28 nM vs. 28
IC50 = 5.0 nM; 32 IC50
nM vs. 30 IC50
=
54 nM vs. 29 IC50 = 0.65 nM; 33 IC50 = 18
=
0.51 nM against D6). These observations
demonstrated that the combination of halogen and OMe groups at
position 1 and 3, respectively, are well tolerated on ring-A,
regardless of the substitutions on aryloxy moiety at position 6 of
ring-B. Shifting the OMe of 28 from position 3 to position 2 as in
34, led to a minimal reduction in antiplasmodial potency (34 IC50
=
17 nM vs. 28 IC50 = 5.0 nM against D6). A nearly 10-fold loss of
activity occurred when the Cl of 34 shifted from position 1 to
position 3 on ring-A as in acridone 35 (35 IC50 = 157 nM vs. 34
IC50 = 17 nM against D6). Complete removal of a halogen substitution
at position 3 on ring-A of acridone 31 as with the analogue 36
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showed a minimal effect on the antiplasmodial activity (36 IC50
=
40 nM vs. 31 IC50 = 28 nM against D6). Interestingly, acridone
compound 37, in which the 3 position of ring-A is substituted with
OMe group, exhibited significantly increased antiplasmodial
potency compared to the corresponding positional isomer 36 (37 IC50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
0.33 nM vs. 36 IC50 = 40 nM against D6). This work suggested
that the OMe group at both positions 1 and 2 on ring-A had adverse
effect on antiplasmodial activity, whereas the OMe group was well
tolerated at the 3 position on ring-A. We next investigated the
effect of halogen and OMe substitutions at positions 2 and 3 on
ring-A. It is noteworthy that the 2,3-disubstituted-acridones
3
8−42 with different aryloxy moieties at the 6 position of ring-
B, containing OMe group at position 3 on ring-A showed superior
antiplasmodial potency with low nanomolar concentrations (IC50
<
2.8 nM across the entire test panel) and diminished cross-
resistance with ATV against Tm90-C2B (ratio of Tm90-C2B IC /D6
50
IC50 < 1.5). Most of the acridone compounds containing OMe group
on ring-A (compounds 31, 32, and 35−42) also displayed diminished
cross-resistance with ATV, compared to the first-generation lead
candidate T31 and corresponding di-halogenated 6-aryloxy-acridones
18, 20, 22, 23, 26 and 27. Taken together, SAR analyses of acridone
compounds 18−42 (Table 1) demonstrated that a halogen in position
1 or 2 and OMe group at position 3 of ring-A, along with an aryloxy
moiety at the 6 position on ring-B are best tolerated.
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3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. In Vitro Blood-Stage Antiplasmodial Activity and
Selectivity of Acridones (18−42)
Cl
O
O
1 1
A
8
B
5
Cl
R
4’
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
R
C H
4
9
N
H
O
N
N
H
O
R2
4
2’
C H
4
9
T31
18-42
blood-stage IC50 (nM) a
cytotoxic
selectivity
index
ity IC50
vs. P. falciparum
a
(nM)
vs.
compd
T31
R1
R2
R3
vs.
HepG2
Cells
HepG2
D6
Dd2
7G8
Tm90-C2B
IC50/D6 IC50
-
-
-
0.022 0.041 0.43
228
1053
16
>54510
>2477727
3435
1
8
9
1-Cl
2-Cl
1-Cl
H
2-Cl
3-Cl
3-Cl
3-Cl
4'-OCF
4'-OCF
4'-OCF
4'-OCF
3
3
3
3
3.2
21
6.8
7.7
0.88
2.4
14
10992
1
14
>34418
>34418
>37340
>1639
20
0.52
2.6
1.2
14
23
>66188
>14361
2
1
2
142
0.004 0.003 0.007
7
2
1-F
3-F
4'-OCF
3
3.2
>245700
>52276595
0
3
23
1-F
1-F
H
3-F
H
4'-Cl
0.010 0.28
0.68
73
12
20
>27953
196
>2795300
816
24
25
26
27
4'-OCF
4'-OCF
4'-OCF
4'-OCF
3
3
3
3
3
3
0.24
3.2
0.06
4.8
0.43
2.2
3-F
3-Cl
3-F
9432
2947
1-F
1-Cl
1-Cl
1-F
1-F
0.028 0.045 0.042 5.6
