Discovery of a Quinoline-4-carboxamide Derivative with a Novel Mechanism of Action, Multistage Antimalarial Activity, and Potent in Vivo Efficacy

Article abstract of DOI:10.1021/acs.jmedchem.6b00723

The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described. This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC₅₀ = 120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED₉₀ values below 1 mg/kg when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism of action, inhibition of translation elongation factor 2 (PfEF2), led to progression of 2 (DDD107498) to preclinical development.

Full text of DOI:10.1021/acs.jmedchem.6b00723

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Article
pubs.acs.org/jmc
Discovery of a Quinoline-4-carboxamide Derivative with a Novel
Mechanism of Action, Multistage Antimalarial Activity, and Potent in
Vivo Efficacy
Beatriz Baragana,† Neil R. Norcross,† Caroline Wilson,† Achim Porzelle,† Irene Hallyburton,†
̃
Raffaella Grimaldi,† Maria Osuna-Cabello,† Suzanne Norval,† Jennifer Riley,† Laste Stojanovski,†
Frederick R. C. Simeons,† Paul G. Wyatt,† Michael J. Delves,‡ Stephan Meister,§ Sandra Duffy,∥
Vicky M. Avery,∥ Elizabeth A. Winzeler,§ Robert E. Sinden,‡ Sergio Wittlin,⊥,# Julie A. Frearson,†
David W. Gray,† Alan H. Fairlamb,† David Waterson,∇ Simon F. Campbell,∇ Paul Willis,∇
Kevin D. Read,*,† and Ian H. Gilbert*,†
†Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee,
DD1 5EH, U.K.
‡Cell and Molecular Biology, Department of Life Sciences, Imperial College, London, SW7 2AZ, U.K.
§School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States
∥Eskitis Institute, Griffith University, Brisbane Innovation Park, Nathan Campus, Brisbane, QLD 4111, Australia
⊥Swiss Tropical and Public Health Institute, Swiss TPH, Socinstrasse 57, 4051 Basel, Switzerland
#University of Basel, CH-4003 Basel, Switzerland
∇Medicines for Malaria Venture, International Centre Cointrin, Entrance G, 3rd Floor, Route de Pre-
́
Bois 20, P.O. Box 1826,
CH-1215, Geneva 15, Switzerland
S
* Supporting Information
ABSTRACT: The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described.
This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed
moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC50
=
120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led
to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED90 values below 1 mg/kg
when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism
of action, inhibition of translation elongation factor 2 (Pf EF2), led to progression of 2 (DDD107498) to preclinical
development.
past decade, the number of deaths from malaria has fallen by
4% per year, and between 2000 and 2015 the number of clinical
cases of malaria has been estimated to have decreased by 40%
where the disease is endemic in Africa.2 However, in recent
years, parasite resistance to artemisinin has been detected in a
INTRODUCTION
■
Malaria is a devastating disease with over 214 million clinical
cases in 2015.1 In that year alone, the World Health
Organization (WHO) estimated 438 000 deaths, mostly
among children under five in Sub-Saharan Africa. The current
malaria control programs that combine preventive measures
with artemisinin combination therapy (ACT) treatment have
proven very effective in reducing the malaria burden. Over the
Received: May 10, 2016
© XXXX American Chemical Society
A
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number of countries in Southeast Asia. For example, in areas
along the Cambodia−Thailand border, Plasmodium falciparum,
the most deadly malaria parasite, has become resistant to most
available antimalarial medicines and the spread of multidrug
resistance is a major concern.3
to lipophilicity, as do aromatic substituents in the carboxamide
(R2) and quinoline (R3) moieties. High numbers of aromatic
rings are associated with unfavorable lipophilicity and poor
compound developability.9
The initial focus was on the R1 and R2 substituents.
Quinoline-4-carboxamides 10−19 were prepared in two steps
from the corresponding isatin (Scheme 1), employing the
Pfitzinger reaction with 1-(p-tolyl)ethanone using potassium
hydroxide as a base in a mixture of ethanol and water at 125 °C
under microwave irradiation to afford the quinoline-4-
carboxylic acid 3.10 Coupling of 3 with the corresponding
amine, using EDC and HOBt in DMF, led to compounds 10−
19 (Scheme 1).
The malaria drug discovery portfolio has dramatically
improved over the past 10 years.4 However, due to the
constant battle against drug resistance and to achieve malaria
elimination, new chemotypes with novel mechanisms of action
are required. New drugs are needed: (1) that are not cross-
resistant to existing drugs; (2) that can be given as a single
dose; (3) that prevent transmission (active against the sexual
stages of the parasite); (4) that can give chemoprotection
(active against liver stages). Recently, we reported the discovery
and profile of 2 (DDD107498),5 a quinoline-4-carboxamide
with excellent pharmacokinetic and antimalarial properties,
including activity against multiple life-cycle stages of the
parasite. Moreover, this compound acts through a novel
mechanism of action for antimalarial chemotherapy, inhibition
of translation elongation factor 2 (PfEF2), which is critical for
protein synthesis.5 In this paper we describe the medicinal
chemistry program that led to the discovery of 2.
The quinoline-4-carboxamide series was identified from a
phenotypic screen of the Dundee protein kinase library6 against
the blood stage of the P. falciparum 3D7 strain. The most active
compound of the original hit series displayed good activity in
vitro against a chloroquine sensitive P. falciparum strain (3D7)
and good selectivity index (>100-fold) against a human cell line
(MRC-5). However, hit compound 1 had a high clogP and
poor aqueous solubility and was metabolically unstable with a
high hepatic microsomal intrinsic clearance (Cli). (Figure 1 and
Table 1).
Replacement of the bromine moiety at R1 with chlorine (10)
or fluorine (11)5 was tolerated without significant loss of
activity while decreasing molecular weight and clogP (Table 1).
However, removal of the halogen altogether in compound 12
led to an 8-fold drop in potency. Although fluorinated
compound 11 was less lipophilic than initial hit 1, it still
showed poor solubility and metabolic stability.
On the basis of these results, we next turned to the R2
position with the aim of reducing the number of aromatic rings.
A range of nonaromatic amines able to duplicate the hydrogen
bonding potential of the 3-pyridyl moiety were evaluated.
Results demonstrated that basicity, lipophilicity, and linker
length were all important for activity (Table 1). Compounds
(17, 18, and 19) with an ethyl linked piperidine or pyrrolidine
retained similar activities to the corresponding 3-pyridyl
derivatives. The replacement of the cyclic amine for a
dimethylamine (13) and the introduction of longer linker
lengths (14) led to drop in potency. Compounds 18 and 195
showed enhanced hepatic microsomal stability and improved
solubility. Furthermore, ligand-lipophilicity efficiency (LLE or
LiPE)11 improved for compound 19 (LLE = 3.2) compared
with the initial hit 1 (LLE = 2.6). Subsequent analogues were
optimized using the ethyl linked pyrrolidine substituent on the
amide at R2, which displayed the best profile in terms of
lipophilicity, activity, and hepatic microsomal stability.
After initial optimization of the R1 and R2 groups, we then
turned our attention to the R3 substituent with the aim of
improving potency while maintaining lipophilicity at a
moderate level (clogP < 3.5). An efficient synthetic route
(Scheme 2) was designed for rapid access to a variety of R3
analogues which involved reaction of 5-fluoroisatin or 5-
chloroisatin with malonic acid in refluxing acetic acid to provide
intermediate 4.12 A one pot chlorination and amide formation
was achieved by treating 2-hydroxyquinoline-4-carboxylic acid 4
with thionyl chloride in the presence of DMF followed by
reaction of the intermediate acid chloride with 2-pyrrolidin-1-
ylethanamine in THF at room temperature. Intermediate 5
underwent aromatic nucleophilic substitution with a range of
amines under microwave irradiation in acetonitrile to afford
compounds 21−30 (Table 2). This route was also used for the
synthesis of derivatives with aromatic R3 substituents where a
Suzuki coupling of intermediate 5 with the appropriate boronic
acid or ester led to compounds 36−39.
Figure 1. Key data for screening hit 1 and preclinical candidate 2. Data
reported previously.5
RESULTS AND DISCUSSION
■
The initial aim of the hit to lead program was to improve
potency (Pf EC50(3D7) < 0.1 μM), aqueous solubility (>100
μM), and metabolic stability (mouse liver microsomes Cli < 5
mL min−1 g−1) of compound 1. Iterative rounds of drug design,
synthesis, and biological evaluation were driven by the
Medicines for Malaria Venture (MMV) compound progression
criteria.7 Initial modifications were directed toward improving
the physicochemical properties particularly reducing lip-
ophilicity. The clogP of the hit was 4.3, which is higher than
average for oral drugs and may contribute to the poor aqueous
solubility and hepatic microsomal instability.8 Several points for
modification on the scaffold were identified that could address
the high lipophilicity: the bromine atom (R1) significantly adds
In general, the replacement of the tolyl substituent by an
array of primary and secondary amines drove lipophilicity down
and also led to improved solubility and hepatic microsomal
stability (Table 2). In terms of potency, small heterocyles like
4-amino-3-methyloxazole 20 and N-methylpiperazine 21 at the
R3 position were not tolerated. However, introduction of the 4-
morpholinopiperidine 24 (EC50 = 0.15 μM, LLE = 4.2) moiety
B
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Table 1. Optimization the R1 and R2 Moieties
a
b
MLM: mouse liver microsomes. Sol: kinetic aqueous solubility. Data for compounds 1, 11, and 19 reported previously.5
a
Scheme 1
a
Conditions: (a) KOH, EtOH/water, 125 °C, microwave, 20 min, 29−58% yield; (b) amine, EDC, HOBt, DMF, room temperature, 16 h, 22−43%
yield.
a
Scheme 2
a
Conditions: (a) malonic acid, acetic acid, reflux, 16 h, 54%; (b) SOCl2, DMF, DCM, reflux, 3 h and then 2-pyrrolidin-1-ylethanamine, THF, room
temperature, 16 h, 27−43% yield; (c) amine, acetonitrile, 170 °C, microwave, 1 h, 7−54% yield; (d) boronic acid or ester, potassium phosphate,
Pd(PPh3)4, DMF/water 3/1, 130 °C, microwave, 30 min, 19−73%.
improved both potency and ligand efficiency compared with
previous lead compound 18. Compound 24 displayed good
aqueous solubility and moderate mouse hepatic microsomal
clearance, which is probably related to the reduction in
lipophilicity (clogP = 2.9).
