Synthesis, Structure-Activity Relationship, and Antimalarial Efficacy of 6-Chloro-2-arylvinylquinolines

Article abstract of DOI:10.1021/acs.jmedchem.0c00858

There is an urgent need to develop new efficacious antimalarials to address the emerging drug-resistant clinical cases. Our previous phenotypic screening identified styrylquinoline UCF501 as a promising antimalarial compound. To optimize UCF501, we herein report a detailed structure-activity relationship study of 2-arylvinylquinolines, leading to the discovery of potent, low nanomolar antiplasmodial compounds against a Plasmodium falciparum CQ-resistant Dd2 strain, with excellent selectivity profiles (resistance index < 1 and selectivity index > 200). Several metabolically stable 2-arylvinylquinolines are identified as fast-acting agents that kill asexual blood-stage parasites at the trophozoite phase, and the most promising compound 24 also demonstrates transmission blocking potential. Additionally, the monophosphate salt of 24 exhibits excellent in vivo antimalarial efficacy in the murine model without noticeable toxicity. Thus, the 2-arylvinylquinolines represent a promising class of antimalarial drug leads.

Full text of DOI:10.1021/acs.jmedchem.0c00858

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Article
Synthesis, structure-activity relationship and
antimalarial efficacy of 6-chloro-2-arylvinylquinolines
Guang Huang, Claribel Murillo Solano, Joel Melendez, Justin Shaw, Jennifer Collins, Robert Banks,
Arash Keshavarzi Arshadi, Rachasak Boonhok, Hui Min, Jun Miao, Debopam Chakrabarti, and Yu Yuan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00858 • Publication Date (Web): 22 Sep 2020
Downloaded from pubs.acs.org on September 22, 2020
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Synthesis, structure-activity relationship and antimalarial efficacy of 6-
chloro-2-arylvinylquinolines
Guang Huang a, †, Claribel Murillo Solano b, †, Joel Melendez , Justin Shaw , Jennifer
b
b
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Collins , Robert Banks , Arash Keshavarzi Arshadi , Rachasak Boonhok d, e, Hui Min d,
b
c
b
d
b,
a,
Jun Miao , Debopam Chakrabarti *, Yu Yuan *
a
Department of Chemistry, University of Central Florida, Orlando, FL 32816, USA
b
Division of Molecular Microbiology, Burnett School of Biomedical Sciences, University
of Central Florida, Orlando, FL 32826, USA
c
Research Program Services, University of Central Florida, Orlando, FL 32816, USA
d
Department of Internal Medicine, Morsani College of Medicine, University of South
Florida, Tampa, FL 33612, USA
e
Department of Medical Technology, School of Allied Health Science, Walailak
University, Nakhon Si Thammarat 80160, Thailand
ABSTRACT
There is an urgent need to develop new efficacious antimalarials to address the emerging
drug-resistant clinical cases. Our previous phenotypic screening identified styrylquinoline
UCF501 as a promising antimalarial compound. To optimize UCF501, we herein report a
detailed structure-activity relationship study of 2-arylvinylquinolines, leading to the
discovery of potent, low nanomolar antiplasmodial compounds against a P. falciparum
CQ-resistant Dd2 strain, with excellent selectivity profiles (RI < 1 and SI > 200). Several
metabolically stable 2-arylvinylquinolines are identified as fast-acting agents that kill
asexual blood stage parasites at the trophozoite phase, and the most promising compound
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4 also demonstrates transmission blocking potential. Additionally, the monophosphate
salt of 24 exhibits excellent in vivo antimalarial efficacy in the murine model without
noticeable toxicity. Thus, the 2-arylvinylquinolines represent a promising class of
antimalarial drug leads.
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KEYWORDS: malaria, antiplasmodial activity, 2-arylvinylquinolines, structure-activity
relationship, fast-acting, parasitocidal, dual-stage activity
INTRODUCTION
Malaria afflicts almost half of the world’s population, causing estimated 228 million
clinical cases and 405,000 deaths in 2018. Predominant mortality was among children
1
below age of five and pregnant women in Africa. Significant progress has been made,
over the last decade, to reduce the global malaria burden using ACTs and long-lasting
insecticide treated nets as well as indoor residual spraying for vector control. However, the
loss of efficacy of ACTs in the CQ-resistant malaria strains underscores the fragility of
gains accomplished in the global effort for malaria eradication. To mitigate this alarming
situation and achieve the eventual elimination of malaria, it is important to develop
inexpensive chemical entities against drug-resistant malaria parasite strains, ideally those
that possess transmission-blocking properties.
Important strategies to develop new antimalarial compounds involve structural re-
engineering of exiting drugs such as CQ, a landmark 4-aminoquinoline compound due to
its efficacy against all types of human malaria parasites, long half-life, low cost and a
2
favorable safety profiles. However, CQ has lost it efficacy due to mutations in the gene
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encoding the DV membrane protein PfCRT, leading to reduced drug accumulation in its
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site of action and ultimately CQ resistance. Based on this premise, numerous CQ
analogues with conformational rigid nitrogen-containing side chains4-6 and CQ hybrids
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CQ‐resistance reverse agents,7-9 CQ-artemisinin,10-12 CQ-synthetic peroxide,13-15 CQ-
(
ferrocene,16, 17 CQ-chalcone,17-19 CQ-N-contained heterocyclic compound,9, 20-22 etc.) have
been developed to overcome the resistance and improve the antimalarial activity.
Piperaquine and Ferroquine are examples of re-engineered CQ analogues. The former
containing two 7-chloro-aminoquinoline moieties is extensively used in Southeast Asia for
2
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prophylaxis and treatment. The latter, a 7-chloroaminoquinoline covalently linked to an
aminoferrocenyl group, is in phase II pilot clinical trials.24, 25 Thus, the quinoline scaffold
is a privileged structure that can be reengineered to design new antimalarial candidates.2, 9,
2
6-28
Within the quinoline class, styrylquinoline derivatives display a wide spectrum of
29
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31
pharmacological properties, such as antileishmanial, anticancer, anti-HIV and anti-
3
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Alzheimer’s activity. Recently, we reported the discovery of styrylquinoline UCF501
3
3
with promising in vitro and in vivo antiplasmodial activity. In particular, it exhibited a
more potent inhibitory activity against CQ-resistant strain than that against CQ-sensitive
strain. From a mechanistic perspective, UCF501 may in fact act differently from CQ, while
its exact molecular target remained elusive. Although a number of analogues were reported
in the previous investigation, a detailed account is highly desirable to elucidate the SAR of
this chemotype. Notably, all of them were less active than the lead compound UCF501.
