Efficient Synthesis of 1,9-Substituted Benzo[h][1,6]naphthyridin-2(1H)-ones and Evaluation of their Plasmodium falciparum Gametocytocidal Activities

Article abstract of DOI:10.1021/acscombsci.7b00119

A novel three-component, two-step, one-pot nucleophilic aromatic substitution (S_NAr)-intramolecular cyclization-Suzuki coupling reaction was developed for the synthesis of benzo[h][1,6]naphthyridin-2(1H)-ones (Torins). On the basis of the new efficiently convergent synthetic route, a library of Torin analogs was synthesized. The antimalarial activities of these compounds were evaluated against asexual parasites using a growth inhibition assay and gametocytes using a viability assay.

Full text of DOI:10.1021/acscombsci.7b00119

Subscriber access provided by University of Florida | Smathers Libraries
Article
Efficient synthesis of 1,9-substituted benzo[h][1,6]naphthyridin-2(1H)-ones
and evaluation of their Plasmodium falciparum gametocytocidal activities
Hao Li, Wei Sun, Xiuli Huang, XIAO LU, Paresma Patel, Myunghoon Kim,
Meghan Orr, Richard Fisher, Takeshi Tanaka, John McKew, Anton Simeonov,
Philip Sanderson, Wei Zheng, Kimberly Willamson, and Wenwei Huang
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00119 • Publication Date (Web): 12 Oct 2017
Downloaded from http://pubs.acs.org on October 18, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Combinatorial Science is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 20
ACS Combinatorial Science
1
2
3
4
5
6
7
8
"For Table of Contents Use Only"
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
Efficient synthesis of 1,9ꢀsubstituted benzo[h][1,6]naphthyridinꢀ
2(1H)ꢀones and evaluation of their Plasmodium falciparum
gametocytocidal activities
a
a
a
a
a
a
#
Hao Li , Wei Sun , Xiuli Huang , Xiao Lu , Paresma R. Patel , Myunghoon Kim , Meghan J.
a
a
b
a
a
⊥
┼
Orr , Richard M. Fisher , Takeshi Q Tanaka , John C. McKew , Anton Simeonov , Philip E.
Sandersona, Wei Zhenga, Kim C. Williamsonc,d, Wenwei Huanga*
1
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 2 of 20
1
2
3
4
5
6
7
8
9
Efficient synthesis of 1,9ꢀsubstituted benzo[h][1,6]naphthyridinꢀ
2(1H)ꢀones and evaluation of their Plasmodium falciparum
gametocytocidal activities
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
a
a
a
a
a
a
#
Hao Li , Wei Sun , Xiuli Huang , Xiao Lu , Paresma R. Patel , Myunghoon Kim , Meghan J.
a
a
b
a
a
⊥
┼
Orr , Richard M. Fisher , Takeshi Q Tanaka , John C. McKew , Anton Simeonov , Philip E.
Sandersona, Wei Zhenga, Kim C. Williamsonc,d, Wenwei Huanga*
aNational Center for Advancing Translational Sciences, National Institutes of Health, Bethesda,
b
MD, 20892, United States Laboratory of Malaria and Vector Research, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United
c
States Department of Biology, Loyola University Chicago, Chicago, IL 60660, United States
dMicrobiology and Immunology Department, Uniformed Services University of the Health
Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814
KEYWORDS: quinoline, benzo[h][1,6]naphthyridinꢀ2(1H)ꢀones, Torin, oneꢀpot reaction,
malaria, gametocytocidal activity.
ABSTRACT: A novel threeꢀcomponent, twoꢀstep, oneꢀpot nucleophilic aromatic substitution
(SNAr)–intramolecular cyclizationꢀSuzuki coupling reaction was developed for the synthesis of
benzo[h][1,6]naphthyridinꢀ2(1H)ꢀones (Torins). Based on the new efficiently convergent
synthetic route, a library of Torin analogs was synthesized. The antimalarial activities of these
compounds were evaluated against asexual parasites using a growth inhibition assay and
gametocytes using a viability assay.
2
ACS Paragon Plus Environment

Page 3 of 20
ACS Combinatorial Science
1
2
3
4
INTRODUCTION
5
6
7
8
Quinolineꢀbased heterocycles have attracted much attention from both synthetic chemists and
medicinal chemists due to their interesting anticancer,1 antimalarial,2 antibacterial,3
antimycobacterial,4 antiꢀinflammatory,5 antihypertensive,6 antiviral,7 and antiꢀoxidant activities.8
Several marketed drugs such as quinine and cabozantinib contain the quinoline scaffold.2a, 2b, 9, 10
Structurally complex tricyclic benzo[h][1,6]naphthyridinꢀ2(1H)ꢀones (Torins) constitute a new
family of quinolines which were not characterized until recently.11 Torin compounds have been
reported as potent inhibitors of mammalian target of rapamycin (mTOR),12 bone marrow kinase
in the X chromosome (BMX),13 and Bruton’s tyrosine kinase (BTK)14 for the treatment of
cancers. Torin 2 (1) (Figure 1) was identified as a promising antimalarial agent effective against
multiple lifeꢀcycle stages of the Plasmodium parasite.11b, 15 In a screen of a collection of 5520
known drugs and biological active compounds, Torin 2 showed an EC50 of 8 nM against stages
IIIꢀV gametocytes and the gametocytocidal activity was decoupled from its mTOR activity.15a In
an in vivo mouse malaria transmission model, we found that Torin 2 completely blocked oocyst
formation in mosquitoes by a single IV dose of 4 mg/kg. Thus, a medicinal chemistry program
was initiated to develop Torin compounds as drug candidates to block malaria transmission.