>35754
798
>1276928
15960
>6953
1161
0.050 0.18
0.28
10
31
28
3-OMe 4'-OCF
3-OMe 4'-OCF
3-OMe 4'-Cl
5.0
0.65
0.51
28
2.9
0.31
0.39
22
18
>34763
755
29
30
31
32
1.6
2.8
79
21
47
548
1074
1-OMe 3-Cl
1-OMe 3-F
1-OMe 3-F
4'-OCF
4'-OCF
4'-Cl
3
3
28
>34768
25903
22462
>34767
>34767
>100000
>37751
>34767
27908
>1242
480
54
32
63
80
33
18
15
82
150
151
137
52
1248
34
1-Cl
2-OMe 4'-OCF
3
3
3
3
3
3
17
24
51
>2045
>221
35
2-OMe 3-Cl
4'-OCF
4'-OCF
157
40
221
57
410
72
36
37
38
39
1-OMe
H
H
>2500
>114397
>93965
21468
3-OMe 4'-OCF
3-OMe 4'-OCF
3-OMe 4'-OCF
0.33
0.37
1.3
0.25
0.83
1.2
1.3
1.61
2.1
1.0
0.58
1.7
2-Cl
2-F
ACS Paragon Plus Environment
17

Page 19 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4
0
2-Cl
2-F
3-OMe 3'-OCF
3-OMe 2'-OCF
3
3
2.5
1.8
2.8
2.4
>34767
14838
>13907
353286
41
42
0.042 0.066 0.076 0.031
4'-
2-Cl
3-OMe
0.69
0.76
1.5
0.85
>33600
>48696
N
O
CQ
-
-
-
-
-
-
15
163
171
208
37577
23160
2505
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ATV
0.10
0.10
0.20
8256
231600
aIC50 values are the average of at least three determinations, each carried
out in triplicate (±10%). CQ: chloroquine; ATV: atovaquone; D6: P. falciparum
CQ sensitive strain; Dd2: MDR P. falciparum strain with Old World genetic
background; 7G8: MDR P. falciparum strain with New World genetic background;
Tm90-C2B: MDR P. falciparum clinical isolate, ATV resistant.
In Vitro Blood-Stage Activity and SAR of 6-Arylamino-Acridones
43−52. Having optimized the impact of various substituents on
antimalarial activity of acridones that contain a combination of
halogen and OMe substitutions on ring-A and an aryloxy moiety at
the 6 position of ring-B, we next investigated the impact of the
arylamino moiety in the place of aryloxy moiety at the 6 position
of ring-B on in vitro blood-stage activity. 1,3-Dihalogenated
acridones 43−45 with either 4-(trifluoromethoxy)phenylamino or 4-
(chloro)phenylamino moiety at the 6 position of ring-B exhibited
excellent potency with low IC50 values against D6 ranging from
0
.0060 nM to 0.55 nM (Table 2). These results were comparable to
the corresponding 6-aryloxy-acridones 20, 22 and 23 (43 IC50 = 0.55
nM vs. 20 IC50 = 0.52 nM; 44 IC50 = 0.20 nM vs. 22 IC50 = 0.0047 nM;
4
5 IC50 = 0.0060 nM vs. 23 IC50 = 0.010 nM against D6). Acridones
6−48 with a halogen at position 1 and OMe group at position 3 on
4
ring-A
and
4-(trifluoromethoxy)phenylamino
and/or
4-
(chloro)phenylamino moiety at position 6 of ring-B also retained
ACS Paragon Plus Environment
18

Journal of Medicinal Chemistry
Page 20 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the potency compared to the corresponding 6-aryloxy-acridones
28−30 (Tables 1 and 2). Significantly, 6-arylamino-acridones 49−52
containing a halogen at position 2 and OMe group at position 3 on
ring-A exhibited optimal antiplasmodial activity at low nanomolar
concentrations (IC50 = 0.81−5.7 nM) and most reduced cross-
resistance with ATV against TM90-C2B. Antiplasmodial potency and
cross-resistance pattern were similar to the corresponding 6-
aryloxy-acridones 38−42 (Tables 1 and 2). Taken together, SAR of
acridone compounds 43−52 demonstrated that a halogen substitution
at either the 1 or 2 position and OMe group at the 3 position on
ring-A and a substituted arylamino moiety at the 6 position on
ring-B are also well tolerated.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Vitro Blood-Stage Activity and SAR of 6-Aryl-Acridones 53−58.