Herein, we describe a comprehensive medicinal chemistry study on arylvinylquinolines
(Fig. 1), report the key structural determinants for the antimalarial activity, and identify
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new compounds that show improved microsomal stability over UCF501. In addition, we
disclose the details of the specific stage action, the rate of killing, and an in vivo evaluation
of the frontrunner compounds.
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Various nitrogen-containing groups
1
R : MeO, H, F or Cl
R4
R3
N
4
n
Absence of arylvinyl moiety
R1
6
2
N
1
R: Phenyl, cyclohexyl, naphthyl, pyridyl
or other heterocycles
n
R2
R
Reduction and removal of the double bond, or
replacement by conjugated double bond
2
R : Electron-donating/withdrawing substituents
Figure 1. SAR strategy around the quinoline scaffold.
RESULTS AND DISCUSSION
Chemistry
The synthesis of compounds 8-37 (Scheme 1 and Table 1) started from commercially
available anilines 1a-d. The reaction of aniline 1a with ethyl acetoacetate 2 in the presence
of acetic acid afforded an imine intermediate, which was converted to hydroxyquinoline
3
4
3
a at elevated temperature. Alternatively, hydroxyquinolines 3a-d were synthesized by
mixing anilines 1a-d with 2 in the presence of PPA.35 The chlorination of
hydroxyquinolines 3a-d with phosphorus oxychloride gave 4-chloroquinolines 4a-d in
quantitative yields, which were then reacted with neat N,N-dimethylaminoalkylamines 5a-
b via nucleophilic substitution to produce the aminoquinolines 6a-6e in excellent yields.
Subsequent olefination of 6a-6e with appropriate aromatic aldehydes 7a-l using p-TsNH2
as a catalyst were carried out in xylene to afford (E)-styrylquinolines 8-37.33
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To study the effect of heterocycles and carbocycles other than benzenoid on the
antiplasmodial potency, 2-arylvinylquinolines 39-57 (Scheme 2 and Table 2) were
synthesized from 2-methylquinolines 6a-c, following the same synthetic sequence as
shown for styrylquinolines 8-37.
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To investigate the influence of the double bond on the antimalarial activity, 2-
pyridylethylquinolines 58 and 59 were prepared in good yields (Scheme 3 and Table 3)
through the reduction of 2-pyridylvinylquinolines (41 and 50) with hydrazine hydrate at 80
°
C.36
To understand the effect of the C4-amino group on the antimalarial potency,
arylvinylquinoline derivatives 62-72, 81-87 and 90-96 were synthesized (Table 4). The
synthetic route of 62-72 is depicted in Scheme 4, using a similar chemistry as described in
Scheme 1. A growing number of studies demonstrated that the isonitrile group displays
good antimalarial activity,37-40 and thus, we prepared compound 64 for the antimalarial
evaluation. Initially, we employed the direct olefination reaction to construct the
arylvinylquinoline motif, but no discernable amount of 64 was detected. Therefore, an
alternative synthetic route was adopted to prepare isonitrile 64 (Scheme 5). In detail, benzyl
bromide 73 was reacted with a stoichiometric amount of triethylphosphite to give the
4
1
phosphonate 74 via an Arbuzov reaction, which was then converted to amine 75 by the
reduction of the nitro group. Isonitrile 76 was then obtained by treatment of amine 75 with
chloroform in the presence of a base. Subsequently, aldehyde 77, derived from selenium
42
dioxide oxidation of 2-methylquinoline 61a, was reacted with phosphonate 76 to afford
isonitrile 64 in moderate yield via a Horner-Wadsworth-Emmons reaction.43
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Diversification of the amino group was achieved through nucleophilic substitutions.
For examples, aminoquinolines 80a-c were prepared by mixing chloroquinoline 4c with
the aminoalcohol followed by mesylation and substitution. With aminoquinolines 80a-c in
hand, 2-arylvinylquinolines 81-87 were obtained by reacting with appropriate aldehydes
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(Scheme 6).
The designed compounds 90-96, containing propylamine and butylamine moieties,
were synthesized in two steps (Scheme 7). First, treatment of quinoline 4c with appropriate
amines 88a-c furnished aminoquinolines 89a-c, and second, aminoquinolines 89a-c were
converted to 90-96 by the corresponding olefination reaction.
Next, we turned our attention to the analogues containing various fluorinated
substituents and conjugated double bonds. The synthesis of arylvinylquinolines 98-114
were commenced from methylquinoline 89b and the appropriate aldehydes 7i or 97a-p
(Scheme 8 and Table 5), using the identical procedures described for compounds 90-96.
To evaluate the importance of the double bond, arylquinolines 119 and 120 were
synthesized from 4-chloroaniline 1c (Scheme 9 and Table 5). Briefly, aniline 1c and ethyl
benzoylacetate 115 or ethyl isonicotinoyl acetate 116 were condensed in the presence of
PPA to give hydroxyquinolines 117a or 117b, which were transformed into
chloroarylquinolines 118a or 118b. Subsequent nucleophilic substitution with 4-
morpholinobutanamine furnished 4-aminoarylvinylquinolines 119 and 120, respectively.