Two approaches to the synthesis of the tricyclic lactamꢀfused quinoline core of Torins have
been reported in the literature. A Povarovꢀtype multicomponent reaction was developed to afford
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
tetrahydrobenzonaphthyridinones
which
were
converted
to
the
corresponding
dihydrobenzonaphthyridinones by DDQ oxidation.16 Gray and coworkers reported a fiveꢀstep
sequence for the synthesis of Torin compounds starting from either 4,6ꢀdichloroquinoline11a, 12b
or 4ꢀchloroꢀ6ꢀbromoquinoline.12a While these methods provide elegant approaches to Torin
derivatives, a synthetic route which diverges at the last two steps would enable the facile
3
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 4 of 20
1
2
3
4
5
6
7
8
9
assembly of a structurally diverse compound library for structureꢀactivity relationship (SAR)
studies. In this paper, we report an efficient synthesis of Torin analogs using a novel oneꢀpot
nucleophilic aromatic substitution (SNAr)–intramolecular cyclizationꢀSuzuki coupling reaction
(Scheme 1b) and the antimalarial activities of a small library of Torin compounds.
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 1. Structure of Torin 2 and two regions proposed for SAR studies
RESULTS AND DISCUSSION
Chemistry. The fiveꢀstep synthesis of Torin compounds reported by Gray and coworkers is
shown in Scheme 1a.12a Reaction of 4ꢀchloroꢀ6ꢀbromoquinoline 2 with anilines gave 3, which
were reduced to alcohols 4 by NaBH4. Oxidation with MnO2 followed by HornerꢀWadsworthꢀ
Emmons olefination and in situ ring closure afforded intermediates 6. Suzuki reaction of 6 with
boronic acids produced the desired compounds 7. Since it is laborious to introduce diversity at
the R2 position using this fiveꢀstep reaction sequence, we proposed an alternative route as
described in Scheme 1b. Using cisꢀα,βꢀunsaturated ester 10 as the point of divergence, we
envisaged Torin analogs 7 could be synthesized by a facile procedure consisting of a
nucleophilic aromatic substitution (SNAr)–intramolecular cyclizationꢀSuzuki coupling sequence.
This new procedure would enable the facile assembly of a library of Torin 2 analogs with diverse
substituents at regions A and B (Figure 1). Compound 10 was prepared from commercially
available 1ꢀ(2ꢀaminoꢀ5ꢀbromophenyl)ethanꢀ1ꢀone 8 in two steps with 46% overall yield through
4
ACS Paragon Plus Environment

Page 5 of 20
ACS Combinatorial Science
1
2
3
4
a VilsmeierꢀHaack reaction17 followed by a StillꢀGennari olefination to selectively form cisꢀα,βꢀ
5
6
7
unsaturated esters.18
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
Scheme 1. (a) Literature procedure for the synthesis of Torin 2 analogs; (b) New route for Torin
2 analogs
The reaction conditions of the oneꢀpot synthesis were initially explored using 3ꢀ
(trifluoromethyl)aniline (11) and 5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyridinꢀ2ꢀamine
(12) as the model substrates. The results were summarized in Table 1. (Z)ꢀEthyl 3ꢀ(6ꢀbromoꢀ4ꢀ
o
chloroquinolinꢀ3ꢀyl)acrylate (10) was first treated with aniline 11 at 180 C for 15 min under
microwave irradiation using 20 mol% HCl as a catalyst followed by a Pd(PPh3)4ꢀcatalyzed
5
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 6 of 20
1
2
o
3
4
Suzuki coupling with boronic ester 12 at 150 C for 10 min. Different solvents were screened
5
6
7
8
(entries 1ꢀ5), and the combination of DMF, i-PrOH, and H2O (2 : 1 : 1) provided the highest
yield of 1 (89%). In the nucleophilic aromatic substitution (SNAr)ꢀintramolecular cyclization
cascade, a catalytic amount of HCl was required for an efficient reaction in the first SNAr step
(entry 5 vs entry 6). When K3PO4 instead of NaHCO3 was used as the base for the Suzuki
coupling, the product yields were further improved (entry 6 vs entry 7) and the use of K3PO4 can
prevent overpressure due to the formation of CO2 under microwave heating. Finally, two
equivalents of K3PO4 were sufficient for the coupling reaction (entry 8) while excess K3PO4 led
to a lower product yield (entry 9). Therefore, the oneꢀpot reaction used 0.2 equivalents of HCl as
the acid catalyst and DMF as the solvent for the nucleophilic aromatic substitution (SNAr)–
intramolecular cyclization step, and the Suzuki coupling was carried out with 2.0 equivalents of
K3PO4 as the base and DMF, iꢀPrOH, and H2O (2 : 1 : 1) as the solvent of choice (entry 8).