To improve the potency of compound 24, we investigated
other aliphatic amines. The introduction of flexibility at R3 with
an aminopropyl morpholine substituent (25) led to a further
improvement in potency against P. falciparum (EC50 = 70 nM)
and lipophilic ligand efficiency (LLE = 5.4), with excellent
C
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Table 2. SAR Study of the R3 Substituent: Amines
a
b
MLM: mouse liver microsomes. Kinetic aqueous solubility.
selectivity against mammalian cells. Compound 25 had good
aqueous solubility and in vitro hepatic microsomal stability
across a range of species (Cli (mL min−1 g−1): mouse 0.8; rat
<0.5; human <0.5) and low plasma protein binding (59%). The
good in vitro DMPK properties of compound 25 translated
into reasonable in vivo pharmacokinetics in mouse (Table 7).
Furthermore, compound 25 afforded oral in vivo activity
(Table 8) in the P. berghei mouse model, with a 93% reduction
of parasitemia when dosed orally at 30 mg/kg once a day for
four consecutive days. An in vivo pharmacokinetic study in
mice for compound 25 showed low clearance, with a moderate
volume of distribution and a resultant good half-life. However,
oral bioavailability was poor (F = 15%). The low systemic
exposure of compound 25 was not attributed to high first-pass
metabolism due to the low in vitro clearance in mouse
microsomes and low in vivo blood clearance but was probably
due to poor permeability as highlighted by results in a PAMPA
assay (Table 6). Preliminary safety profiling of compound 25
showed a weak affinity to the hERG ion channel (16%
inhibition at 11 μM) and an oral maximum tolerated dose
(MTD) greater than 300 mg/kg b.i.d. for 4 days. With an
attractive overall profile, compound 25 was identified as a key
molecule to declare early lead status for this series, according to
the MMV compound development criteria.7
Moving into lead optimization, our focus was to improve
potency, permeability, and bioavailability through structural
modifications while retaining good physicochemical properties.
Reducing the flexibility of compound 25 by shortening the
linker length of the aminoalkylmorpholine moiety at R3 was
tolerated (26). More promising was the 17-fold improvement
(EC50 = 4 nM) on antiplasmodial activity obtained when the
linker was extended from three to four carbons (27).
Compound 27 displayed excellent lipophilic ligand efficiency
(LLE = 6.2). This improvement on in vitro potency led to
enhanced in vivo efficacy (Table 8) with an ED90 of 2.6 mg/kg.
In addition with compound 27, one out of three mice went to
cure at 4 × 30 mg/kg (q.d. po). Mouse in vivo
pharmacokinetics showed a longer half-life than the early lead
25 as a result of lower in vivo clearance and a slightly higher
volume of distribution (Table 7). Despite having improved in
vivo potency and half-life, oral bioavailability was still poor,
presumably still due to poor permeability (PAMPA Pe = 2 nm/
s).
D
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Modifications of our lead compound (25) focused on
modulation of basicity, with the aim of improving permeability.
Specifically, we envisaged that lowering basicity would reduce
protonation at physiological pH, increase passive permeability,
and ultimately improve bioavailability. Early lead 25 has two
basic groups, a pyrrolidine (R2) and a morpholine (R3) with
calculated pKa values of 8.5 and 7.0 respectively. We first
investigated the effect that modifications on the morpholine
group at R3 had on in vitro activity and permeability. We found
that the morpholine oxygen was crucial for antiparasitic activity
and replacement of the morpholino group by piperidine (28)
was not tolerated. In contrast, the morpholine nitrogen was not
essential for potency and removal, as exemplified by
compounds 29 and 30, was well tolerated leading to single
digit nanomolar potency against P. falciparum. Moreover, the
removal of the basic group at R3 had not only improved activity
but increased permeability more than 20-fold (30, Pe = 48 nm/
s). Furthermore, improved in vitro permeability also translated
in vivo, with an increase in oral bioavailability in mice (F =
23%). Despite its shorter half-life, compound 30 was efficacious
in the P. berghei mouse model with an ED90 of 1 mg/kg and
achieved a level of in vivo efficacy that met the MMV late lead
criteria. However, compound 30 showed very poor rat in vivo
exposure that was explained by high intrinsic clearance in rat
hepatocytes (Cli = 3 mL min−1 g−1) which was subsequently
confirmed by high hepatic extraction (76%) in a rat hepatic
portal vein study. As for other compounds in this series, in vitro
hepatic microsomal clearance was consistently low across
species (Table 6).
After establishing a link between basicity, PAMPA perme-
ability, and oral bioavailability for the series, we focused on
modulating the pKa of the pyrrolidine group, the stronger of the
two basic groups on early lead 25. Taking into account previous
SAR showing that basicity and linker length were important for
activity, we designed a short array of analogues with decreasing
basicity.13 Predicted pKa ranged from 5.0 for 3-difluoropyrro-
lidine 35 to 7.9 for 4-fluoropiperidine 31 compared with a
predicted pKa of 8.5 for the lead pyrrolidine 25 (Table 3). As
expected, a reduction of basicity resulted in up to 50-fold
improvement in permeability. However, potency was dramat-
ically reduced, highlighting the importance of the basic
pyrrolidine nitrogen at the R2 position.
Table 3. Modulating Basicity of the Amide Substituent
a
b
Calculated pKa using ChemAxon software. Controls: atenolol, 0.7
nm/s; propanolol, 159 nm/s.
Furthermore, in vivo efficacy studies with compound 2
demonstrated complete cure at 4 × 30 mg/kg po q.d. in a P.
berghei mouse model, with an ED90 of 0.1−0.3 mg/kg (Table
8).
We developed short, four-step synthetic routes for synthesis
of 2 and 42 which did not involve the use of palladium catalysis
(Scheme 3). Two approaches were employed to synthesize the
methyl ketone 8 depending on the availability of commercial
starting materials. In the first route, nucleophilic displacement
of commercially available 4-(bromomethyl)benzonitrile with
morpholine using trimethylamine as a base in DCM gave
intermediate 6 which was then converted into the desired
methyl ketone 8 by reaction with methylmagnesium bromide
followed by an acidic quench. In the second route, radical
bromination of commercially available 1-(2-chloro-4-
methylphenyl)ethanone with NBS using catalytic amounts of
benzoyl peroxide in dichlorobenzene afforded compound 7,
which was subsequently reacted with morpholine to yield the
methyl ketone 8. As described earlier, a Pfitzinger reaction of 5-
fluoroisatin with the appropriate methyl ketone led to acid 9.
The final amides were prepared using 2-chloro-4,6-dimethoxy-
1,3,5-triazine (CDMT) as coupling agent and N-methylmor-
pholine in DCM at room temperature. Details of other
synthetic routes for individual compounds are described in the
Supporting Information.
As highlighted above, a basic group is not required for
activity at R3. Thus, derivatives of 2 with either reduced basicity
(40, 41, and 42) or a nonbasic substituent (38, 43, 44) at R3
showed excellent potency with the exception of amide 39. The
introduction of a conformationally restricted bridge amide as
exemplified by compound 39 is detrimental for potency,
possibly because it does not allow rotation of the morpholine
group to adopt the optimal orientation for binding. The
importance of the orientation of the morpholine substituent for
activity is also highlighted by compound 45. In this case, a
change of the methylmorpholine group from para to the meta
position led to a weakly active compound.
Our attention then turned back to the R3 position, with the
aim of further improving permeability and bioavailability across
species. Previous SAR had shown that a methyl group at the
para position of an aromatic ring at C-2 was tolerated, so we
therefore expanded the range of substituents to include larger
groups while maintaining moderate lipophilicity. This strategy
also had the advantage of reducing the number of H-bond
donors and molecular flexibility, two key factors that can
modulate permeability. Early examples of substitution at R3
showed that introduction of amines, such as dimethylamine
(36) and morpholine (37), reduced lipophilicity and was
tolerated in terms of potency compared to compounds 11 and
19 (Table 4). Taking into account the leap in potency observed
for 25 with a morpholine attached through a flexible linker at
C-2, we prepared compound 2 with a carbon spacer between
the phenyl ring and the morpholine to allow improved rotation.
The introduction of the benzyl morpholine at R3 (compound
2) resulted in a 70-fold increase in potency against Pf (3D7)
(EC50 = 1 nM) while retaining good ligand efficiency (LLE =
5.9), more than 30 fold increase in permeability (PAMPA Pe =
73 nm/s) and excellent bioavailability in mice (F = 74%).