Scheme 1. Synthesis of 6-substituted 2-styrylquinolines 8-37 α
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N
n
OH
Cl
HN
N
R1
a
R1
R1
c
R1
b
d
NH2
N
N
4a-d
1
1a: R = MeO
3a-d
1
6
a: R = MeO, n = 1
1
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b: R = F
1
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b: R = F, n = 1
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c: R = Cl
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c: R = Cl, n = 1
1
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d: R = H
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d: R = H, n = 1
1
6e: R = Cl, n = 2
N
n
HN
R1
2
R = H (7a), 4/2/3-NO (7b-d), 4-OMe (7e), 3,4-diOMe (7f), 3,4,5,-triOMe
2
(
7g), 4-F(7h), 4-CF (7i), 4-N,N-dimethylamino (7j), 2/3-F (7k-l)
3
N
R2
8
-37
αReagents and conditions: a) method A for 3a: i. p-anisidine 1a, ethyl acetoacetate 2, acetic
acid, anhydrous magnesium sulfate, ethanol, 90 °C, 6 h; ii. Dowtherm, 270 °C, 30 min,
4
5
3% for 2 steps; method B for 3a-d: anilines 1a-d, ethyl acetoacetate 2, PPA, 150 °C, 2 h,
7-68%; b) phosphorus oxychloride, 105 °C, h, 85-90%; c) N,N-
2
dimethylaminoalkylamines 5a-b, 130 °C, 24 h, 87-93%; d) aromatic aldehydes 7a-l, p-
TsNH , xylene, 130 °C, 12 h, 60-89%.
2
Scheme 2. Synthesis of 6-substituted 2-arylvinylquinolines 39-57 α
N
N
HN
HN
R1
R1
a
N
N
R
6
a-6c
39-57
H
N
N
N
S
N
O
S
N
R =
N
N
a
c
g
h
b
e
k
f
d
N
OMe
j
i
α
Reagents and conditions: a) p-TsNH , aldehydes 38a-k, xylene, 130 °C, 12 h, 48-86%.
2
Scheme 3. Synthesis of 6-substituted 2-alkylquinolines 58-59 α
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58 R = MeO
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1 R = MeO
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0 R = Cl
59 R = Cl
αReagents and conditions: a) hydrazine hydrate, EtOH, 80 °C, 36 h, 81-83%.
Scheme 4. Synthesis of 4-substituted-arylvinylquinolines 62-72 α
R3 R4
N
R3 R4
N
Cl
N
Cl
Cl
N
Cl
a
b
R2
X
N
N
62-72
6
1a-f
4
c
HN
N
N
N
N
R3 R4
N
N
N
HN
=
N
f
N
e
O
a
c
d
b
2
R = 4F and H; X = C or N
αReagents and conditions: a) amines 60a-f ,130 °C, EtOH, 12 h, 73-92%; b) aldehydes 7a,
h or 38c, p-TsNH , xylene, 130 °C, 12 h, 48-89%.
7
2
Scheme 5. Synthesis of isonitrile styrylquinoline 64 α
OEt
P
OEt
P
OEt
P
Br
EtO
EtO
EtO
O
O
O
a
c
b
NC
7
NO2
NO2
74
NH2
75
6
e
73
64
O
N
d
6
1a
Cl
N
CHO
77
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α
Reagents and conditions: a) triethylphosphite, reflux, 20 h, 88%; b) Pd/C, H , MeOH,
2
overnight, 75%; c) 50% NaOH, CHCl , tetrabutylammonium bromide, DCM, rt to 40 °C,
3
2 h, 80%; d) SeO 1,4-dioxane, 80 °C, 6 h, 32%; e) potassium tert-butoxide, anhydrous
2
,
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DMF, rt, 1 h, 58%.
Scheme 6. Synthesis of 4-aminoarylvinylquinolines 81-87 α
R3
OH
OMs
c
Cl
HN
HN
HN
Cl
a
Cl
Cl
Cl
b
N
N
N
N
N
78
4
c
79
3
8
0a: R =
O
R3
HN
3
Cl
8
0b: R =
N
N
d
X = C or N
R2
X
2
R = 4-F or H
3
8
0c: R =
N
N
8
1-87
α
Reagents and conditions: a) 2-aminoethanol, EtOH, 130 °C, 48 h, 97%; b) MsCl, Et N,
3
THF, 0 °C, 1 h, 72%; c) K CO , amines, anhydrous CH CN, reflux, overnight, 80-94%; d)
2
3
3
aldehydes 7a, 7h or 38c, p-TsNH , xylene, 130 °C, 12 h, 79-91%.
2
Scheme 7. Synthesis of 4-aminoarylvinylquinolines 90-96 α
R3
n
R3
HN
Cl
HN
n
Cl
Cl
a
Cl
b
R2
X
N
N
X = C and N
N
4
c
90-96
2
R = 4-F, H and 4-CF
3
3
89a: n= 2 R =
N
O
89b: n= 3
3
N
O
R =
89c: n= 3
3
R =
N
N
αReagents and conditions: a) aliphatic amines 88a-c, 130 °C, 24 h, 86-93%; b) aldehydes
7a, 7h, 7j or 38c, p-TsNH , xylene, 130 °C, 12 h, 67-88%.
2
Scheme 8. Synthesis of 4-morpholinobutylaminoarylvinylquinolines 98-114 α
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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
O
O
N
N
HN
HN
Cl
Cl
a
n = 0 or 1
N
N
9b
n
R
8
98-114
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
α
Reagents and conditions: a) aldehydes 7i and 97a-p, p-TsNH , xylene, 130 °C, 12 h, 52-
2
9
2%.
Scheme 9. Synthesis of 4-morpholinobutylaminoarylquinolines 119-120 α
O
N
OH
Cl
NH2
Cl
HN
Cl
Cl
c
Cl
b
a
N
N
N
X
118a-b
X
X
1
19 X= C
120 X= N
1
17a X= C
1c
117b X= N
αReagents and conditions: a) ethyl benzoylacetate 115 or ethyl isonicotinoyl acetate 116,
50 °C, PPA, 6 h; b) phosphorus oxychloride, 105 °C, 2 h, 20-45% for two steps; c) 4-
1
morpholinobutanamine, 130 °C, 24 h, 43-49%.
Biology
In vitro Antiplasmodial Activity and Cytotoxicity
The SAR studies were focused on improving the in vitro activity of 2-
arylvinylquinolines against the CQ-resistant Pf Dd2 strain.
SAR1 Targets. A series of styrylquinoline analogues 8-34 bearing various C6
1
2
substituents (R ) and benzenoid substituents (R ) were evaluated for their in vitro activity
against Dd2 strain (Table 1).