Table 1. Optimization of the reaction conditions for the twoꢀstep oneꢀpot reactiona
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
6
ACS Paragon Plus Environment

Page 7 of 20
ACS Combinatorial Science
1
2
3
4
5
6
7
8
solvent
(step 1)
iꢀPrOH
MW temp
(oC)
HCl
(equiv)
0.2
solvent
(step 2)
base
(equiv)
yieldb
(%)
31
entry
DMF + H2O
NaHCO3 (2.0)
1
180
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
DMF + H2O
NaHCO3 (2.0)
2
3
4
5
6
DMF
180
180
180
180
180
0.2
0.2
0.2
0.2
0
28
81
50
89
53
CH3CN
iꢀPrOH + H2O NaHCO3 (2.0)
iꢀPrOH + H2O NaHCO3 (2.0)
iꢀPrOH + H2O NaHCO3 (2.0)
iꢀPrOH + H2O NaHCO3 (2.0)
iꢀPrOH
DMF
DMF
iꢀPrOH + H2O
K3PO4 (2.0)
7
8
9
DMF
DMF
DMF
180
100
100
0
78
90
83
0.2
0.2
iꢀPrOH + H2O
iꢀPrOH + H2O
K3PO4 (2.0)
K3PO4 (3.0)
aReactions were carried out with 10 (0.2 mmol), 11 (0.3 mmol), and 12 (0.4 mmol) in a mixed
solvent using Pd(PPh3)4 (0.01 mmol) as the catalyst. bIsolated yields.
With the optimized conditions in hand, the substrate scope of the oneꢀpot synthesis was then
investigated. A wide variety of Torin compounds were successfully prepared as shown in Table
2. This new method has a broad substrate scope and excellent functional group compatibility.
Functional groups including amine, amide, pyridine, phenol, aryl halides, ketone, and free
carboxylic acid were wellꢀtolerated. Both electron donating and electron withdrawing
substituents on the aromatic rings in region A and B worked well in the reaction.
SAR studies. Preliminary SAR studies on both inhibition of asexual parasite growth and
gametocytocidal activity were carried out and results are summarized in Table 2. The
gametocytocidal activity of Torin analogs are generally 2ꢀ to 5ꢀfold weaker than their
corresponding activities in inhibiting growth of asexual parasites. Since a key objective of this
7
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 8 of 20
1
2
3
4
5
6
7
8
9
program was to develop malaria transmission blocking agents, our SAR discussion will focus on
the gametocytocidal activity of these Torin 2 analogs. We first explored the SAR in region A of
Torin 2 by holding the 3ꢀtrifluoromethylphenyl group constant at the R2 position. A series of
pyridyl derivatives (13ꢀ25) with different substituents at the R1 position were synthesized. We
found that the 6ꢀaminoꢀ3ꢀpyridyl at the R1 position is optimal for the gametocytocidal activity.
Capping the amino group with an acetyl (13) resulted in 2.9ꢀfold loss of potency, while
replacement of the amino group with piperidine (14) let to 159ꢀfold decrease in potency. Shifting
the 6ꢀaminoꢀ3ꢀpyridyl (1) to 5ꢀaminoꢀ3ꢀpyridyl (15) led to a 10ꢀfold reduction in potency, and
removal of the 2ꢀamino substitution (16) resulted in a 6ꢀfold loss of activity. Exchanging the
pyridyl moiety of the 6ꢀaminoꢀ3ꢀpyridyl with a phenyl (17) caused a 2ꢀfold reduction in potency.
Unsubstituted 3ꢀpyridyl and 4ꢀpyridyl had similar activity (16 vs 18). 5ꢀ(2ꢀpyridonyl) (19) and 6ꢀ
trifluromethylꢀ3ꢀpyridyl (20) resulted in 7ꢀ to 21ꢀfold loss of activity, respectively. Consistent
with our previous observation15d, the 6ꢀmethylꢀ3ꢀpyridyl (21) showed good potency with an EC50
of 73 nM. Shifting the 6ꢀmethylꢀ3ꢀpyridyl to 5ꢀmethylꢀ3ꢀpyridyl (22) led to a 45ꢀfold reduction
in potency. Both 4ꢀtrifluoromethyl (24) and 4ꢀmethyl (25) derivatives had weak or no activity in
the gametocyte assay, suggesting that substituents are not tolerated at the 4 position.
Additionally, substitution of 6ꢀaminoꢀ3ꢀpyridyl with 2ꢀaminoꢀ5ꢀpyrimidinyl (26) gave similar
potency, while N,Nꢀdimethylꢀ2ꢀaminoꢀ5ꢀpyrimidinyl (27), and simple 5ꢀpyrimidinyl (29)
resulted in loss of activity. Fiveꢀmembered heteroaryls (30-32), bicyclic heteroaryls (33-36),
substituted phenyls (37ꢀ44), and 1,2,3,6ꢀtetrahydropyridinyl (45) at the R1 position all led to less
active compounds.