After establishing the halogen and OMe substitutions pattern at
the 1, 3 and 2, 3 positions of ring-A, and aryloxy and arylamino
groups at the
6
position of ring-B of acridones, we next
investigated whether the aryl substitutions at the 6 position on
ring-B are tolerated. We synthesized and screened a series of 6-
aryl-acridones 53−58 (Table 2) with halogens at both positions 1
and 3, and a combination of halogen and OMe substitutions at either
position 1 and 3 or positions 2 and 3 on ring-A. All the 6-aryl-
acridones 53−58 showed great in vitro antiplasmodial potency with
low nanomolar concentrations (IC50 = 0.54−3.5 nM against D6),
similar to the SAR and cross-resistance trend observed with the
ACS Paragon Plus Environment
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Page 21 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
corresponding 6-aryloxy-acridones and 6-arylamino-acridones. In
general, SAR analyses of the in vitro blood-stage antiplasmodial
activity with acridones 18−58 (Tables 1 and 2) unambiguously
demonstrated that a halogen either at position 1 or 2 and OMe group
at position 3 on ring-A and the aryloxy, arylamino and aryl
moieties at position 6 on ring-B have an important role on potent
antiplasmodial activity, as well as on mitigating cross-resistance
with ATV.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. In Vitro Blood-Stage Antiplasmodial Activity and
Selectivity of Acridones (43−58)
O
O
1 1
A
8
B
5
1 1
A
8
B
5
R
R
4
R
’
3
N
H
N
N
H
R2
R2
4
4
H
2’
R3
43-52
53-58
blood-stage IC50 (nM) a
cytotoxic
selectivity
index
ity IC50
vs. P. falciparum
a
(nM)
vs.
compd
R1
R2
R3
vs.
HepG2
D6
Dd2
7G8
Tm90-C2B
HepG2
Cells
IC50/D6 IC50
43
1-Cl
1-F
3-Cl
3-F
4'-OCF
4'-OCF
3
3
0.55
0.20
1.2
4.6
71
35
10365
18845
28895
4
4
5
0.12
0.73
5779
3036
0.006
4
1-F
3-F
4'-Cl
0.020 0.016 30
506000
0
46
1-Cl
1-F
3-OMe 4'-OCF
3-OMe 4'-OCF
3
3
14
26
51
47
>34846
>10000
>2489
4
7
8
0.049 0.12
0.60
2.3
>204081
0.007 0.003 0.005
4
1-F
3-OMe 4'-Cl
2.9
3010
430000
0
0
5
49
2-F
2-F
2-F
3-OMe 4'-OCF
3-OMe 3'-OCF
3-OMe 2'-OCF
3
3
3
1.8
2.0
4.0
3.3
5.4
2.8
>36216
>36216
>36216
>20120
>44711
>30180
5
0
1
0.81
1.2
1.5
2.4
2.2
5.4
5
4'-
5
2
3
2-Cl
1-F
3-OMe
3-F
5.7
2.2
9.2
3.5
9.2
59
5.7
138
30722
27266
5390
N
O
5
Cl
12393
ACS Paragon Plus Environment
20

Journal of Medicinal Chemistry
Page 22 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
4
1-F
1-F
1-F
1-F
2-Cl
-
3-F
3-F
OCF
H
3
0.54
0.60
1.3
1.8
3.5
15
0.75
1.3
3.0
1.9
6.7
163
0.10
21
19
>25556
31972
>28266
22223
25614
37577
23160
>47326
53287
>21743
12346
7318
55
17
117
22
56
57
58
3-OMe Cl
3.2
7.5
15
3-OMe OCF
3
2.0
2.1
208
8256
3-OMe
N
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CQ
-
-
-
-
171
0.20
2505
ATV
-
0.10
231600
aIC50 values are the average of at least three determinations, each carried
out in triplicate (±10%). CQ: chloroquine; ATV: atovaquone; D6: P. falciparum
CQ sensitive strain; Dd2: MDR P. falciparum strain with Old World genetic
background; 7G8: MDR P. falciparum strain with New World genetic background;
Tm90-C2B: MDR P. falciparum clinical isolate, ATV resistant.