1
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9
2
In the C6-methoxy series, compound 9 (R = 4-NO , EC = 28.6 ± 0.9 nM) exhibited
2
50
2
slightly higher inhibitory activity than the unsubstituted compound 8 (R = H, EC = 41.2
5
0
± 5.3 nM). Moving the nitro group from the para-position to ortho- or meta-position led to
decreased activity, as seen with compound 10 (EC = 56.3 ± 8.1 nM) and 11 (EC = 49.5
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
5
0
50
±
4.0 nM). Replacement of the nitro group with 4-methxoy, 3,4-dimethoxy and 3,4,5-
trimethxoy groups afforded compounds 12-14, respectively, which were less potent than
compound 9.
The introduction of a fluorine atom at the C6 position improved antiplasmodial
2
activity over the corresponding methoxylated analogues. For instance, compound 16 (R =
2
H, EC = 21.0 ± 2.1 nM) and compound 21 (R = 3,4,5-trimethoxy, EC = 38.6 ± 1.8 nM)
5
0
50
showed almost 2-fold greater activity than the counterparts 8 and 14. Another noteworthy
observation was that fluorinated analogue 20 with a 3,4-dimethoxy group exhibited
approximately 4.5-fold improved activity as compared to compound 13.
Substitution of the fluorine atom by a chlorine atom led to further enhancement of the
antiplasmodial activity. Chlorostyrylquinolines bearing a fluoro or trifluoromethyl group
on the benzene ring showed potent activity against Dd2 strain. Among these analogues,
2
compound 29 (R = 4-F) was the most active one with an EC value of 4.8 ± 2.0 nM,
5
0
which was almost 14-fold more potent than UCF501. Compared with compound 29,
compound 24 and 31 demonstrated slightly lower inhibitory activity with EC values of
50
10.9 ± 1.9 and 5.9 ± 1.4 nM, respectively. However, altering the fluorine atom from the
para position to ortho position led to 5-fold decrease in activity as observed in compound
1
3
0 (EC = 26.0 ± 0.9 nM). For all styrylquinolines (R = -OMe, -F and -Cl), the
5
0
2
introduction of electron-rich groups (R ) proved to be detrimental to the antiplasmodial
1
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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
potency, as seen with compounds 12-14, 19-21 and 25-28, which were less active relative
2
to their analogues (R = H and 4-NO ).
2
1
Removal of the C6-substituent (R = H) caused a significant drop in the antiplasmodial
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
1
activity. For example, compound 32 (R = H, EC = 80.7 ± 22.4 nM) exhibited a much
5
0
1
1
1
lower activity than the corresponding analogues 8 (R = MeO), 16 (R = F) and 22 (R =
Cl). Therefore, the general trend of the substituents at the C6 position in the order of
improved potency is H < OMe < F < Cl.
We also briefly investigated the spacing parameter for the C4 amino side-chain of
styrylquinolines. As shown in Table 1, significant loss of potency was observed for the
compounds containing a dimethylaminobutyl group. It appeared that, for styrylquinolines,
dimethylaminoethylamine was a superior side-chain to dimethylaminobutylamine.
SAR2 Targets. Having determined the impacts of dimethylaminoalkyl and halogen
substituents on the antiplasmodial activity, we next tested if the replacement of the phenyl
ring (R) with heterocycles and non-benzenoid carbocycles would affect the inhibitory
activity against Dd2 strain (Table 2). Substitutions of the benzenoid motif by five-
membered aromatic heterocycles including furan (compounds 39, 44 and 47), thiophene
(
compounds 40, 45 and 48) and thiazole (compound 49) led to the reduced antiplasmodial
activity. However, replacement of the phenyl group with a 4-pyridyl group (compounds
1, 46 and 50) retained the antiplasmodial potency. Importantly, the position of nitrogen
4
atom significantly influenced the antiplasmodial activity. For instance, 4-
pyridylvinylquinoline 50 was approximately 2-fold more active than compound 51 bearing
2
-pyridylvinyl group. This manifestation became more evident for methoxylated
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pyridylvinylquinolines since the activity difference was up to 30-fold, e.g. compound 41
(
R = 4-pyridyl, EC = 33.6 ± 5.8 nM) and 42 (R = 2-pyridyl, EC = 1032.8 ± 232.9 nM).
50 50
Moreover, chlorinated arylvinylquinolines were more potent than the corresponding
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
1
fluorinated and methoxylated analogues in this series. For instance, compound 47 (R = Cl,
1
EC = 37.0 ± 4.3 nM) showed a 2-fold higher activity than compounds 39 (R = MeO,
5
0
1
EC = 88.7 ± 2.3 nM) and 44 (R = F, EC = 82.6 ± 9.4 nM). Accordingly, this work
50
50
provided further supports that the chlorine atom at the C6 position was superior to fluorine
atom and methoxy substituent for the antiplasmodial potency.
In addition, we found that replacement of pyridine by other heterocycles such as
pyrimidine, indole and quinoline resulted in a marked loss of potency, as seen with the
corresponding arylvinylquinolines 53 (EC = 155.0 ± 11.6 nM), 54 (EC = 95.9 ± 6.7 nM)
5
0
50
and 55 (EC = 281.3 ± 40.3 nM). Unfortunately, our search for compounds more active
5
0
than 22 by incorporating carbocycles, such as naphthalene and saturated cyclohexane, to
the vinylquinoline scaffold was unsuccessful.
SAR3 Targets. Compared with 4-aminoquinolines, e.g. CQ, a unique feature of our
lead compound is the vinyl group that bridges the quinoline core and the aromatic ring. In
this series, we intended to assess the impacts of the double bond on antiplasmodial activity.