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
Having established an SAR profile of region A, we next turned our attention to region B and
kept the 6ꢀaminoꢀ3ꢀpyridyl group constant at the R1 position. Substituted aryls were investigated
8
ACS Paragon Plus Environment

Page 9 of 20
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
for their structureꢀactivity relationship (46ꢀ55). Removal of the 3ꢀtrifluoromethyl led to a 2ꢀfold
loss of potency (1 vs 46). 3ꢀMethyl (49) was slightly more potent than Torin 2 with an EC50 of
20 nM. Sterically bulky 3ꢀisopropyl substitution (51) resulted in a 7ꢀfold loss of activity
suggesting large substituents are not tolerated at the 3 position of the phenyl group in region B.
2ꢀMethyl (47) was about 7ꢀfold less potent than 3ꢀmethyl analog (49). Both 2ꢀ and 3ꢀmethoxy
substituted analogs (48 and 50) are less active than Torin 2. Interestingly, 3ꢀcarboxyl acid (52)
and 3ꢀtetrazole (53) had weak or no gametocytocidal activity. Finally, hydroxyl substitution at
both 3 and 4 positions (54 and 55) could be tolerated, but less active than Torin 2.
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
Table 2. Torin derivatives and their biological activities
asexual EC50
(nM)a, b
gametocytocidal
EC50 (nM)a, c
compound
R1
R2
12
55
35
1
103
13
5215
5564
14
9
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 10 of 20
1
2
3
4
5
6
7
8
155
72
357
216
15
16
17
18
19
20
21
22
23
24
9
N
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
43
78
92
246
128
155
17
246
754
73
1962
3989
26430
3338
7772
inactive
10
ACS Paragon Plus Environment

Page 11 of 20
ACS Combinatorial Science
1
2
3
4
5
6
7
8
5096
16
10760
33
25
26
27
28
29
30
31
32
33
34
9
H2N
N
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
N
1856
748
359
80
3318
1404
452
169
286
326
1020
21
765
1037
3667
99
11
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 12 of 20
1
2
3
4
5
6
7
8
148
1134
229
252
1813
657
35
36
37
38
39
40
9
S
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
2310
292
5066
392
392
547
18700
15680
inactive
19960
41
42
1734
736
3358
3769
43
44
12
ACS Paragon Plus Environment

Page 13 of 20
ACS Combinatorial Science
1
2
3
4
5
6
15720
18390
45
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
24
50
63
46
47
145
69
8
200
20
48
49
63
98
126
247
50
51
6513
8485
52
26560
39
inactive
111
53
54
13
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 14 of 20
1
2
3
4
5
6
24
43
55
7
8
9
a
b
Average EC50 from triplicated measurements; Chloroquine (EC50 = 16 nM) was used as a
control for the asexual parasites growth inhibition assay; c Both methylene blue (EC50 = 256 nM)
and primaquine (EC50 = 1288 nM) were used as controls for the gametocyte viability assay.
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
CONCLUSION
A novel oneꢀpot nucleophilic aromatic substitution (SNAr)–intramolecular cyclizationꢀSuzuki
coupling was developed for an efficient synthesis of Torin derivatives. Using this new method,
diverse substitution patterns can be introduced to both region A and region B in a oneꢀpot
procedure based on a common intermediate 10, which greatly facilitates compound library
synthesis. The newly developed synthetic route toward Torin derivatives offers several
advantages over current methods such as shorter reaction time, more general substrate scope,
broader functional group compatibility, and higher efficiency. This new method provides an
important synthetic tool for optimizing Torin compounds for the treatment of malaria and its
transmission. A preliminary SAR study showed that Torin analogs have promising
gametocytocidal activities which could lead to the discovery of novel compounds as malaria
transmission blocking agents.
EXPERIMENTAL PROCEDURES
General experimental details. All commercially available reagents, compounds, and solvents
were purchased and used without further purification. Column chromatography on silica gel was
performed on RediSep column using the Teledyne Isco CombiFlash Rf system. Preparative
purification was performed on a Waters semiꢀpreparative HPLC. The column used was a
14
ACS Paragon Plus Environment

Page 15 of 20
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase
consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of
10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was
triggered by UV detection (220 nm).
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
1H spectra were recorded using an INOVA 400 MHz spectrometer (Varian). Samples were
analyzed on an Agilent 1200 series LC/MS using a Zorbax Eclipse XDBꢀC18 reverse phase (5
micron, 4.6 x 150 mm) column and a flow rate of 1.1 mL/min. The mobile phase was a mixture
of acetonitrile and H2O each containing 0.05% trifluoroacetic acid. LC Method A, a gradient of
4% to 100% acetonitrile over 7 minutes was used during analytical analysis. LC Method B, a
gradient of 4% to 100% acetonitrile over 3 minutes was used during analytical analysis. High
resolution mass spectrometry was recorded on Agilent 6210 TimeꢀofꢀFlight LC/MS system.