In Vitro Blood-Stage Activity and SAR of 7-Aryloxy- and
Arylamino-Acridones 68−80. We next investigated whether the
aryloxy and arylamino substitutions at position 7 on ring-B are
tolerated for antiplasmodial activities. We synthesized a series
of acridones 68−80, where ring-A is substituted with either two
halogens or a combination of halogen and methoxy groups, and ring-B
is substituted with various (trifluoromethoxy)phenyloxy and
(
trifluoromethoxy)phenylamino moieties and screened for their in
vitro blood-stage antiplasmodial activity (Table 3). Of the 7-
aryloxy-acridones 68−71, acridones 68 and 69 with di-fluoro
substitutions at positions 1 and 3 and 1-fluoro and 3-methoxy
substitutions on ring-A, respectively, exhibited the greatest
antiplasmodial potency (IC50 < 0.69 nM against D6). However, the
potency of compound 68 significantly declined compared to the
corresponding 6-aryloxy-acridone 22 (68 IC50 = 0.12 nM vs. 22 IC50
= 0.0047 nM against D6). Interchange of fluorine and methoxy groups
ACS Paragon Plus Environment
21

Page 23 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
between positions 1 and 3 on ring-A as in 70 resulted in a 25-fold
decrease in potency (70 IC50 = 17 nM vs. 69 IC50 = 0.69 nM against
D6). Similarly, shifting of fluorine at position 1 of 69 to position
2, as in 71, the potency significantly declined (> 100-fold). It
was observed that acridones 69−71 with a methoxy group on ring-A
exhibited less cross-resistance with ATV than the di-fluoro
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
substituted 7-aryloxy-acridone 68 (the ratio of Tm90-C2B IC /D6
50
IC50 for 69−71 = 1.9−14 vs. 68 = 167), again suggesting the
importance of the methoxy group on ring-A. Next, we prepared a
series of acridones 72−80 with di-halogens at positions 1 and 3
and a combination of halogen and methoxy group at positions 2 and
3
on ring-A and various (trifluoromethoxy)phenylamino moieties at
position 7 on ring-B. All of these 7-arylamino-acridones 72−80,
with the exception of 74 (IC50 = 0.048 nM against D6), displayed
moderate antiplasmodial activity (IC50 = 29−1474 nM against D6)
than the corresponding 7-aryloxy-acridones and 6-arylamino-
acridones. These findings indicate that the arylamino groups at
position 7 of ring-B were detrimental to optimal antimalarial
activities.
Table 3. In Vitro Blood-Stage Antiplasmodial Activity and
Selectivity of Acridones (68−80)
O
O
1 1
A
8
B
5
2’
1 1
A
8
B
5
H
2’
R
R
O
N
R3
4’
R3
4’
N
H
N
H
R2
R2
4
4
68-71
72-80
ACS Paragon Plus Environment
22

Journal of Medicinal Chemistry
Page 24 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
blood-stage IC50 (nM) a
vs. P. falciparum
cytotoxic
ity IC
selectivity
index
vs.
HepG2
IC50/D6 IC50
R1
R2
R3
compd
50
a
(
nM)
vs.
D6
Dd2
7G8
Tm90-C2B
HepG2
Cells
68
1-F
1-F
1-OMe 3-F
3-OMe 4'-OCF
3-F
4'-OCF
3
3
3
3
3
3
3
3
3
3
3
3
3
0.12
0.69
17
3.3
1.3
17
4.1
1.3
35
20
>37199
>309992
>52364
>2125
>1010
>643
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
69
3-OMe 4'-OCF
4'-OCF
9.8
157
187
92
>36131
>36131
>100000
>37289
>37289
18673
70
71
72
73
2-F
1-F
1-F
1-F
1-Cl
1-Cl
2-F
2-F
2-Cl
2-Cl
-
99
164
16
130
41
3-F
4'-OCF
3'-OCF
2'-OCF
4'-OCF
3'-OCF
58
3-F
29
17
32
41
>1286
389021
>89
74
3-F
0.048 0.047 0.73
23
7
5
6
3-Cl
3-Cl
385
1474
45
1402
1690
91
1733
750
156
286
166
141
171
0.20
748
3017
139
130
78
>34495
>34495
>36216
>36216
>34846
>34846
37577
7
>23
77
3-OMe 4'-OCF
3-OMe 3'-OCF
3-OMe 4'-OCF
3-OMe 3'-OCF
>805
78
79
80
100
82
145
83
>362
>425
78
56
55
>447
CQ
-
-
15
163
208
2505
ATV
-
-
-
0.10
0.10
8256
23160
231600
aIC50 values are the average of at least three determinations, each carried
out in triplicate (±10%). CQ: chloroquine; ATV: atovaquone; D6: P. falciparum
CQ sensitive strain; Dd2: MDR P. falciparum strain with Old World genetic
background; 7G8: MDR P. falciparum strain with New World genetic background;
Tm90-C2B: MDR P. falciparum clinical isolate, ATV resistant.