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Table 4. SAR Study of the R3 Substituent: Aromatic Groups
P. falciparum (3D7) (47, EC50 = 87 nM). Finally, it is possible
to reduce the size of the ring on the R2 substituent and retain
activity as shown by compounds 48 and 49. Compound 49
showed excellent in vivo activity in the P. berghei mouse model
at 4 × 10 mg/kg and 4 × 3 mg/kg. (Table 8). However, none
of these compounds offered a particular advantage to
compound 2.
Although in vitro DMPK data and in vivo efficacy in the P.
berghei model were comparable for compound 2 and the
fluorinated derivative 41, oral bioavailability in rat (33% for 41
vs 84% for 2) was lower and rat intravenous elimination half-life
(4 h for 41 vs 10 h for 2) was shorter for 41 (Table 9).
Therefore, compound 2 showed the best overall profile for
further progression from this novel quinoline-4-carboxamide
series (Table 9 and Figure 2). Compound 2 fulfilled the efficacy
and DMPK requirements for a late lead according to the MMV
criteria and, following further profiling, was selected as a
preclinical candidate by MMV. The studies required to profile 2
for candidate selection have been described elsewhere.5
We also profiled key compounds of this series for their
activity against different life stages of the malaria parasite life
cycle (Table 10). Following a mosquito bite (blood meal),
sporozoites are injected into the skin and migrate in the
bloodstream to the liver, where they invade liver hepatocytes
and then develop into liver schizonts. Compounds active
against liver schizonts can potentially prevent disease develop-
ment and be used in chemoprotection. Compounds 2, 27, 30,
and 38 showed low nanomolar activity against the live schizont
forms of Plasmodium yoelli.14 Several compounds were also
tested in vitro against P. falciparum late stage (IV−V)
gametocytes. Stage V gametocytes, typically insensitive to
antimalarial drugs, are infectious to mosquitoes and responsible
for the transmission of the disease. 4-Quinolinecarboxamides,
in particular compounds 2 and 30, are potent antigametocy-
tocidal (stage IV−V) with nanomolar activities.15 The ability of
compounds of this series to block transmission was further
tested in the P. berghei ookinete development assay, which
simulates in vitro the first 24 h of parasite development in the
mosquito midgut, from mature gametocyte transformation into
gametes, through fertilization and to mature ookinete develop-
ment. Compounds 2, 27, 30 and 38 showed nanomolar
potency in this assay.14b
Finally, compounds 2 and 30 were tested against several
Plasmodium falciparum drug resistant strains (K1, W2, 7G8,
TM90C2A, D6, and V1/S) showing similar levels of activity
across strains14b (Table 11).
a
Calculated pKa using ChemAxon software. Experimental pKa using
b
potentiometric titration is shown in parentheses. Data for this
compound reported previously.5
CONCLUSION
■
On the basis of their excellent potency, good permeability,
microsomal stability, and solubility (Table 6), compounds 2,
40, 41, 43, and 44 were progressed for efficacy studies in mice
dosing at 1 mg/kg for 4 days (Table 8). Compounds 40, 43,
and 44 showed excellent activity at this low dose, with
reductions of parasitemia above 99%. Compound 41 showed
the best survival time (14 days) comparable with 2.
We have evolved a malaria phenotypic hit series, which started
with moderate in vitro activity and selectivity but suboptimal
metabolic stability and physicochemical properties, into an early
lead compound 25 with improved potency, ligand efficiency,
metabolic stability, and in vivo efficacy. The lead optimization
phase focused on improving low oral bioavailability caused by
the poor permeability of the early lead. The most advanced
quinoline-4-carboxamides showed exceptional in vitro and in
vivo activities, with a reduction of parasitemia of more than
99% when administered at low doses (4 × 1 mg/kg, 4 days, po)
in the P. berghei mouse model. In addition to potent
intraerythrocyte activity, compounds in this series showed
similar potency against liver schizonts, gametocytes, and
ookinetes in vitro. Furthermore, a combination of in vitro
and in vivo activities across the different stages of the malaria
Once an optimal R3 substituent had been identified, we
turned our attention back to the R2 substituent to see if it was
possible to improve the profile of compound 2 (Table 5). As
highlighted before, lowering basicity at R2 results in a reduction
in potency. The replacement of the pyrrolidine for a
morpholine in compound 46 led to a 12-fold drop in potency.
The amide NH is also important for activity, as capping with a
methyl group resulted in an 87-fold decrease in potency against
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a
Scheme 3
a
Conditions: (a) morpholine, Et3N, DCM, 16 h, 72% yield; (b) MeMgBr, toluene, reflux, 4 h and then a 10% aqueous HCl, reflux, 1 h, 70% yield;
(c) NBS, benzoyl peroxide, dichlorobenzene, 140 °C, 16 h, 70% yield; (d) morpholine, K2CO3, acetonitrile, 40 °C, 16 h, 64% yield; (e) 5-
fluoroisatin, KOH, EtOH, 120 °C, microwave, 20 min, 30−76% yield; (f) amine, CDMT, N-methylmorpholine, DCM, 20−61% yield.
Table 5. SAR Study of the R2 Substituent
this profile, compound 2 has the potential for single dose
treatment of malaria as part of a combination therapy, together
with transmission blocking potential (TCP2 and TCP3b).7 It
also has the potential for chemoprotection (TCP4).7
Compound 2 was active against parasites that show resistance
to currently used antimalarials and has a novel mode of action
through inhibition of elongation factor 2 (PfeEF2), which is
involved in protein synthesis. The favorable potency, selectivity,
DMPK properties, efficacy, safety profile, and novel mechanism
of action support the progression of 2 toward clinical
development.
EXPERIMENTAL SECTION
■
Chemistry. General. Solvents and reagents were purchased from
commercial suppliers and used without further purification. Dry
solvents were purchased in Sure/Seal bottles stored over molecular
sieves. Reactions using microwave irradiation were carried out in a
Biotage Initiator microwave. Normal phase TLCs were carried out on
precoated silica plates (Kieselgel 60 F254, BDH) with visualization via
UV light (UV 254/365 nm) and/or ninhydrin solution. Flash
chromatography was performed using Combiflash Companion Rf
(Teledyne ISCO) and prepacked silica gel columns purchased from
a
Calculated pKa using ChemAxon software.
Grace Davison Discovery Science or SiliCycle. Mass-directed
preparative HPLC separations were performed using a Waters
parasite life cycle demonstrated the potential of this novel
HPLC (2545 binary gradient pumps, 515 HPLC make-up pump,
quinoline-4-carboxamide series to meet a number of the
2767 sample manager) connected to a Waters 2998 photodiode array
MMV’s malaria target candidate profiles (TCPs), as previously
and a Waters 3100 mass detector. Preparative HPLC separations were
performed with a Gilson HPLC (321 pumps, 819 injection module,
215 liquid handler/injector) connected to a Gilson 155 UV/vis
detector. On both instruments, HPLC chromatographic separations
described.7
Compound 2 has been extensively profiled5 in vitro and in
vivo and displays activity against multiple life-cycle stages of the
parasite with a long half-life in preclinical animal studies. With
were conducted using Waters XBridge C18 columns, 19 mm × 100
Figure 2. Mean blood concentration time profile of compounds 2 and 41 following intravenous or oral administration to male Sprague Dawley rats.
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Table 6. Key in vitro DMPK Data for Selected Analogues
a
b
c
d
e
f
compd PAMPA Pe (nm/s) Sol. (μM) MLM Cli (mL min−1 g−1
)
RLM Cli (mL min−1 g−1
)
HLM Cli (mL min−1 g−1
)
PPB (%) hERG IC50 (μM)
25
27
30
2.3
2
232
217
0.8
2.0
<0.5
<0.5
<1
59
49
77
63
75
>11
>11
>11
16
48
232
2
0.8
1
g
2
73
29
216
<0.5
1.9
≤0.8
≤1.1
38
40
41
42
43
44
46
47
48
49
109
11
63
>208
208
1.0
132
<0.5
0.5
<0.5
1.5
2
>201
171
56
14
49
<0.5
2.0
0.8
0.6
>217
>210
>210
<0.5
<0.5
2
137
49
75
71
0.5
a
b
c
d
Controls: atenolol, 0.2−4.6 nm/s; propanolol, 103−159 nm/s. MLM: mouse liver microsome. RLM: rat liver microsome. HLM: human liver
e
f
g
microsome. Mouse plasma protein binding. Measured using IonWorks Patch Clamp electrophysiology. Data for this compound reported
previously.5
Table 7. In vivo Pharmacokinetic Profile in Mice of Key Compounds
intravenous at 3 mg/kg
oral at 10 mg/kg
compd
Clb (mL min−1 kg−1
)
Vdss (L/kg)
T1/2 (h)
Cmax (ng/mL)
AUC (ng·min/mL)
Tmax (h)
F (%)
25
27
30
14
4
3
3.2
315
579
193
92922
176115
72728
2
15
16
23
74
3.5
7.4
12.5
2.9
2
34
0.5
a
b
b
b
c
c
c
2
12
15
16
90
179272
1
a
b
c
Data for this compound reported previously.5 iv dose: 1 mg/kg. po dose: 3 mg/kg.