As illustrated in Table 3, in the absence of arylvinyl group, aminoquinolines 6a, 6c and 6e
were inactive against the Dd2 strain. Even with an aromatic group, the saturated analogues
showed almost an order of magnitude weaker activity than the vinyl analogues, as
demonstrated by pyridylethylquinoline 58 (EC = 708.7 ± 58.2 nM) vs 41 (EC = 38.8 ±
5
0
50
4
.7 nM) and pyridylethylquinolines 59 (EC = 259.0 ± 15.5 nM) vs 50 (EC = 28.8 ± 5.0
50 50
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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
nM). Therefore, these data indicated that the absence of the 2-arylvinyl moiety severely
reduced the antiplasmodial activity, which was in line with recent studies.29, 44
SAR4 Targets. Having identified the optimal substituent at the C6 position and the
aromatic motif (phenyl and pyridyl) at C2 position, we shifted our focus on exploring
nitrogen-containing groups at C4 position. As a result, the first subset of analogues 62-72
were prepared by incorporating morpholine, pyrrolidine, 1-(2-pyridyl)piperazine, 4-
piperidinoaniline and bipiperidine directly to the arylvinylquinoline scaffold, and they
were screened for their antiplasmodial activity (Table 4). These compounds generally
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
showed moderate to low activity against the Dd2 strain, with EC values ranging from
50
428.0 ± 15.0 to 6753.3 ± 1076.0 nM. Unexpectedly, the isonitrile compound 64 did not
show significant antiplasmodial activity (EC > 1000 nM). Meanwhile, we found that the
5
0
1
arylvinylquinoline (R = 4-NC) containing N,N-dimethylaminoethylamino group was less
potent than compound 29 (data not shown). Compound 72 bearing a 2-picolylmethylamine
moiety, the most active compound in this subset, had an EC value of 155.5 ± 34.5 nM.
5
0
Inspired by the above-mentioned results, the second subset of analogues 81-96 were
synthesized by attaching different alkylamines. These analogues displayed promising
activities with EC values below 100 nM, and the majority of them were more potent than
5
0
the positive control UCF501. The nitrogen atom spacing was also screened in this subset
of analogues. For the morpholinylalkylamine series, arylvinylquinolines with a
tetramethylene linker showed a much higher activity than that of di- or trimethylene linkers
as demonstrated by the most potent compounds 92 (EC = 2.4 ± 1.1 nM) and 93 (EC =
5
0
50
9
.9 ± 1.3 nM), displaying almost a 2-fold and 13-fold improved potency as compared with
the counterparts 81 and 82, respectively. However, this linker length preference was
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inconclusive for the N-methylpiperazinylalkylamine series. For example, compound 96,
containing a 4-carbon linker, was slightly more active compared to the corresponding
analogue 87. Whereas compound 86 bearing a 2-carbon linker was nearly 2.5-fold more
potent than analogue 95.
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
SAR5 Targets. After C4-substitution optimization, we used arylvinylquinolines
containing a 4-morpholinobutanamine motif (92 and 93) as benchmark molecules for
further investigation. In this series, we focused on the C2 modification by fluorine-
containing aromatics. Given the wide use of fluorine substitution in drug discovery to
improve biological activity, permeability, pharmacokinetic issues,45-47 and in light of
morpholine as a privileged structure with advantageous physicochemical, biological, and
48
metabolic properties, it was expected to produce synergistic effects when these two
motifs were combined. For this reason, we prepared arylvinylquinolines 98-114 with the
optimal 4-morpholinobutanamine at the C4 position to evaluate their capability to suppress
the growth of the Dd2 strain. The results are summarized in Table 5. Compound 100 with
a 3-trifluoromethyl group showed comparable activity to its para positional isomer 98,
which was 3-fold more potent than the ortho isomer 99 (EC = 103.6 ± 7.2 nM). This
50
result provided additional evidence that introducing ortho-substituents at the phenyl ring
may compromise the antiplasmodial potency. Replacement of the 4-trifluoromethyl group
by 4-trifluoromethoxy substituent afforded compound 101 with a slightly decreased
activity. A noteworthy observation was that the introduction of di-substituted fluorinated
phenyl ring caused a significant loss of antiplasmodial potency as observed in analogues
1
02-105. We also observed that compounds 105 and 104 were nearly 7-fold and 2.5-fold
more active than 103, respectively, confirming that the fluorine substituent at the para
1
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1
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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
position of phenyl ring was favorable to improve the antiplasmodial activity. Further
fluorination on the phenyl ring dramatically reduced the activity, rendering compounds
106-108 less active than the benchmark molecule 92. Collectively, the SAR study
demonstrated that monofluorination at the para-position is the best choice within current
scope of screening and any positional deviation or excessive fluorination is
disadvantageous for the antiplasmodial potency. Additionally, these results suggested that
there is limited space available for the target interactions, with the substituent ortho-
position and steric effects being particularly sensitive.
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
Deviating from our expectations, the introduction of a fluorine atom at the pyridine
ring did not increase the antiplasmodial activity. For instance, compound 109 with a 3-
fluoro substituent (EC = 79.6 ± 11.8 nM) was almost 7.5-fold less active than the
5
0
counterpart 93 (Table 4, EC = 9.9 ± 1.3 nM). Interestingly, both pyridylvinylquinolines
5
0
109 and 111 demonstrated much weaker inhibitory activity than compound 110 (EC =
5
0
5
4.6 ± 16.5 nM). Additionally, among this series, compound 112 bearing 3-fluoropyridine
moiety displayed the weakest inhibitory effect on the Dd2 strain with an EC value of
5
0
1
97.1 ± 16.5 nM. Again, these results implied that the ortho substitution at the aromatic
ring could intervene the target interactions that were quite sensitive to the steric effect and
the orientation of the aromatic ring.
Analogues with a different number of double bonds (113-114 and 119-120) were also
assessed for their in vitro antiplasmodial activity. Compounds 113 and 114 with two double
bonds exhibited a much lower activity than the benchmark compounds, implying that a
single double bond is a better choice for improving the antimalarial potency. Additional
support for the crucial role of vinyl group came from the evaluation of analogues without
1
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any double bond. As shown in Table 5, a loss of activity was observed in arylquinolines
1
19 and 120 (EC > 400 nM). Thus, we concluded that a single double bond between the
50
quinoline core and the aromatic ring is required for the antiplasmodial activity, highlighting
the uniqueness of our chemical scaffold.