General procedure for synthesis of Torin derivatives. To a 2 mL microwave tube (sharp
bottom) charged with ethyl (Z)ꢀ3ꢀ(6ꢀbromoꢀ4ꢀchloroquinolinꢀ3ꢀyl)acrylate 10 (68 mg, 0.2 mmol)
in DMF (0.5 mL) was added a substituted aniline (0.3 mmol, 1.5 eq) and HCl (4M in dioxane,
o
0.01 mL, 0.2 eq). The reaction was heated under microwave irradiation at 100 C for 15 min.
After cooling down, DMF (0.5 mL) and iꢀPrOH (0.5 mL) were added followed by aq. K3PO4
solution (0.8 M, 0.5 mL, 2.0 eq). The mixture was stirred for 5 min. Nitrogen gas was bubbled
through the reaction solution for 1ꢀ2 min, and then Pd(PPh3)4 (11.5 mg, 0.01 mmol, 0.05 eq) and
a boronic acid (0.4 mmol, 2.0 eq) were added. The reaction was heated under microwave
o
irradiation at 150 C for 10 min. The mixture was filtered through Celite and the filtrate was
purified by reverse phase HPLC. The pure fractions were dried down by a lyophilizer to afford
the desired Torin derivatives 7.
15
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 16 of 20
1
2
3
4
5
6
7
8
9
Biology: Stage III–V gametocytes were enriched using Percoll density gradient centrifugation
as described previously19. Briefly, 2.5 ꢁL/well incomplete medium was dispensed into each well
of 1,536ꢀwell plates using the Multidrop Combi followed by 23 nL compound transfer using the
NXꢀTR Pintool (WAKO Scientific Solutions, San Diego, CA). Then, 2.5 ꢁL/well of gametocytes
was dispensed with a seeding density of 20,000 cells/well using the Multidrop Combi. The assay
plates were incubated for 72 h at 37 °C with 5% CO2. After addition of 5 ꢁL/well of 2X
AlamarBlue dye (Life Technologies, cat. no. DAL1100), the plates were incubated for 24 h at 37
°C with 5% CO2 and then were read in a fluorescence detection mode (Ex = 525 nm, Em = 598
nm) on a ViewLux plate reader (PerkinElmer).
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
Asexual parasites of P. falciparum strain 3D7 were cultured as described previously.20 Drug
activity in inhibiting growth of asexual stage parasites was tested using a SYBR Green assay as
described previously.21 Briefly, parasites were diluted to 0.5% parasitemia in complete culture
medium with 2% hematocrit and drugs were diluted in DMSO (≤0.5%). The preꢀdiluted parasites
were dispensed into a 1536ꢀwell plate (2.5 ꢁL/well). After 72 h incubation under the standard
culture condition, 5 ꢁL/well of lysis buffer containing SYBR Green I was added to the parasite
culture and incubated for 30 min at room temperature. The fluorescence of each well was
measured at 520 nm following excitation at 490 nm using a ViewLux platereader (PerkinElmer).
ASSOCIATED CONTENT
Supporting Information. Experimental procedures, 1H NMR, HRMS, and LCꢀMS spectra for
all new compounds. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
16
ACS Paragon Plus Environment

Page 17 of 20
ACS Combinatorial Science
1
2
3
4
Corresponding Author
5
6
7
8
9
* Telephone: 301ꢀ217ꢀ5740; eꢀmail: huangwe@mail.nih.gov
ORCID
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
Wenwei Huang: 0000ꢀ0002ꢀ7727ꢀ9287
Present Addresses
# US Food & Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD 20993
┼ Faculty of Medicine/Graduate School of Medicine, Kagawa University, 761ꢀ0793 Japan
⊥ Lumos Pharma 4200 Marathon Drive, Suite 200, Austin, TX 78756
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
The Intramural Research Programs of the National Center for Advancing Translational Sciences
and National Institute of Allergy and Infectious Diseases, and Public Health Service grants
AI101396 and AI114761 (KW) from the National Institute of Allergy and Infectious Diseases.
ACKNOWLEDGMENTS
This work was supported by the Intramural Research Programs of the National Center for
Advancing Translational Sciences and National Institute of Allergy and Infectious Diseases, and
Public Health Service grants AI101396 and AI114761 (KW) from the National Institute of
Allergy and Infectious Diseases. We thank Paul Shinn, Danielle VanLeer, Misha Itkin, Zina
17
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 18 of 20
1
2
3
4
5
6
7
8
Itkin, and Crystal McKnight from the National Center for Advancing Translational Sciences for
preparing compound plates. We also thank the analytical chemistry group from the National
Center for Advancing Translational Sciences for analytical supports. TQT is a JSPS Research
Fellow in Biomedical and Behavioral Research at the NIH.
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
REFERENCES
(1) (a) Solomon, V. R.; Lee, H. Quinoline as a privileged scaffold in cancer drug discovery.