Table 8. In vivo Oral Activity in the P. berghei Mouse Model Peter’s Test
4 × 30 mg/kg
4 × 10 mg/kg
4 × 3 mg/kg
4 × 1 mg/kg
compd activity (%) survival (days) cure activity (%) survival (days) cure activity (%) survival (days) cure activity (%) survival (days) cure
25
27
30
2
93.0
99.8
99.9
99.9
7
22
10
>30
1/3
3/3
99.7
99.9
99.9
15
8
96.0
98.0
99.9
9
48.0
90.0
99.9
99.8
99.9
99.0
<40
6.0
7
6
>30
3/3
25
2/3
14
11.0
14
7
40
41
43
44
49
99.1
20.3
1/3
99.2
9.3
Table 9. In vivo Pharmacokinetic Parameters in Male Sprague Dawley Rat
intravenous at 1 mg/kg
oral at 3 mg/kg
compd
Clb (mL min−1 kg−1
)
Vdss (L/kg)
15
T1/2 (h)
Cmax (ng/mL)
Tmax (h)
AUC (ng·min/mL)
F (%)
a
b
b
b
2
18
32
10
4
180
4
200542
84
33
41
10
29
8
30400
a
b
Data for this compound reported previously.5 po dose: 5 mg/kg.
mm, 5 μm particle size, using 0.1% ammonia in water (solvent A) and
resolution mass spectroscopy (HRMS) was performed using a Bruker
Daltonics MicroTof mass spectrometer. LC−MS analysis and
chromatographic separation were conducted with a Bruker Daltonics
MicrOTOF mass spectrometer or an Agilent Technologies 1200 series
HPLC connected to an Agilent Technologies 6130 quadrupole LC/
MS, where both instruments were connected to an Agilent diode array
detector. The column used was a Waters XBridge column (50 mm ×
2.1 mm, 3.5 μm particle size), and the compounds were eluted with a
gradient of 5−95% acetonitrile/water + 0.1% ammonia. All final
compounds showed chemical purity of ≥95% as determined by the
1
acetonitrile (solvent B) as mobile phase. H NMR, 19F NMR spectra
were recorded on a Bruker Avance DPX 500 spectrometer (1H at
500.1 MHz, 19F at 470.5 MHz) or a Bruker Avance DPX 300 (1H at
300 MHz). Chemical shifts (δ) are expressed in ppm recorded using
the residual solvent as the internal reference in all cases. Signal splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), broadened (br), or a combination thereof.
Coupling constants (J) are quoted to the nearest 0.1 Hz. Low
resolution electrospray (ES) mass spectra were recorded on a Bruker
Daltonics MicrOTOF mass spectrometer run in positive mode. High
H
DOI: 10.1021/acs.jmedchem.6b00723
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Journal of Medicinal Chemistry
Article
7.70 (m, 1H), 7.52 (dd, J = 4.8, 8.2 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H),
2.46 (s, 3H) ppm; LC−MS m/z 340 (M + H)+.
Table 10. Activity against Plasmodium Life Cycle Stages
Pf (3D7)
compd EC50 (nM)
Py liver stage
EC50 (nM)
Pf GAM IV−V
a
Pb pokinete
EC50 (nM)
6-Chloro-N-(3-(dimethylamino)ethyl)-2-(p-tolyl)quinoline-4-
carboxamide (13). Prepared using general procedure B. Product was
purified by column chromatography using a 4 g silica gel cartridge.
Solvent A: DCM. Solvent B: 20% MeOH in DCM. Gradient: 3 min
hold 100% A, 15 min ramp to 50% B, 2 min hold at 50% B. Fractions
containing product were combined and concentrated to dryness under
EC50 (nM)
27
30
2
4
6
1
4
18
1
104
39
5
14
b
b
1
24
5
38
4
152
15
1
reduced pressure to obtain 13. Yield, 27% (50 mg); H NMR (500
a
Run in duplicate. Reference controls: pyronaridine EC50 = 3108 nM,
MHz, CDCl3) 8.20 (d, J = 2.4 Hz, 1H), 8.08−8.05 (m, 1H), 7.99 (d, J
= 8.2 Hz, 2H), 7.84 (s, 1H), 7.64 (dd, J = 2.2, 9.0 Hz, 1H), 7.33−7.28
(2H, m), 7.06 (1H, s), 3.65 (q, J = 5.6 Hz, 2H), 2.62 (t, J = 5.9 Hz,
2H), 2.45 (s, 3H), 2.33 (s, 6H); LC−MS m/z 368 (M + H)+.
6-Chloro-N-(3-(dimethylamino)propyl)-2-(p-tolyl)quinoline-
4-carboxamide (14). Prepared using general procedure B. Yield, 28%
(53 mg); 1H NMR (500 MHz, CDCl3) δ 8.51 (br s, 1H), 8.34 (d, 1H,
J = 2.4 Hz), 8.08 (d, 1H, J = 9.0 Hz), 8.04 (d, 2H, J = 8.2 Hz), 7.92 (s,
1H), 7.65 (dd, 1H, J = 2.1, 9.0 Hz), 7.30 (d, 2H, J = 8.2 Hz), 3.66 (q,
2H, J = 5.6 Hz), 2.51 (t, 2H, J = 5.6 Hz), 2.42 (s, 3H), 2.19 (s, 6H),
b
tafenoquine EC50 = 5250 nM, artemisinin EC50 = 0.8 nM. Data
reported previously.5
Table 11. Activity against Plasmodium falciparum Resistant
a
Strains
EC50 (nM)
compd
NF5
K1
W2
7G8
TM90C2A
D6
V1/S
30
2
0.5
0.3
0.8
0.4
0.6
0.4
0.7
0.4
0.5
0.4
0.7
0.4
0.9
0.7
+
1.84−1.79 (m, 2H) ppm; HRMS for C22H25ClN3O (M + H) calcd
382.1681, found 382.1673.
a
Data for compound 2 have been previously reported.5
6-Chloro-N-(2-morpholinoethyl)-2-(p-tolyl)quinoline-4-car-
boxamide (15). Prepared using general procedure B. Yield, 22% (45
mg); 1H NMR (500 MHz, DMSO-d6) δ 8.88 (t, J = 5.6 Hz, 1H), 8.31
(d, J = 2.4 Hz, 1H), 8.24−8.22 (m, 2H), 8.23 (s, 1H), 8.19 (s, 1H),
8.14 (d, J = 9.0 Hz, 1H), 7.85 (dd, J = 2.4, 9.0 Hz, 1H), 7.40 (d, J = 8.0
Hz, 2H), 3.65 (t, J = 4.5 Hz, 4H), 3.52 (q, J = 6.4 Hz, 2H), 2.57−2.54
(m, 2H), 2.41 (s, 3H) ppm; LC−MS m/z 410 (M + H)+.
6-Chloro-N-(2-(4-methylpiperazin-1-yl)ethyl)-2-(p-tolyl)-
quinoline-4-carboxamide (16). Prepared using general procedure
UV chromatogram (190−450 nm) obtained by LC−MS analysis.
Unless otherwise stated herein reactions have not been optimized.
General Procedure A: Preparation of 2-(p-Tolyl)quinilone-4-
carboxylic Acids (3). To a mixture of the corresponding isatin (5
mmol) in ethanol (10 mL) were added the corresponding
acetophenone (5 mmol), water (10 mL), and potassium hydroxide
(2.80 g, 50 mmol). The reaction mixture was heated under microwave
irradiation at 125 °C for 20 min. The resulting dark red colored
solution was diluted with water (50 mL) and acidified by adding HCl
(2 M, 30 mL). The resulting precipitate (yellow to ochre in color) was
collected by filtration, washed with water (50 mL), ethyl acetate (100
mL), and dichloromethane (25 mL). The remaining solid was used in
the next step without further purification.
1
B. Yield, 27% (55 mg); H NMR (500 MHz, DMSO-d6) δ 8.30 (d,
1H, J = 2.4 Hz), 8.23 (d, J = 8.2 Hz, 2H), 8.18−8.13 (m, 2H), 7.85
(dd, J = 2.4, 9.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 3.54−3.48 (m, 2H),
2.55 (t, J = 6.4 Hz, 4H), 2.52−2.50 (m, 4H), 2.41 (s, 5H), 2.22 (s, 3H)
ppm; LC−MS m/z 408 (M + H)+.
6-Chloro-N-(2-(piperidin-1-yl)ethyl-2-(p-tolyl)quinoline-4-
carboxamide (17). Prepared using general procedure B. Yield, 27%
(55 mg); 1H NMR (500 MHz, DMSO-d6) δ 8.93 (t, J = 5.6 Hz, 1H),
8.39 (d, J = 2.4 Hz, 1H), 8.33−8.31 (m, 2H), 8.27 (s, 1H), 8.23 (d, J =
9.0 Hz, 1H), 7.94 (dd, J = 2.4, 9.0 Hz, 1H), 7.49 (d, J = 8.1 Hz, 2H),
3.60 (q, J = 6.4 Hz, 2H), 2.56−2.49 (m, 7H), 1.67−1.63 (m, 4H),
1.53−1.49 (m, 2H) ppm; LC−MS m/z 408 (M + H)+.
6-Chloro-N-(2-(pyrrolidin-1-yl)ethyl-2-(p-tolyl)quinoline-4-
carboxamide (18). Prepared using general procedure B. Yield, 43%
(85 mg); 1H NMR (500 MHz, DMSO-d6) δ 8.87 (t, J = 5.7 Hz, 1H),
8.30 (d, J = 2.7 Hz, 1H), 8.22 (d, J = 8.8 Hz, 2H), 8.17 (s, 1H), 8.12
(d, J = 10.0 Hz, 1H), 7.82 (dd, J = 2.7, 8.8 Hz, 1H), 7.38 (d, J = 8.1
Hz, 2H), 3.50 (q, J = 5.7 Hz, 2H), 2.65 (t, J = 7.8 Hz, 2H), 2.56−2.54
(m, 4H), 2.30 (s, 3H), 1.73 (m, 4H) ppm; HRMS for C23H25ClN3O
(M + H)+ calcd 394.1681, found 394.1663.