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 1. Antiplasmodial activity of SAR1 targets against Dd2 strain
N
HN
n
R1
N
R2
Compd
R1
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
F
n
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
R2
H
EC50 (nM)
41.2 ± 5.3
28.6 ± 0.9
56.3 ± 8.1
49.5 ± 4.0
43.6 ± 2.0
187.3 ± 13.6
74.2 ± 8.8
32.9 ± 5.1
21.0 ± 2.1
30.9 ± 5.9
30.9 ± 5.5
37.8 ± 8.7
41.1 ± 0.6
38.6 ± 1.8
22.4 ± 2.0
28.7 ± 3.8
10.9 ± 1.9
8
9
4-NO
2-NO
3-NO
2
2
2
1
1
1
1
1
1
1
0
1
2
3
4
5
6
4-OMe
3,4-diOMe
3,4,5-triOMe
4-F
H
17
18
19
F
4-NO
4-F
2
F
F
4-OMe
3,4-diOMe
3,4,5-triOMe
H
2
2
2
2
2
0
1
2
3
4
F
F
Cl
Cl
4-NO
4-CF
2
Cl
3
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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
2
2
5
6
Cl
Cl
Cl
Cl
1
1
4-OMe
34.8 ± 7.9
38.9 ± 7.8
44.4 ± 3.6
88.3 ± 2.2
3,4-diOMe
27
28
1
1
3,4,5-triOMe
4-N,N-
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
dimethylamino
2
3
3
3
3
3
3
3
3
9
0
1
2
3
4
5
6
7
Cl
Cl
Cl
H
1
1
1
1
1
1
2
2
2
4-F
2-F
3-F
H
4.8 ± 2.0
26.0 ± 0.9
5.9 ± 1.4
80.7 ± 22.4
47.9 ± 9.5
25.5 ± 7.1
68.0 ± 9.0
52.5 ± 2.0
51.2 ± 1.6
67.0 ± 8.0
174.0 ± 15.5
H
4-NO
4-F
2
H
Cl
Cl
Cl
H
4-NO
4-F
2
UCF501
CQ
Table 2. Antiplasmodial activity of SAR 2 targets against Dd2 strain
N
HN
R1
N
R
Compd
R1
R
EC50 (nM)
88.7 ± 2.3
O
3
4
9
0
MeO
S
MeO
80.0 ± 7.0
1
8
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4
1
MeO
38.8 ± 4.7
1032.8 ± 232.9
71.0 ± 27.5
N
42
MeO
N
4
3
MeO
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
N
O
S
4
4
4
4
4
4
5
6
7
8
F
F
82.6 ± 9.4
110.9 ± 29.1
36.1 ± 2.1
F
N
O
S
Cl
Cl
Cl
37.0 ± 4.3
29.7 ± 4.6
49
S
239.7 ± 30.6
N
5
0
Cl
Cl
Cl
28.8 ± 5.0
67.5 ± 1.9
33.6 ± 5.8
N
51
N
5
5
2
3
N
Cl
Cl
N
N
155.0 ± 11.6
95.9 ± 6.7
5
4
H
N
5
5
5
6
Cl
Cl
281.3 ± 40.3
373.0 ± 19.0
N
OMe
5
7
Cl
394.0 ± 40.0
1
9
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2
3
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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 3. Antiplasmodial activity of SAR 3 targets against Dd2 strain
N
HN
n
R1
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
N
R2
Compd
R1
MeO
Cl
n
1
1
2
1
R2
EC50 (nM)
5516.0 ± 571.3
731.6 ± 107.1
1245.0 ± 139.2
38.8 ± 4.7
6
a
Me
Me
Me
6
6
c
e
Cl
4
5
5
5
1
8
0
9
MeO
N
N
N
N
MeO
Cl
1
1
1
708.7 ± 58.2
28.8 ± 5.0
Cl
259.0 ± 15.5
Table 4. Antiplasmodial activity of SAR 4 targets against Dd2 strain
R1
Cl
R2
N
X
Compd
R1
X
C
R2
EC50 (nM)
6
6
2
3
4-F
6753.3 ± 1076.0
N
N
O
O
N
H
3590.0 ± 870.0
2
0
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5
C
4-NC
1007.6 ± 84.2
N
O
C
N
C
N
C
N
C
C
4-F
H
428.0 ± 15.0
1185.0 ± 175.0
3300.0 ± 60.0
2570.0 ± 610.0
666.7 ± 116.7
793.3 ± 81.3
486.7 ± 69.5
155.5 ± 34.5
N
66
N
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
6
6
6
7
8
9
4-F
H
N
N
N
N
N
N
N
4-F
H
HN
HN
70
N
7
7
1
2
4-F
4-F
N
N
HN
N
8
8
8
1
2
3
C
N
4-F
H
24.2 ± 0.8
O
O
N
N
NH
136.9 ± 14.6
NH
NH
NH
NH
C
C
N
C
4-F
H
92.3 ± 5.8
106.9 ± 8.4
67.7 ± 11.6
15.3 ± 3.4
N
84
N
N
8
8
5
6
H
4-F
N
N
N
N
NH
NH
NH
8
9
7
0
N
C
H
53.3 ± 8.5
40.8 ± 5.6
O
4-F
N
2
2
1
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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
9
9
9
1
2
3
O
O
O
C
C
N
C
C
N
4-CF
4-F
H
3
84.8 ± 6.3
2.4 ± 1.1
9.9 ± 1.3
58.9 ± 8.8
43.0 ± 7.5
33.4 ± 5.4
N
2
NH
NH
NH
N
3
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
N
3
94
95
96
N
H
N
NH
3
N
N
4-F
H
N
3
NH
NH
N
3
Table 5. Antiplasmodial activity of SAR 5 targets against Dd2 strain
O
N
NH
Cl
N
R
n
Compd
n
1
R
EC50 (nM)
98
30.7 ± 5.2
CF3
99
1
1
1
103.6 ± 7.2
F3C
1
1
00
01
33.9 ± 1.4
56.6 ± 7.6
CF3
OCF3
2
2
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8
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1
1
02
03
1
1
CF3
108.0 ± 8.0
CF3
F
185.3 ± 34.1
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
F
1
1
1
04
05
06
1
1
1
72.6 ± 17.2
25.5 ± 6.1
F
F
F
F
F
F
587.5 ± 255.5
F
F
1
1
07
08
1
1
196.7 ± 39.7
130.1 ± 34.9
F
F
F
F
F
1
09
1
F
79.6 ± 11.8
N
1
1
1
10
11
12
1
1
1
54.6 ± 16.5
73.1 ± 11.9
197.1 ± 81.2
F
F
N
N
F
N
1
1
13
14
2
2
96.5 ± 24.4
F
142.7 ± 18.6
2
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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
1
19
0
0
422.7 ± 62.7
796.3 ± 161.3
120
N
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
Upon completion of the SAR study on Dd2 strain, several bioactive