In Vitro Blood-Stage Activity and SAR of 6-and 7-Benzyloxy
Acridones 86−93, 99−102 and 107−109. Our detailed SAR explorations
around ring-A and ring-B of acridones led to valuable insight of
the structural features that are required for potent antimalarial
activity. Last, we sought to explore whether the substitution of
benzyloxy group in place of aryloxy group on ring-B is tolerated.
A series of novel acridones 86−93, 99−102 and 107−109, in which
ring-B is substituted with a variety of benzyloxy moieties at
ACS Paragon Plus Environment
23

Page 25 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
either position 6 or 7 and ring-A is substituted with either
halogens or a combination of halogen and methoxy groups, were
generated and tested for their in vitro blood-stage antimalarial
activity (Table 4). Remarkably, all of these benzyloxy-acridones
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
86−93, 99−102 and 107−109 showed the highest antiplasmodial
activities with many compounds exhibiting picomolar IC50 values
against D6, regardless of the substitution pattern on the benzyloxy
moieties of ring-B and halogen and methoxy substitutions on ring-A.
These results unequivocally demonstrated that the benzyloxy
moieties at positions 6 and 7 of ring-B play a vital role in
enhancing antiplasmodial potency. Notably, the 6-benzyloxy-
acridones 99−102 with OMe group on ring-A, exhibited equal or
comparable potency across the entire P. falciparum test panel
(Table 4) with diminished cross-resistance compared to the
corresponding benzyloxy-acridones lacking the OMe group on ring-
A. Overall, the detailed SAR of the second-generation acridones
(Tables 1−4) suggested that aryl, aryloxy, arylamino and benzyloxy
moieties at position 6 on ring-B, and halogens and OMe group at
either the 1 and 3 or 2 and 3 positions on ring-A play key roles
in enhancing in vitro blood-stage antimalarial potency with
balanced cross-resistance pattern.
Table 4. In Vitro Blood-Stage Antiplasmodial Activity and
Selectivity of Acridones (86−93, 99−102 and 107−109)
ACS Paragon Plus Environment
24

Journal of Medicinal Chemistry
Page 26 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4’
R3
O
O
R1
1
A
4
8
B
5
1 1
A
8
R
O
2’
B
5
2’
N
H
O
N
R2
R2
4
R3
4’
H
8
6-93 and 99-102
107-109
blood-stage IC50 (nM) a
cytotoxic
ity IC
(nM)
vs.
HepG2
Cells
selectivity
index
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
R2
R3
compd
vs. P. falciparum
50
a
vs.
HepG2
D6
Dd2
7G8
Tm90-C2B
IC50/D6 IC50
8
6
7
1-F
3-F
H
0.031
0.046 0.54
0.006
45
>29644
>956258
4'-
OCF
8
1-F
3-F
0.0040
0.020 7.2
>23735
>5933750
3
3
5
4'-
SCF
88
1-F
1-F
1-F
1-F
1-F
1-Cl
2-Cl
2-Cl
2-F
2-F
1-F
3-F
3-F
3-F
3-F
3-F
2-Cl
0.013
0.042
0.054
0.084
0.0030
0.033
0.19
0.078 0.16
3.8
772
59384
89
4'-CF
'-
3
0.063 0.045 8.0
15617
371833
3
90
91
92
0.17
0.21
0.070 2.5
>23735
>24672
>24672
>24711
>22232
>23052
>100000
>100000
>23735
>439537
>293714
>8224000
>748818
>117010
>288150
>66667
OCF
3
3'-CF
2'-CF
4'-Cl
3
3
0.13
15
0.004 0.006
4.3
21
0
3
93
0.038 0.28
4'-
99
3-OMe
0.14
0.14
1.9
0.17
0.45
2.9
0.27
0.43
2.8
10.5
65
OCF
3
1
00
01
3-OMe 4'-CF
'-
3
3
3
0.08
4
1
3-OMe
1.5
OCF
3
102
3-OMe 4'-CF
3.2
3.2
7.1
>31250
4'-
1
07
08
3-F
0.17
0.42
0.25
>139618
OCF
3
1
1-F
1-F
-
3-F
3-F
-
4'-CF
0.41
0.069
15
0.60
0.19
163
0.48
2.4
189
235
208
8256
>24672
>26898
37577
23160
>60175
>389826
2505
109
CQ
4'-Cl
-
-
171
ATV
-
-
0.10
0.10
0.20
231600
aIC50 values are the average of at least three determinations, each carried
out in triplicate (±10%). CQ: chloroquine; ATV: atovaquone; D6: P. falciparum
CQ sensitive strain; Dd2: MDR P. falciparum strain with Old World genetic
background; 7G8: MDR P. falciparum strain with New World genetic background;
Tm90-C2B: MDR P. falciparum clinical isolate, ATV resistant.