6-Chloro-2-(p-tolyl)quinolone-4-carboxylic Acids (3, R1 = Cl).
1
Prepared using general procedure A. Yield, 58% (861 mg); H NMR
(500 MHz, DMSO-d6) δ 8.82 (d, J = 2.3 Hz, 1H), 8.44 (s, 1H), 8.19
(d, J = 8.2 Hz, 2H), 8.13 (d, J = 9.0 Hz, 1H), 7.82 (dd, J = 2.3, 9.0 Hz,
1H), 7.38 (d, J = 8.0 Hz, 2H), 2.41 (s, 3H) ppm; LC−MS m/z 298 (M
+ H)+.
2-(p-Tolyl)quinolone-4-carboxylic Acids (3, R1 = H). Prepared
using general procedure A. Yield, 29% (384 mg); 1H NMR (500 MHz,
DMSO-d6) δ 8.65 (d, 1H, J = 8.5 Hz), 8.45 (s, 1H), 8.22 (d, 2H, J =
8.2 Hz), 8.16 (d, 1H, J = 7.8 Hz), 7.85 (ddd, 1H, J = 1.4, 6.9, 8.4 Hz),
7.70 (ddd, 1H, J = 1.3, 6.9, 8.3 Hz), 7.39 (d, 2H, J = 7.9 Hz), 2.41 (s,
3H) ppm; LC−MS m/z 251 (M + H)+.
General Procedure B: Preparation of 2-(p-Tolyl)quinolone-4-
carboxamides (10−18). To a solution of quinolinecarboxylic acid 3
(0.5 mmol) in DMF (3 mL) was added diisopropylethylamine (0.07
mL, 0.75 mmol) followed by EDC (144 mg, 0.75 mmol) and HOBt
(101 mg, 0.75 mmol). After 5 min the corresponding amine (1 mmol)
was added followed by DMF (2 mL). The reaction mixture was stirred
at room temperature overnight. The reaction was diluted with water
(50 mL) and brine (10 mL) and extracted with ethyl acetate (30 mL).
Solvents were removed, and the resulting off white solid was washed
with dichloromethane and filtered. Amides that were soluble in
dichloromethane were purified by column chromatography on silica.
6-Chloro-N-(pyridine-3-yl)-2-(p-tolyl)quinoline-4-carboxa-
mide (10). Prepared using general procedure B. Yield, 25% (47 mg);
1H NMR (500 MHz, DMSO-d6) δ 11.06 (br s, 1H), 8.96 (d, J = 2.0
6-Fluoro-2-hydroxy-quinoline-4-carboxylic Acid (4, R1 = F).
A stirred suspension of 5-fluoroisatin (10.00 g, 61 mmol) and malonic
acid (18.91 g, 182 mmol) in acetic acid (400 mL) was refluxed for 16
h. Acetic acid was removed under reduced pressure, and the residue
was suspended in water (400 mL), filtered, and washed with water
(300 mL) to give a brown solid. The solid was stirred in NaHCO3
saturated aqueous solution (800 mL), and the insoluble material was
filtered off. The filtrate was acidified to pH 1−2 with concentrated
HCl, and the resulting precipitate was filtered, washed with water (300
mL), and dried. The resulting pale yellow solid was used directly for
synthesis of 4 without further purification. Yield, 54% (10 g); 1H NMR
(500 MHz; DMSO-d6) δ 2.08 (s, 1.5 H), 7.01 (s, 1H), 7.35−7.37 (m,
0.5 H)*, 7.48−7.49 (m, 2H), 7.64−7.68 (m, 0.9H)*, 8.01−8.04 (m,
1H), 12.29 (brs, 1H), 13.38 (brs, 0.7H)* ppm; LC−MS purity 63%,
m/z 208 (M + H)+; * corresponds to impurity.
Hz, 1H), 8.48 (s, 1H), 8.39 (dd, J = 1.4, 4.8 Hz, 1H), 8.30−8.25 (m,
4H), 8.18 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 2.4, 9.0 Hz, 1H), 7.47 (dd, J
= 4.8, 8.3 Hz, 1H), 7.41 (d, J = 8.3 Hz, 2H), 2.41 (s, 3H) ppm; HRMS
6-Chloro-2-hydroxyquinoline-4-carboxylic Acid (4, R1 = Cl).
A stirred suspension of 6-chloroisatin (10.00 g, 55 mmol) and malonic
acid (17.00 g, 165 mmol) in acetic acid (400 mL) was refluxed for 16
h. Acetic acid was removed under reduced pressure, and the residue
was suspended in water (400 mL), filtered, and washed with water
+
for C22H17ClN3O (M + H) calcd 374.1055, found 374.1051.
N-(Pyridine-3-yl)-2-(p-tolyl)quinoline-4-carboxamide (12).
1
Prepared using general procedure B. Yield, 41% (69 mg); H NMR
(500 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.02 (d, J = 1.9 Hz, 1H),
8.44−8.43 (m, 2H), 8.34−8.31 (m, 3H), 7.92−7.89 (m, 1H), 7.73−
I
DOI: 10.1021/acs.jmedchem.6b00723
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Journal of Medicinal Chemistry
Article
(300 mL) to give a gray solid. The solid was stirred in NaHCO3
saturated aqueous solution (800 mL), and the insoluble material was
filtered off. The filtrate was acidified to pH 1−2 with concentrated
HCl, and the precipitate was filtered, washed with water, and dried.
The resulting pale yellow solid was used for the synthesis of 4 without
further purification. Yield, 54% (9.5 g, 42 mmol); 1H NMR (500
MHz; DMSO-d6) δ 7.00 (s, 1H), 7.42 (d, 1H, J = 8.8 Hz), 7.58 (d, 0.3
H, J = 8.9 Hz)*, 7.60−7.62 (m, 1.3 H), 7.81 (dd, 0.3H, J = 2.3 Hz, J =
8.9 Hz)*, 8.29 (d, 1H, J = 2.4 Hz), 12.28 (brs, 1H), 13.22 (brs, 0.3
H)* ppm; LC−MS purity 65%, m/z 224 (M + H)+; * corresponds to
impurity.
2-Chloro-6-fluoro-N-(2-pyrrolidin-1-ylethyl)quinoline-4-car-
boxamide (5, R1 = F). To a stirred suspension of 6-fluoro-2-
hydroxyquinoline-4-carboxylic acid (4, R1 = F) (10.00 g, 48 mmol) in
anhydrous DCM (350 mL) were added anhydrous DMF (7 mL) and
thionyl chloride (14 mL, 193 mmol) under argon at room
temperature. The mixture was refluxed for 3 h and then allowed to
cool to room temperature. The solvents were removed under reduced
pressure, and the residue was dissolved in anhydrous THF (350 mL)
under argon. 2-Pyrrolidin-1-ylethanamine (18 mL, 145 mmol) was
added, and the reaction was stirred at room temperature for 16 h.
Solvents were removed under vacuum and the residue was partitioned
between NaHCO3 saturated aqueous solution (250 mL) and DCM (2
× 200 mL). The organic layers were combined, dried over MgSO4,
filtered, and evaporated under reduced pressure. The crude product
was purified by column chromatography using a 120 g silica gel
cartridge. Solvent A: DCM. Solvent B: 10% MeOH−NH3 in DCM.
Gradient: 2 min hold 100% A followed by 18 min ramp to 30% B and
then 15 min hold at 30% B. Fractions containing product were
combined and concentrated to dryness under reduced pressure to
obtain the desired compound as an off-white solid. Yield, 27% (4.59
g); 1H NMR (500 MHz; CDCl3) δ 8.07 (dd, J = 5.4, 9.2 Hz, 1H), 7.99
(dd, J = 2.8, 9.7 Hz, 1H), 7.56 (ddd, J = 2.8, 7.9, 9.2 Hz, 1H), 7.53 (s,
1H), 6.88 (brs, 1H), 3.65−3.69 (m, 2H), 2.81 (t, J = 5.5 Hz, 2H), 2.64
(brs, 4H), 1.83−1.85 (m, 4H) ppm; 19F NMR (407.5 MHz; CDCl3) δ
−110.03 ppm; LC−MS m/z 322 (M + H)+.
silica cartridge using A (DCM) and B (10% MeOH−NH3 in DCM).
Fractions containing product were pooled together to obtain 20 as a
white solid. Yield, 13% (10 mg); 1H NMR (500 MHz, CDCl3) δ 7.94
(s, 1H), 7.62−7.57 (m, 2H), 7.32 (dd, J = 2.8, 8.2 Hz, 1H), 6.88 (s,
1H), 5.68−5.63 (m, 1H), 3.80 (d, J = 4.6 Hz, 2H), 2.95 (t, J = 5.3 Hz,
2H), 2.80 (s, 4H), 2.27 (s, 3H), 1.77 (s, 4H) ppm; 19F NMR (407.5
MHz; CDCl3) δ −117.06 ppm; LC−MS m/z 384 (M + 1).