arylvinylquinolines were selected for further antiplasmodium activity assay using the CQ-
sensitive 3D7 strain (Table S1 and Table 6). Methoxylated styrylquinolines (8 and 13-14)
bearing dimethylaminoethylamine moiety exhibited much stronger inhibitory activity
against CQ-sensitive 3D7 strain than CQ-resistant Dd2 strain (RI ≥ 1.5), whereas all
fluorinated and chlorinated analogues were more potent against Dd2 strain than 3D7 (RI <
1), except for compounds 28 (RI = 1.3), indicative of no cross-resistance induced by these
vinylquinolines between the CQ-resistant and CQ-sensitive parasites. By contrast, RI of
CQ is almost 10. In this series, the SAR trends observed for the 3D7 strain were similar to
that observed for the Dd2 strain. For instance, compound 30 with a 2-fluoro group (EC =
5
0
5
4
5.9 ± 9.5 nM) showed remarkably diminished potency as compared to the corresponding
-fluoro (compound 29, EC = 8.7 ± 0.5 nM) or 3-fluoro analogues (compound 31, EC
50
5
0
= 23.0 ± 2.8 nM). Besides, removal of methoxy group or fluorine atom at C6 position of
styrylquinoline scaffold resulted in the decreased activity, as seen with compounds 32 vs
8
and 32 vs 16. In addition, 2-pyridylvinylquinoline 51 were less potent than its
corresponding isomers 50 and 52 against 3D7 strain. All pyridylvinylquinolines
demonstrated highly potent activity against Dd2 strain as compared to 3D7 strain (RI < 1),
as seen with compounds 41, 46 and 50-52. Interestingly, substitution of pyridine in
compound 50 by other nitrogen-containing heterocycles (e.g. pyrimidine, indole and
quinoline) and carbocycles (e.g. naphthalene and cyclohexane) led to respective
2
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compounds 53-57, which displayed significantly stronger growth-inhibitory effect on 3D7
strain than Dd2 strain (RI ≥ 1.3) (Table S1). These results indicated that, for R group,
phenyl and pyridyl are favorable to overcome cross-resistance.
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
A similar observation was that arylvinylquinolines (64 and 67-72) devoid of the
methylene linker exhibited moderate to low activity against the 3D7 strain with EC values
5
0
in the ranger of 155.0 ± 6.0 to 2643.1 ± 214.8 nM (Table S1). By comparison, incorporation
of morpholinoalkylamino or N-methylpiperazinylalkylamino group to the
arylvinylquinoline scaffold markedly enhanced the antiplasmodial activity against 3D7
strain. These results further confirmed that the introduction of flexible alkylamine moiety
on the arylvinylquinoline scaffold was beneficial to the antiplasmodium activity. It was
worth noting that, among this subset, arylvinylquinolines (106-107, 110 and 112)
containing 4-morpholinobutanamino motif showed significantly improved inhibitory
activity against 3D7 strain relative to Dd2 strain. In addition, compound 110 was almost
5
-fold more potent than compound 112 against 3D7 strain, further corroborating the result
that the positional effect of nitrogen atom and fluorine atom on the pyridyl ring
dramatically influenced the antiplasmodial activity.
Selected compounds were also evaluated for their cytotoxicity against HepG2 cells
(Table 6). In most cases the cytotoxicity profiles of these compounds correlated well with
their antiplasmodial activity, as demonstrated by compounds 24 vs 29, 29 vs 72 vs 81, 72
vs 81 vs 86, 81 vs 92, 86 vs 96, 93 vs 96 and 98 vs 105. For instance, compound 92
containing a tetramethylene linker exhibited nearly 6-fold increase in inhibitory activity
against the 3D7 strain and a 4.8-fold increase in cytotoxicity relative to compound 81.
Nevertheless, the cytotoxic activity of compound 24 was comparable to compound 98,
2
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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
although compound 24 demonstrated more potent antiparasitic activity. Additionally, we
observed that the position of fluorine atom at the phenyl ring has a marginal effect on their
cytotoxicity, as seen with compounds 29-31. Notably, all compounds tested showed high
selectivity profiles (SI > 120), especially for compounds 24, 29 and 92 (SI > 1000),
indicating good safety windows.
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 6. Selectivity and resistance indices of synthesized analogues.