Curr. Med. Chem. 2011, 18, 1488ꢀ1508; (b) Kumar, S.; Bawa, S.; Gupta, H. Biological activities
of quinoline derivatives. Mini Rev. Med. Chem. 2009, 9, 1648ꢀ1654; (c) Kumar, E. V.; Etukala,
J. R.; Ablordeppey, S. Y. Indolo[3,2ꢀb]quinolines: synthesis, biological evaluation and structure
activityꢀrelationships. Mini Rev. Med. Chem. 2008, 8, 538ꢀ554.
(2) (a) Anthony, M. P.; Burrows, J. N.; Duparc, S.; Moehrle, J. J.; Wells, T. N. The global
pipeline of new medicines for the control and elimination of malaria. Malar. J. 2012, 11, 316; (b)
Barnett, D. S.; Guy, R. K. Antimalarials in Development in 2014. Chem. Rev. 2014, 11221ꢀ
11241; (c) Biamonte, M. A.; Wanner, J.; Le Roch, K. G. Recent advances in malaria drug
discovery. Bioorg. Med. Chem. Lett. 2013, 23, 2829ꢀ2843; (d) Schrader, F. C.; Barho, M.;
Steiner, I.; Ortmann, R.; Schlitzer, M. The antimalarial pipelineꢀꢀan update. Int. J. Med.
Microbiol. 2012, 302, 165ꢀ171; (e) Leven, M.; Held, J.; Duffy, S.; Tschan, S.; Sax, S.; Kamber,
J.; Frank, W.; Kuna, K.; Geffken, D.; Siethoff, C.; Barth, S.; Avery, V. M.; Wittlin, S.;
Mordmuller, B.; Kurz, T. Blood schizontocidal and gametocytocidal activity of 3ꢀhydroxyꢀN'ꢀ
arylidenepropanehydrazonamides: a new class of antiplasmodial compounds. J. Med. Chem.
2014, 57, 7971ꢀ7976; (f) Bray, P. G.; Ward, S. A.; O'Neill, P. M. Quinolines and artemisinin:
chemistry, biology and history. Curr. Top. Microbiol. Immunol. 2005, 295, 3ꢀ38.
(3) (a) Eswaran, S.; Adhikari, A. V.; Shetty, N. S. Synthesis and antimicrobial activities of novel
quinoline derivatives carrying 1,2,4ꢀtriazole moiety. Eur. J. Med. Chem. 2009, 44, 4637ꢀ4647;
(b) Mahamoud, A.; Chevalier, J.; DavinꢀRegli, A.; Barbe, J.; Pages, J. M. Quinoline derivatives
as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes
isolates. Curr. Drug Targets 2006, 7, 843ꢀ847.
(4) Keri, R. S.; Patil, S. A. Quinoline: a promising antitubercular target. Biomed. Pharmacother.
2014, 68, 1161ꢀ1175.
(5) (a) Ko, T. C.; Hour, M. J.; Lien, J. C.; Teng, C. M.; Lee, K. H.; Kuo, S. C.; Huang, L. J.
Synthesis of 4ꢀalkoxyꢀ2ꢀphenylquinoline derivatives as potent antiplatelet agents. Bioorg. Med.
Chem. Lett. 2001, 11, 279ꢀ282; (b) Bekhit, A. A.; ElꢀSayed, O. A.; Aboulmagd, E.; Park, J. Y.
Tetrazolo[1,5ꢀa]quinoline as a potential promising new scaffold for the synthesis of novel antiꢀ
inflammatory and antibacterial agents. Eur. J. Med. Chem. 2004, 39, 249ꢀ255; (c) Lavrado, J.;
Moreira, R.; Paulo, A. Indoloquinolines as scaffolds for drug discovery. Curr. Med. Chem. 2010,
17, 2348ꢀ2370.
(6) (a) Zhang, C. B.; Cui, X.; Hong, L.; Quan, Z. S.; Piao, H. R. Synthesis and positive inotropic
activity
of
Nꢀ(4,5ꢀdihydroꢀ[1,2,4]triazolo[4,3ꢀa]quinolinꢀ7ꢀyl)ꢀ2ꢀ(piperazinꢀ1ꢀyl)acetamide
derivatives. Bioorg. Med. Chem. Lett. 2008, 18, 4606ꢀ4609; (b) Ferlin, M. G.; Chiarelotto, G.;
Antonucci, F.; Caparrotta, L.; Froldi, G. Mannich bases of 3Hꢀpyrrolo[3,2ꢀf]quinoline having
vasorelaxing activity. Eur. J. Med. Chem. 2002, 37, 427ꢀ434.
(7) Strekowski, L.; Mokrosz, J. L.; Honkan, V. A.; Czarny, A.; Cegla, M. T.; Wydra, R. L.;
Patterson, S. E.; Schinazi, R. F. Synthesis and quantitative structureꢀactivity relationship analysis
18
ACS Paragon Plus Environment

Page 19 of 20
ACS Combinatorial Science
1
2
3
4
5
6
7
8
of 2ꢀ(aryl or heteroaryl)quinolinꢀ4ꢀamines, a new class of antiꢀHIVꢀ1 agents. J. Med. Chem.
1991, 34, 1739ꢀ1746.