General Procedure C: Preparation of 2-Amino-4-carbox-
amides (21−30). A solution of 5 (1 equiv) and the corresponding
amine (3 equiv) in acetonitrile (2.5 mL) in a microwave vial was
heated at 170 °C for 1 h under microwave irradiation. Solvents were
removed and product was purified by column chromatography on 4 g
silica cartridges using A (DCM) and B (10% MeOH−NH3 in DCM).
6-Fluoro-2-(4-methylpiperazin-1-yl)-N-(2-(pyrrolidin-1-yl)-
ethyl)quinoline-4-carboxamide (21). Prepared using general
procedure C starting from 5 (R1 = F) (0.2 mmol, 65 mg), light
1
yellow solid. Yield, 26% (20 mg); H NMR (500 MHz, CDCl3) δ
7.72−7.64 (m, 2H), 7.34−7.29 (m, 1H), 7.13 (s, 1H), 6.83 (s, 1H),
3.76 (dd, J = 5.0, 5.0 Hz, 4H), 3.68−3.62 (m, 2H), 2.78 (dd, J = 5.9,
5.9 Hz, 2H), 2.61−2.54 (m, 8H), 2.38 (s, 3H), 1.81−1.78 (m, 4H)
ppm; 19F NMR (407.5 MHz; CDCl3) δ −118.04 ppm; LC−MS m/z
386 (M + 1).
2-(5,6-Dihydroimidazo[1,2-a]pyrazine-7(8H)-yl)-6-fluoro-N-
(2-(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (22). Pre-
pared using general procedure C starting from 5 (R1 = F) (0.6
mmol, 96 mg), white solid. Yield, 18% (15 mg); 1H NMR (500 MHz,
CDCl3) δ 7.78−7.71 (m, 2H), 7.39−7.34 (m, 1H), 7.23 (s, 1H),
7.13−7.11 (m, 1H), 7.04−7.03 (m, 1H), 6.88−6.87 (m, 1H), 4.88 (s,
2H), 4.28 (t, J = 5.4 Hz, 2H), 4.14 (t, J = 5.4 Hz, 2H), 3.67−3.62 (m,
2H), 2.77 (t, J = 6.0 Hz, 2H), 2.60−2.57 (m, 4H), 1.82−1.77 (m, 4H)
ppm; 19F NMR (407.5 MHz; CDCl3) δ −117.16 ppm; LC−MS m/z
409 (M + 1).
6-Fluoro-2-(4-morpholinopiperidin-1-yl)-N-(2-(pyrrolidin-1-
yl)ethyl)quinoline-4-carboxamide (23). Prepared using general
procedure C starting from 5 (R1 = F) (0.2 mmol, 65 mg), yellow solid.
1
Yield, 22% (20 mg); H NMR (500 MHz, CDCl3) δ 7.71−7.64 (m,
2H), 7.35−7.30 (m, 1H), 7.14 (s, 1H), 6.77 (s, 1H), 4.55 (d, J = 13.2
Hz, 2H), 3.74 (t, J = 4.7 Hz, 4H), 3.66−3.62 (m, 2H), 2.98−2.93 (m,
2H), 2.77 (t, J = 6.0 Hz, 2H), 2.61−2.57 (m, 8H), 2.52−2.43 (m, 1H),
1.80 (t, J = 6.6 Hz, 4H), 1.61−1.50 (m, 2H) ppm; 19F NMR (407.5
MHz; CDCl3) δ −118.31 ppm; LC−MS m/z 456 (M + 1).
6-Chloro-2-(4-morpholinopiperidin-1-yl)-N-(2-(pyrrolidin-1-
yl)ethyl)quinoline-4-carboxamide (24). Prepared using general
procedure C starting from 5 (R1 = Cl) (0.2 mmol, 70 mg), yellow
solid. Yield, 31% (28 mg); 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J =
2.2 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.50 (dd, J = 2.4, 8.8 Hz, 1H),
7.15 (s, 1H), 6.63−6.63 (m, 1H), 4.59 (d, J = 13.2 Hz, 2H), 3.75 (t, J
= 4.6 Hz, 4H), 3.66 (q, J = 5.6 Hz, 2H), 3.05−2.98 (m, 2H), 2.78 (dd,
J = 5.8, 5.8 Hz, 2H), 2.63- 2.58 (m, 8H), 2.53−2.47 (m, 1H), 2.00 (d, J
= 12.5 Hz, 2H), 1.82 (dd, J = 3.2, 6.5 Hz, 4H), 1.65−1.53 (m, 2H)
ppm; LC−MS m/z 473 (M + 1).
6-Fluoro-2-((3-morpholinopropyl)amino)-N-(2-(pyrrolidin-1-
yl)ethyl)quinoline-4-carboxamide (25). Prepared using general
procedure C starting from 5 (R1 = F) (0.75 mmol, 241 mg), off white
solid. Yield, 34% (110 mg); 1H NMR (500 MHz, CDCl3) δ 7.52−7.48
(m, 2H), 7.19−7.16 (m 1H), 6.36−6.35 (m 1H), 6.02 (s, 1H), 3.64
(br s, 4H), 3.56 (q, J = 5.6 Hz, 2H), 3.22 (br s, 2H), 2.70 (t, J = 5.9
Hz, 2H), 2.53 (br s, 4H), 2.35 (br s, 6H), 1.71 (br s, 4H), 1.65 (br s,
2H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −119.32 ppm; LC−MS
m/z 430 (M + 1).
2,6-Chloro-N-(2-pyrrolidin-1-ylethyl)quinoline-4-carboxa-
mide (5, R1 = Cl). To a stirred suspension of 6-chloro-2-
hydroxyquinoline-4-carboxylic acid (3) (8.50 g, 38 mmol) in
anhydrous DCM (250 mL) were added anhydrous DMF (2 mL)
and thionyl chloride (11 mL, 152 mmol) at room temperature under
argon. The mixture was refluxed for 3 h and then allowed to cool to
room temperature. The solvents were removed under reduced
pressure, and the residue was dissolved in anhydrous THF (300
mL) under argon. 2-Pyrrolidin-1-ylethanamine (14 mL, 114 mmol)
was added, and the reaction was stirred at room temperature for 16 h.
Solvents were removed under vacuum and the residue was partitioned
between NaHCO3 saturated aqueous solution (250 mL) and DCM (2
× 250 mL). The organic layers were combined, dried over MgSO4,
filtered, and evaporated under reduced pressure. The crude was
purified by column chromatography using a 120 g silica gel cartridge.
Solvent A: DCM. Solvent B: 10% MeOH−NH3 in DCM. Gradient: 2
min hold 100% A followed by 18 min ramp to 30% B and then 15 min
hold at 30% B. The relevant fractions were combined and
concentrated to dryness under reduced pressure to obtain the desired
product as an off-white solid. Yield, 43% (5.6 g); 1H NMR (500 MHz;
CDCl3) δ 8.27 (d, J = 2.3 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.70 (dd,
J = 2.3,9.0 Hz, 1H), 6.83 (brs, 1H), 7.48 (s, 1H), 3.64 (dt, J = 5.2, 11.6
Hz, 2H), 2.74−2.77 (m, 2H), 2.57−2.60 (m, 4H), 1.78−1.81 (m, 4H)
ppm; LC−MS m/z 338 (M + H)+.
6-Fluoro-2-((3-methylisoxazol-5-yl)amino-N-(2-(pyrrolidin-
1-yl)ethyl)quinoline-4-carboxamide (20). To a solution of 5 (R1 =
F) (65 mg, 0.2 mmol) and 5-amino-3-methylisoxazole (39 mg, 0.4
mmol), BINAP (33 mg, 0.05 mmol) in dioxane (4 mL) were added
sodium tert-butoxide (38 mg, 0.4 mmol) and palladium acetate (9 mg,
0.04 mmol). Reaction was heated at 115 °C overnight in a sealed tube.
Reaction crude was filtered through Celite, and the filtrate was
partitioned between water (5 mL) and DCM (25 mL). Organic phase
was dried over MgSO4, and solvents were removed under reduce
pressure. Product was purified by column chromatography on a 4 g
6-Fluoro-2-((3-morpholinoethyl)amino)-N-(2-(pyrrolidin-1-
yl)ethyl)quinoline-4-carboxamide (26). Prepared using general
procedure C starting from 5 (R1 = F) (0.3 mmol, 88 mg), yellow solid.
1
Yield, 29% (33 mg); H NMR (500 MHz, CDCl3) δ 7.69−7.65 (m,
2H), 7.31 (ddd, J = 2.9, 8.2, 9.1 Hz, 1H), 6.91−6.89 (m, 1H), 6.76 (s,
1H), 3.74 (dd, J = 4.6, 4.6 Hz, 4H), 3.65−3.53 (m, 4H), 2.75 (t, J = 5.9
Hz, 2H), 2.66−2.55 (m, 6H), 2.54−2.48 (m, 4H), 1.81−1.79 (m,
4H)ppm; 19F NMR (407.5 MHz; CDCl3) δ −118.87 ppm; LC−MS
m/z 416 (M + 1).
6-Chloro-2-((4-morpholinobutyl)amino)-N-(2-(pyrrolidin-1-
yl)ethyl)quinoline-4-carboxamide (27). Prepared using general
J
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1
procedure C starting from 5 (R1 = Cl) (0.3 mmol, 100 mg), white
solid. Yield, 54% (50 mg); 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J =
20.4 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.49 (dd, J = 2.4, 9.0 Hz, 1H),
6.82 (s, 1H), 6.66 (s, 1H), 3.75 (t, J = 4.7 Hz, 4H), 3.68−3.63 (m,
2H), 3.44 (q, J = 6.2 Hz, 2H), 2.78 (t, J = 5.9 Hz, 2H), 2.62 (br s, 4H),
2.48−2.39 (m, 8H), 1.84−1.79 (m, 4H), 1.73−1.61 (m, 2H) ppm;
LC−MS m/z 460 (M + 1).