R1
Cl
R2
N
X
Compd
R1
X
C
N
R2
Antiplasmodium activity EC50 (nM)
Cytotoxic activity EC50 (nM)
Dd2
3D7
RI a
0.6
0.6
0.5
0.3
0.5
HepG2
SI b
1031
1110
186
24
29
4-CF
4-F
2-F
3-F
H
3
10.9 ± 1.9
4.8 ± 2.0
26.0 ± 0.9
5.9 ± 1.4
28.8 ± 5.0
16.8 ± 0.8
8.7 ± 0.5
55.9 ± 9.5
23.0 ± 2.8
56.6 ± 11.3
11235.3 ± 1855.0
5331.6 ± 964.9
4827.3 ± 1072.0
4777.4 ± 1588.0
6110.6 ± 1046.0
19760.6 ± 3272.0
N
NH
3
3
5
7
0
1
0
2
810
212
155.5 ± 34.5 248.3 ± 35.1 0.6
127
N
H
N
81
24.2 ± 0.8
15.3 ± 3.4
41.8 ± 12.7
15.7 ± 2.9
0.6
1.0
17204.6 ± 3299.0
3195.2 ± 490.8
711
209
O
N
NH
C
4-F
8
6
N
N
NH
NH
92
C
N
N
4-F
H
2.4 ± 1.1
9.9 ± 1.3
33.4 ± 5.4
6.9 ± 1.3
20.8 ± 4.4
36.1 ± 11.3
0.3
0.5
0.9
3547.0 ± 384.1
5670.1 ± 1162.0
8377.7 ± 2576.0
1478
561
O
N
2
9
9
3
6
H
251
N
N
2
NH
2
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8
C
4-CF
3
30.7 ± 5.2
25.5 ± 6.1
67.0 ± 8.0
174.0 ± 15.5
73.8 ± 9.7
27.6 ± 1.2
119.0 ± 3.0
17.8 ± 5.5
0.4
0.9
0.6
9.8
10178.2 ± 2391.0
6135.2 ± 1222.0
7019.1 ± 244.5
10430.0 ± 860.0
332
241
105
60
O
N
2
NH
1
05
3,4-diF
UCF501
-
-
CQ
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
a
RI = Dd2 EC50 / 3D7 EC50; b SI = HepG2 EC50 / Dd2 EC50
Metabolic Stability and Preliminary Metabolite Identification.
To assess the metabolic stability, selected compounds listed in Table 7 were subjected
to an in vitro microsomal turnover assay with mouse liver microsomal preparations. This
assay determines the percentage of the parent compound residues after 60 min incubation
(
Table 7). We identified that compounds bearing the 4-trifluoromethyl group on the phenyl
ring exhibited a better metabolic stability than that with 4-fluoro group, as demonstrated
by compounds 24 (t1/2 = 104.2 min) vs 29 (t1/2 = 55.2 min) and 92 (t1/2 = 22.2 min) vs 98
(
t1/2 = 64.9 min). The intrinsic stability of the N, N-dimethylaminoethylamino moiety was
superior to the 2-pyridinemethanamino or 2-(4-methylpiperaziyl)ethanamino groups, e.g.
compounds 29 vs 72 (t1/2 = 65.3 min) and 86 (t1/2 = 60.3 min). The morpholine motif
appeared to be the most labile group upon enzymatic degradation as seen with compound
92 which had the shortest half-life time (t1/2 = 22.2 min) in this series. Additionally, we
observed that replacing the phenyl ring with pyridine in arylvinylquinoline rendered the
compound more susceptible to hepatic metabolism, such as in the case of compounds 50
(
t1/2 = 22.3 min) and compound 93 (t1/2 = 14.4 min). This suggested that the pyridyl ring
had no advantages over benzenoids in the microsomal stability. The assay indicated that
the C4 amino side-chain and the arylvinyl group significantly influenced compound
metabolic stability.
2
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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
In the context of the present study, the metabolite mixture was analyzed by LC-MS
after 15, 30, 60 min incubations. N-dealkylation from the tertiary terminal amine and
oxidation of arylvinylquinoline scaffold appeared to be the major pathways for the
metabolic decomposition, which is consistent with the previous studies on 4-
aminoquinolines metabolism,4, 8, 49 and P450 mediated oxidation in liver microsomes.8, 50
For example, the microsomal metabolites of UCF501 were primarily derived from N-
deethylation of the tertiary amine (monodeethyl UCF501, UCF501-M1), O-demethylation
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
(UCF501-M2), oxidation of styrylquinoline (UCF501-M3) and the reduction of the nitro
group (three minor metabolites, Figure S1). The tentatively assigned metabolites of
compound 29 included monodemethylated (29-M1), bidemethylated (29-M2) and
oxidized products (29-M3) (Figure S3). The same decomposition pathway was not
observed for compound 24 containing an identical C4 side-chain (Figure S2), which could
explain the different metabolic stability profiles of 24, 29 and UCF501. Although N-
demethylation was the primary metabolic route for compound 24, its half-life time was
acceptable as compared to related compounds in other literature reports.8, 27, 51, 52
Demethylated (86-M1) and oxidized products (86-M2) were identified as the possible
metabolites of compound 86 (Figure S4). The metabolic instability of arylvinylquinolines
9
2, 93, 98 and 105 was largely attributed to the breakage of the morpholine ring
alkanolamine metabolite M1 and primary amine metabolite M2). Among this series,
compound 93 was the most susceptible to hepatic metabolism, and oxidative metabolites
3-M3 and 93-M4 were observed in addition to ring-opening metabolites M1 and M2
Figure S5).
Table 7. In vitro metabolism in mouse liver microsomes
(
9
(
2
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Page 29 of 117
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compd
% remaining after 60 min
Projected t1/2 (min)
Clint (µL/min/mg)
2
2
4
9
67.1
47.1
38.2
15.5
52.9
50.2
15.4
5.6
104.2
55.2
43.2
22.3
65.3
60.3
22.2
14.4
64.9
25.8
13.3
25.1
32.1
62.2
21.2
23.0
62.4
96.2
21.4
53.7
33.4
-
31
50
72
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
8
9
9
9
6
2
3
8
52.7
19.9
36.7
1
05
UCF501
41.5
7.8a
56.2a
4
a
Values reported from the initial study.33
2
-Arylvinylquinolines Blocks Trophozoite Stage in Pf Asexual Life Cycle
To understand the antiplasmodial activity of 2-arylvinylquinolines, we decided to
establish the developmental stage specific action of the most promising compounds 24, 29
and 86 by microscopic and flow cytometric analysis.33, 53 Tightly synchronized cultures
were exposed to 5 × EC concentration of compound 24 at 6, 18, 30 and 42 h post-invasion
50
(HPI) of the merozoites. DHA (50 nM), atovaquone (6.6 nM), and DMSO vehicle were
included as controls. Microscopic analysis of Giemsa-stained-thin smears and flow
cytometric evaluation were performed at 12 h intervals. As seen in Figure 2A, the untreated
control cultures underwent normal cell cycle progress through trophozoite (18 HPI), early
schizont (30 HPI), late schizont/segmenter (42 HPI) and reinvaded ring (54 HPI) with an
increased peak height (Figure 2A-B). In contrast to untreated cultures, the cycle
2
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