(8) Orhan Puskullu, M.; Tekiner, B.; Suzen, S. Recent studies of antioxidant quinoline
derivatives. Mini Rev. Med. Chem. 2013, 13, 365ꢀ372.
(9) Kurzrock, R.; Sherman, S. I.; Ball, D. W.; Forastiere, A. A.; Cohen, R. B.; Mehra, R.; Pfister,
D. G.; Cohen, E. E. W.; Janisch, L.; Nauling, F.; Hong, D. S.; Ng, C. S.; Ye, L.; Gagel, R. F.;
Frye, J.; Muller, T.; Ratain, M. J.; Salgia, R. Activity of XL184 (Cabozantinib), an Oral Tyrosine
Kinase Inhibitor, in Patients With Medullary Thyroid Cancer. J Clin Oncol 2011, 29, 2660ꢀ2666.
(10) (a) Leroy, D.; Campo, B.; Ding, X. C.; Burrows, J. N.; Cherbuin, S. Defining the biology
component of the drug discovery strategy for malaria eradication. Trends Parasitol. 2014, 30,
478ꢀ490; (b) Baumann, M.; Baxendale, I. R. An overview of the synthetic routes to the best
selling drugs containing 6ꢀmembered heterocycles. Beilstein J. Org. Chem. 2013, 9, 2265ꢀ2319;
(c) DechyꢀCabaret, O.; BenoitꢀVical, F. Effects of antimalarial molecules on the gametocyte
stage of Plasmodium falciparum: the debate. J. Med. Chem. 2012, 55, 10328ꢀ10344; (d)
Burrows, J. N.; van Huijsduijnen, R. H.; Mohrle, J. J.; Oeuvray, C.; Wells, T. N. Designing the
next generation of medicines for malaria control and eradication. Malar. J. 2013, 12, 187.
(11) (a) Liu, Q.; Chang, J. W.; Wang, J.; Kang, S. A.; Thoreen, C. C.; Markhard, A.; Hur, W.;
Zhang, J.; Sim, T.; Sabatini, D. M.; Gray, N. S. Discovery of 1ꢀ(4ꢀ(4ꢀpropionylpiperazinꢀ1ꢀyl)ꢀ3ꢀ
(trifluoromethyl)phenyl)ꢀ9ꢀ(quinolinꢀ3ꢀyl)benz o[h][1,6]naphthyridinꢀ2(1H)ꢀone as a highly
potent, selective mammalian target of rapamycin (mTOR) inhibitor for the treatment of cancer. J.
Med. Chem. 2010, 53, 7146ꢀ7155; (b) Hanson, K. K.; Ressurreicao, A. S.; Buchholz, K.;
Prudencio, M.; HermanꢀOrnelas, J. D.; Rebelo, M.; Beatty, W. L.; Wirth, D. F.; Hanscheid, T.;
Moreira, R.; Marti, M.; Mota, M. M. Torins are potent antimalarials that block replenishment of
Plasmodium liver stage parasitophorous vacuole membrane proteins. Proc. Natl. Acad. Sci. U. S.
A. 2013, 110, E2838ꢀ2847.
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
(12) (a) Liu, Q.; Wang, J.; Kang, S. A.; Thoreen, C. C.; Hur, W.; Ahmed, T.; Sabatini, D. M.;
Gray,
N.
S.
Discovery
of
9ꢀ(6ꢀaminopyridinꢀ3ꢀyl)ꢀ1ꢀ(3ꢀ
(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridinꢀ2( 1H)ꢀone (Torin2) as a potent, selective,
and orally available mammalian target of rapamycin (mTOR) inhibitor for treatment of cancer. J.
Med. Chem. 2011, 54, 1473ꢀ1480; (b) Liu, Q.; Wang, J.; Kang, S. A.; Thoreen, C. C.; Hur, W.;
Choi, H. G.; Waller, D. L.; Sim, T.; Sabatini, D. M.; Gray, N. S. Discovery and optimization of
potent and selective benzonaphthyridinone analogs as small molecule mTOR inhibitors with
improved mouse microsome stability. Bioorg. Med. Chem. Lett. 2011, 21, 4036ꢀ4040.
(13) Liu, F.; Zhang, X.; Weisberg, E.; Chen, S.; Hur, W.; Wu, H.; Zhao, Z.; Wang, W.; Mao, M.;
Cai, C.; Simon, N. I.; Sanda, T.; Wang, J.; Look, A. T.; Griffin, J. D.; Balk, S. P.; Liu, Q.; Gray,
N. S. Discovery of a selective irreversible BMX inhibitor for prostate cancer. ACS Chem. Biol.
2013, 8, 1423ꢀ1428.
(14) Wu, H.; Wang, W.; Liu, F.; Weisberg, E. L.; Tian, B.; Chen, Y.; Li, B.; Wang, A.; Wang,
B.; Zhao, Z.; McMillin, D. W.; Hu, C.; Li, H.; Wang, J.; Liang, Y.; Buhrlage, S. J.; Liang, J.;
Liu, J.; Yang, G.; Brown, J. R.; Treon, S. P.; Mitsiades, C. S.; Griffin, J. D.; Liu, Q.; Gray, N. S.