Yield, 20% (24 mg); H NMR (500 MHz, CDCl3) δ 8.36−8.33 (m,
2H), 8.22 (dd, J = 5.4, 9.2 Hz, 1H), 8.04−7.99 (m, 2H), 7.91 (d, J =
8.5 Hz, 2H), 7.58 (ddd, J = 2.8, 8.0, 9.2 Hz, 1H), 6.98 (s, 1H), 3.79−
3.69 (m, 6H), 3.07 (t, J = 4.7 Hz, 4H), 2.81 (t, J = 5.9 Hz, 2H), 2.62
(d, J = 5.4 Hz, 4H), 1.84−1.79 (m, 4H) ppm; 19F NMR (407.5 MHz;
CDCl3) δ −109.76 ppm; LC−MS m/z 513 (M + H)+.
6-Fluoro-2-(4-(morpholine-4-carbonyl)phenyl)-N-(2-(pyrroli-
din-1-yl)ethyl)quinoline-4-carboxamide (39). Prepared using
general procedure D starting from 5 (R1 = F) (0.25 mmol, 80 mg),
6-Fluoro-2-((2-(piperidin-1-yl)amino)-N-(2-(pyrrolidin-1-yl)-
ethyl)quinoline-4-carboxamide (28). Prepared using general
procedure C starting from 5 (R1 = F) (0.26 mmol, 84 mg), off
white solid. Yield, 9% (10 mg); 1H NMR (500 MHz, CDCl3) δ 7.61−
7.57 (m, 2H), 7.26 (ddd, J = 3.0, 8.6, 8.6 Hz, 1H), 6.70 (s, 1H), 6.67
(s, 1H), 5.44−5.42 (m, 1H), 3.57−3.44 (m, 4H), 2.66 (dd, J = 6.0, 6.0
Hz, 2H), 2.53−2.47 (m, 4H), 2.44−2.28 (m, 6H), 1.74−1.69 (m, 4H),
1.54−1.45 (m, 5H) ppm; 19F NMR (407.5 MHz; CDCl3) δ −119.16
ppm; LC−MS m/z 414 (M + 1).
1
yellow solid. Yield, 73% (87 mg); H NMR (500 MHz, CDCl3) δ
8.18−8.14 (m, 3H), 7.96−7.94 (m, 2H), 7.54−7.50 (m, 3H), 7.10 (dd,
J = 4.9, 4.9 Hz, 1H), 3.76−3.72 (m, 6H), 3.69−3.66 (m, 4H), 2.78 (t, J
= 6.0 Hz, 2H), 2.61−2.59 (m, 4H), 1.81−1.79 (m, 4H) ppm; 19F
NMR (407.5 MHz; CDCl3) δ −110.76 ppm; LC−MS m/z 477 (M +
H)+.
1-(4-(Bromomethyl)-2-chlorophenyl)ethanone (7). A mixture
of 1-(2-chloro-4-methylphenyl)ethanone (1.6 g, 9.50 mmol) and
chlorobenzene (60 mL) was prepared at rt and N-bromosuccinimide
(NBS) (1.86 g, 10.44 mmol) added followed by benzoyl peroxide
(∼1.5 mg, 0.005 mmol, catalytic amount), and the mixture was heated
to 140−145 °C for 16 h. The mixture was then cooled to rt, diluted
with toluene (50 mL), and filtered through a Celite pad. The pad was
washed with toluene (2 × 50 mL) and the filtrate concentrated under
reduced pressure and purified by column chromatography (0−10%
EtOAc/hexanes) to afford 1-(4-(bromomethyl)-2-chlorophenyl)-
6-Fluoro-N-(2-(pyrrolidin-1-yl)ethyl)-2-((2-(tetrahydro-2H-
pyran-4-yl)ethyl)amino)quinoline-4-carboxamide (29). Pre-
pared using general procedure C starting from 5 (R1 = F) (0.32
1
mmol, 100 mg), white solid. Yield, 7% (6 mg); H NMR (500 MHz,
CDCl3) δ 7.61−7.55 (m, 2H), 7.23 (ddd, J = 2.4, 7.8, 9.7 Hz, 1H),
6.75 (br s, 1H), 6.59 (s, 1H), 4.74 (br s, 1H), 3.89 (dd, J = 3.8, 11.2
Hz, 2H), 3.55 (q, J = 5.6 Hz, 2H), 3.39−3.27 (m, 4H), 2.68 (t, J = 5.9
Hz, 2H), 2.50 (d, J = 5.4 Hz, 4H), 1.74−1.69 (m, 4H), 1.62−1.48 (m,
5H), 1.33−1.21 (m, 2H) ppm; 19F NMR (407.5 MHz; CDCl3) δ
−118.84 ppm; LC−MS m/z 415 (M + 1).
1
ethanone (1.65 g, 6.65 mmol, 70%) as a yellow oil. H NMR (500
MHz; CDCl3) δ 2.65 (s, 3H), 4.43 (s, 2H), 7.34 (dd, 1H, J = 1.3, 8.0
Hz), 7.46 (s, 1H), 7.54 (d, 1H, J = 7.9 Hz) ppm.
6-Chloro-N-(2-(pyrrolidin-1-yl)ethyl)-2-((2-(tetrahydro-2H-
pyran-4-yl)ethyl)amino)quinoline-4-carboxamide (30). Pre-
pared using general procedure C starting from 5 (R1 = Cl) (1.5
1-(2-Chloro-4-(morpholinomethyl)phenyl)ethanone (8, X =
Cl). A mixture of 1-(4-(bromomethyl)-2-chlorophenyl)ethanone (1.65
g, 6.65 mmol) and acetonitrile (25 mL) was prepared at rt and stirred
under nitrogen. Potassium carbonate (1.10g 7.98 mmol) was then
added followed by morpholine (0.695 mL, 695 mg, 7.98 mmol), and
the mixture was stirred at rt. After 2 h, TLC showed presence of
product and starting material. Mixture was then heated under nitrogen
to 40 °C for 16 h, cooled to room temp, filtered to remove excess
carbonate and filtrate concentrated under reduced pressure. Mixture
was then diluted in DCM (30 mL), washed with water (2 × 10 mL),
filtered through a phase separator and filtrate concentrated under
reduced pressure. Mixture was purified by column chromatography
(40−100% ethyl acetate/hexane) to afford 1-(2-chloro-4-
(morpholinomethyl)phenyl)ethanone (1.08 g, 4.25 mmol, 64%) as a
1
mmol, 500 mg), yellow solid. Yield, 31% (200 mg); H NMR (500
MHz, CDCl3) δ 8.06 (br s, 1H), 8.05 (d, J = 2 0.4 Hz, 1H), 7.51 (d, J
= 9.0 Hz, 1H), 7.37 (dd, J = 2.4, 8.8 Hz, 1H), 7.17 (m, 1H), 4.94 (t, J
= 5.0 Hz, 1H), 3.88 (dd, J = 3.8, 11.2 Hz, 2H), 3.74 (q, J = 5.4 Hz,
2H), 3.46 (q, J = 6.6 Hz, 2H), 3.33−3.27 (m, 2H), 3.16−3.06 (m,
6H), 2.03−1.98 (m, 3H), 1.63−1.49 (m, 6H), 1.32−1.21 (m, 2H)
ppm; LC−MS m/z 431 (M + 1).
General Procedure D: Suzuki Coupling on Intermediate 5.
To a solution of 5 (1 equiv) in DMF/water 3/1 (4 mL) in a
microwave vial were added potassium phosphate (3 equiv), the
corresponding boronic acid (3 equiv), and Pd(PPh3)4 (3 mol %). The
reaction was degassed by bubbling nitrogen through the mixture for 5
min and then heated under microwave irradiation at 130 °C for 30
min. Reaction mixture was filtered through Celite. Celite was washed
with DCM. Filtrate was partitioned between water (5 mL) and DCM
(25 mL × 2). Organic phase was dried over MgSO4, and solvents were
evaporated under reduced pressure. Product was purified by column
chromatography on 4 g silica cartridges using A (DCM) and B (20%
MeOH−NH3 in DCM) and the following gradient: 3 min hold 100%
A, 15 min ramp to 30% B, 4 min hold at 30% B.