In Vivo Blood-Stage Efficacy in a Rodent Malaria Model. In vivo
antimalarial blood-stage efficacy was determined using a well-
established murine 4-day suppression model against P. yoelii.14, 21,
22
Sixty-two acridone compounds were tested in this in vivo model
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Page 27 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
using CQ as a reference drug and the results are summarized in
Table 5. The in vivo data are expressed as ED50 and ED90 values. The
animals were considered cured if they survive 28 days after the
infection without detectable bloodstream parasites. Most of the
acridone derivatives exhibited excellent blood-stage oral efficacy
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(ED50 and ED90 values are shown Table 5). Most significantly,
eighteen acridone compounds demonstrated superior blood-stage
efficacy compared to first-generation lead compound T31, with ED50
values less than 1 mg/kg/d. Acridones 20, 22, 26, 27, 29 and 88
were curative out to day 28 in this model after treatment of either
10 mg/kg × 4 days or 30 mg/kg × 4 days, i.e., much lower dosages
than required for T31 (80 mg/kg × 4 days) to achieve a cure.
Notably, acridones 18 and 22 were curative out to day 28 in the
same model at 5 and 3 mg/kg × 4 days, respectively. No overt
clinical toxicity or behavior change was observed in mice treated
with these acridones. In vitro blood-stage activity (Tables 1-4)
mostly correlates well with in vivo blood-stage efficacy (Table
5), with the exceptions of compounds 23, 28, 41, 42, 47, 48, 54,
55, 56, 68, 74, 86, 87, 88, 92, 93, 99, and 109 where excellent in
vitro blood-stage activity did not translate into superior in vivo
blood-stage efficacy. Our down-selection process for lead
candidates at this stage was primarily driven by superior in vivo
efficacy.
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Journal of Medicinal Chemistry
Page 28 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. In Vivo Blood-Stage Antimalarial Efficacy against P.
yoelii in Mice
blood-stage efficacy vs. P.
yoelii (4 days)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compd
ED50
ED90 (mg/kg/d)
(
mg/kg/d)
T31
8a
1.0
1.6
0.82
1.9
6.8
3.6
0.32
9.8
0.86
6.3
0.098
7.2
>30
0.32
12
1
0.52
1.8
19
21
4.5
0b
2
1.8
2
2c,d
0.18
5.0
2
3
4
5
2
0.55
3.7
2
2
6c
27b
0.045
2.1
28
>30
2
9b,c
0.28
3.6
30
31
>30
>30
>30
8.9
7.5
>30
>30
14
3
2
3
23
3
5.4
34
2.8
35
36
37
>30
>30
1.8
38
0.75
1.3
3.9
7.3
3.6
>30
>30
9.8
0.79
2.2
>30
2.3
7.4
6.7
>30
39
40
41
0.16
>30
42
>30
4
3
4
0.23
0.23
0.96
>30
4
45
46
47
48
1.1
4.1
49
3.7
50
6.8
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Page 29 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5
5
5
5
5
5
1
2
3
4
5
6
6.1
>30
10
>30
>30
19
27
>30
28
15
17
32
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
57
9.2
8.9
1.6
0.30
>30
4.6
3.9
5.4
7.2
0.34
0.36
0.28
0.17
0.15
0.38
8.0
6.1
2.8
>10
>10
0.15
<5.0
9.5
1.5
14.7
16
58
68
69
1.8
1.0
>30
6.6
5.8
6.9
18
70
7
3
4
7
77
86
87
0.42
0.45
7.7
0.63
0.56
0.47
15
8
8c
89
90
91
92
93
99
12
100
13
101
>10
>10
1.5
<5.0
16
102
107
108
109
CQ
3.3
acurative at 5 mg/kg/d; bcurative at 30 mg/kg/d; ccurative at 10 mg/kg/d;
d
curative at 3.0 mg/kg/d.
In Vitro Liver-Stage Antimalarial Activity and SAR of Second-
Generation Acridones. In vitro liver-stage activity was assessed
utilizing luciferase-expressing P. berghei sporozoite infected
human hepatocyte HepG2 cells14, 23, 24 and the results are summarized
in Table 6. A number of second-generation acridones exhibited
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