Discovery of a potent, covalent BTK inhibitor for Bꢀcell lymphoma. ACS Chem. Biol. 2014, 9,
1086ꢀ1091.
(15) (a) Sun, W.; Tanaka, T. Q.; Magle, C. T.; Huang, W.; Southall, N.; Huang, R.; Dehdashti, S.
J.; McKew, J. C.; Williamson, K. C.; Zheng, W. Chemical signatures and new drug targets for
gametocytocidal drug development. Sci. Rep. 2014, 4, 3743; (b) Derbyshire, E. R.; ZuzarteꢀLuis,
V.; Magalhaes, A. D.; Kato, N.; Sanschagrin, P. C.; Wang, J.; Zhou, W.; Miduturu, C. V.;
19
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 20 of 20
1
2
3
4
5
6
7
8
9
Mazitschek, R.; Sliz, P.; Mota, M. M.; Gray, N. S.; Clardy, J. Chemical interrogation of the
malaria kinome. Chembiochem 2014, 15, 1920ꢀ1930; (c) Mott, B. T.; Eastman, R. T.; Guha, R.;
Sherlach, K. S.; Siriwardana, A.; Shinn, P.; McKnight, C.; Michael, S.; LacerdaꢀQueiroz, N.;
Patel, P. R.; Khine, P.; Sun, H.; Kasbekar, M.; Aghdam, N.; Fontaine, S. D.; Liu, D.; Mierzwa,
T.; MathewsꢀGriner, L. A.; Ferrer, M.; Renslo, A. R.; Inglese, J.; Yuan, J.; Roepe, P. D.; Su, X.
Z.; Thomas, C. J. Highꢀthroughput matrix screening identifies synergistic and antagonistic
antimalarial drug combinations. Sci. Rep. 2015, 5, 13891; (d) Patel, P. R.; Sun, W.; Kim, M.;
Huang, X.; Sanderson, P. E.; Tanaka, T. Q.; McKew, J. C.; Simeonov, A.; Williamson, K. C.;
Zheng, W.; Huang, W. In vitro evaluation of imidazo[4,5ꢀc]quinolinꢀ2ꢀones as gametocytocidal
antimalarial agents. Bioorg. Med. Chem. Lett. 2016, 26, 2907ꢀ2911.
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
(16) (a) VicenteꢀGarcia, E.; Catti, F.; Ramon, R.; Lavilla, R. Unsaturated lactams: new inputs for
povarovꢀtype multicomponent reactions. Org. Lett. 2010, 12, 860ꢀ863; (b) Di Pietro, O.; Viayna,
E.; VicenteꢀGarcia, E.; Bartolini, M.; Ramon, R.; JuarezꢀJimenez, J.; Clos, M. V.; Perez, B.;
Andrisano,
V.;
Luque,
F.
J.;
Lavilla,
R.;
MunozꢀTorrero,
D.
1,2,3,4ꢀ
Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheralꢀtoꢀmidgorgeꢀsite
inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies.
Eur. J. Med. Chem. 2014, 73, 141ꢀ152.
(17) Maluleka, M. M.; Mphahlele, M. J. 6,8ꢀDibromoꢀ4ꢀchloroquinolineꢀ3ꢀcarbaldehyde as a
synthon in the development of novel 1,6,8ꢀtriarylꢀ1Hꢀpyrazolo[4,3ꢀc]quinolines. Tetrahedron
2013, 69, 699ꢀ704.
(18) Still, W. C.; Gennari, C. Direct Synthesis of ZꢀUnsaturated Esters ꢀ a Useful Modification of
the HornerꢀEmmons Olefination. Tetrahedron Lett. 1983, 24, 4405ꢀ4408.
(19) Tanaka, T. Q.; Williamson, K. C. A malaria gametocytocidal assay using oxidoreduction
indicator, alamarBlue. Mol. Biochem. Parasitol. 2011, 177, 160ꢀ163.
(20) Sun, W.; Huang, X.; Li, H.; Tawa, G.; Fisher, E.; Tanaka, T. Q.; Shinn, P.; Huang, W.;
Williamson, K. C.; Zheng, W. Novel lead structures with both Plasmodium falciparum
gametocytocidal and asexual blood stage activity identified from high throughput compound
screening. Malar. J. 2017, 16, 147.
(21) (a) Eastman, R. T.; Pattaradilokrat, S.; Raj, D. K.; Dixit, S.; Deng, B.; Miura, K.; Yuan, J.;
Tanaka, T. Q.; Johnson, R. L.; Jiang, H.; Huang, R.; Williamson, K. C.; Lambert, L. E.; Long,
C.; Austin, C. P.; Wu, Y.; Su, X. Z. A class of tricyclic compounds blocking malaria parasite
oocyst development and transmission. Antimicrob. Agents Chemother. 2013, 57, 425ꢀ435; (b)
Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe, M. Simple and inexpensive
fluorescenceꢀbased technique for highꢀthroughput antimalarial drug screening. Antimicrob.
Agents Chemother. 2004, 48, 1803ꢀ1806.
20
ACS Paragon Plus Environment