1
yellow oil. H NMR (500 MHz; CDCl3) δ 2.44 (brs, 4H), 2.65 (s,
3H), 3.49 (s, 2H), 3.72 (t, J = 4.6 Hz, 4H), 7.29 (dd, J = 1.5, 7.9 Hz,
1H,), 7.43 (brs, 1H), 7.55 (d, J = 7.9 Hz, 1H) ppm; LCMS m/z 254
[M + H]+
2-(2-Chloro-4-(morpholinomethyl)phenyl)-6-fluoroquino-
line-4-carboxylic Acid (9, X = Cl). A mixture of 5-fluoroisatin (7.02
mg, 4.25 mmol) and 1-(2-chloro-4-(morpholinomethyl)phenyl)-
ethanone (1.08 g, 4.25 mmol) was prepared in EtOH/water (1:1)
(10 mL) and then KOH (2.40 g, 42.50 mmol) added and the mixture
heated in microwave, 125 °C, 20 min. The mixture was then diluted
with water (10 mL), acidified to pH 3 with 2 M HCl, stirred for 16 h at
rt and the resulting precipitate filtered, washed with water (2 × 10
mL), and concentrated under reduced pressure to afford 2-(2-chloro-
4-(morpholinomethyl)phenyl)-6-fluoroquinoline-4-carboxylic acid
(503 mg, 1.25 mmol, 30%) as an orange solid. 1H NMR (500
MHz; CDCl3) δ 2.54 (brs, 4H), 3.64 (s, 4H), 3.70 (brs, 2H), 7.50 (d, J
= 8.1 Hz, 1H), 7.62 (s, 1H), 7.73 (bd, J = 7.9 Hz, 1H), 7.84 (dt, J =
2.9, 8.2 Hz, 1H,), 8.24 (dd, J = 5.8, 9.2 Hz, 1H), 8.56 (dd, J = 2.9, 11.0
Hz, 1H) ppm; LCMS m/z 399 [M − H]−.
2-(2-Chloro-4-(morpholinomethyl)phenyl)-6-fluoro-N-(2-
(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (42). A mixture
of 2-(2-chloro-4-(morpholinomethyl)phenyl)-6-fluoroquinoline-4-car-
boxylic acid (303 mg, 0.76 mmol) in DCM (6 mL) was prepared at rt,
and N-methylmorpholine (0.166 mL, 153 mg, 1.51 mmol) and 2-
chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (159 mg, 0.91 mmol)
were added, and the mixture was stirred for 1 h in a sealed vial. 2-
(Pyrrolidin-1-yl)ethanamine (0.143 mL, 130 mg, 1.13 mmol) was then
added and the mixture stirred in a sealed vial for 17 h. The mixture was
2-(4-(Dimethylamino)phenyl)-6-fluoro-N-(2-(pyrrolidin-1-yl)-
ethyl)quinoline-4-carboxamide (36). Prepared using general
procedure D starting from 5 (R1 = F) (0.2 mmol, 65 mg), yellow
solid. Yield, 25% (20 mg); 1H NMR (500 MHz, CDCl3) δ 8.13−8.07
(m, 3H), 7.91−7.87 (m, 2H), 7.47 (ddd, J = 2.9, 8.1, 9.2 Hz, 1H), 6.96
(s, 1H), 6.85−6.82 (m, 2H), 3.70 (q, J = 5.7 Hz, 2H), 3.08 (s, 6H),
2.83 (t, J = 6.0 Hz, 2H), 2.67−2.64 (m, 4H), 1.86−1.81 (m, 4H) ppm;
19F NMR (407.5 MHz; CDCl3) δ −113.18 ppm; LC−MS m/z 407 (M
+ H)+.
6-Fluoro-2-(4-morpholinophenyl)-N-(2-(pyrrolidin-1-yl)-
ethyl)quinoline-4-carboxamide (37). Prepared using general
procedure D starting from 5 (R1 = F) (0.5 mmol, 150 mg), yellow
solid. Yield, 19% (43 mg); 1H NMR (500 MHz, CDCl3) δ 8.15−8.07
(m, 3H), 7.91−7.87 (m, 2H), 7.52−7.47 (m, 1H), 7.03 (d, J = 8.8 Hz,
2H), 6.89 (t, J = 4.5 Hz, 1H), 3.92 (t, J = 4.8 Hz, 4H), 3.68 (q, J = 5.7
Hz, 2H), 3.30 (t, J = 4.8 Hz, 4H), 2.79 (t, J = 6.0 Hz, 2H), 2.60−2.62
(m, 4H), 1.81 (t, J = 3.3 Hz, 4H) ppm; 19F NMR (407.5 MHz;
CDCl3) δ −112.39 ppm; LC−MS m/z 449 (M + H)+.
6-Fluoro-2-(4-(morpholinosulfonyl)phenyl)-N-(2-(pyrrolidin-
1-yl)ethyl)quinoline-4-carboxamide (38). Prepared using general
procedure D starting from 5 (R1 = F) (0.23 mmol, 75 mg), white solid.
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then diluted with DCM (5 mL), and the organic layers were washed
with water (2 × 3 mL) and filtered through a phase separator and the
organic layers concentrated under reduced pressure and purified by
column chromatography (0−10% 7 M NH3 in MeOH/DCM) to
afford an off white solid. Analysis by 1H NMR showed impurities, and
the mixture was further purified by MDAP to produce 2-(2-chloro-4-
(morpholinomethyl)phenyl)-6-fluoro-N-(2-(pyrrolidin-1-yl)ethyl)-
quinoline-4-carboxamide (228 mg, 0.46 mmol, 61%) as an off-white
3.66 (m, 6H), 3.59 (s, 2H), 3.06 (t, J = 5.8 Hz, 2H), 2.49 (s, 4H),
2.23−2.18 (m, 1H), 0.54−0.49 (m, 2H), 0.39−0.35 (m, 2H); LC−MS
m/z 449 (M + H)+.
N-(2-(Azetidin-1-yl)ethyl)-6-fluoro-2-(4-(morpholinomethyl)-
phenyl)quinoline-4-carboxamide (49). To a suspension of 6-
fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-carboxylic acid (9,
X = H)5 (100 mg,0.27 mmol) in anhydrous DCM (10 mL) were
added N-methylmorpholine (0.06 mL, 0.54 mmol, 2 equiv) and 2-
chloro-4,6-dimethoxy-1,3,5-triazine CDMT (57 mg, 0.32 mmol, 1.2
equiv). The reaction mixture was stirred at room temperature for 1 h.
2,2-Dimethoxyethanamine (28 mg,0.27 mmol) was added and the
reaction mixture stirred at room temperature for 2.5 days. The mixture
was then diluted with DCM (10 mL), water (10 mL) was added, and
the layers were separated. The aqueous portion was extracted with
further DCM (10 mL). The combined DCM extracts were evaporated
in vacuo. The residue was dissolved in DCM and purified by silica (12
g), eluting with 0−100% EtOAc/hexane and then 0−50% (10%
MeOH in DCM/DCM) to afford N-(2,2-dimethoxyethyl)-6-fluoro-2-
[4-(morpholinomethyl)phenyl]quinoline-4-carboxamide as a yellow
gum (109 mg, 79% yield). A solution of N-(2,2-dimethoxyethyl)-6-
fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-carboxamide (109
mg,0.24 mmol) in 1,4-dioxane (5 mL) was treated with conc. HCl (1
mL) and stirred at room temperature for 1.5 h. The mixture was
neutralized with saturated sodium bicarbonate portionwise and then
exracted with EtOAc (2 × 20 mL). The combined EtOAc extracts
were evaporated in vacuo to afford 6-fluoro-2-[4-(morpholinomethyl)-
phenyl]-N-(2-oxoethyl)quinoline-4-carboxamide as a yellow gum (78
mg, 71% yield). A mixture of 6-fluoro-2-[4-(morpholinomethyl)-
phenyl]-N-(2-oxoethyl)quinoline-4-carboxamide (78 mg,0.19 mmol),
azetidine (32 mg,0.57 mmol) in DCM (5 mL) was stirred for 15 min
in a stoppered flask at room temperature. Sodium triacetoxyborohy-
dride (56 mg,0.26 mmol) was then added, and the reaction mixture
was stirred at room temperature overnight. The reaction mixture was
partitioned between water (10 mL) and DCM (10 mL) and the
aqueous extracted with further DCM (10 mL). The combined DCM
extracts were evaporated in vacuo. The residue was dissolved in DMF
and purified by mass directed autoprep, 5−95% MeCN, basic, to afford
a yellow gum. The sample was freeze-dried to afford 49 as a cream
colored solid (18 mg, 14% yield); 1H NMR (500 MHz, CDCl3) δ 8.21
(dd, J = 5.4, 9.2 Hz, 1H), 8.13 (d, J = 8.4 Hz, 2H), 8.00−7.97 (m, 2H),
7.58−7.52 (m, 3H), 6.73−6.72 (m, 1H), 3.76 (t, J = 4.6 Hz, 4H), 3.61
(s, 2H), 3.57−3.53 (m, 2H), 3.28 (t, J = 7.0 Hz, 4H), 2.72 (t, J = 5.8
Hz, 2H), 2.51 (s, 4H), 2.14−2.07 (m, 2H); LC−MS m/z 449 (M +
H)+.
1
solid. H NMR (500 MHz; CDCl3) δ 1.76−1.79 (m, 4H), 2.49 (brs,
4H), 2.57 (brs, 4H), 2.75 (t, J = 6.0 Hz, 2H), 3.55 (s, 2H), 3.65 (q, J =
5.3 Hz, 2H), 3.74 (t, J = 4.6 Hz, 4H), 6.82 (brs, 1H), 7.40 (dd, J = 1.6,
7.9 Hz, 1H), 7.52−7.57 (m, 2H), 7.68 (d, J = 7.9 Hz, 1H), 7.89 (s,
1H), 8.05 (dd, J = 2.8, 10.0 Hz, 1H), 8.19 (dd, J = 5.5, 9.2 Hz, 1H)
ppm; LCMS m/z 497 [M + H]+.
6-Fluoro-N-(2-morpholinoethyl)-2-(4-morpholinomethyl)-
phenyl)quinoline-4-carboxamide (46). A solution of 6-fluoro-2-
(4-(morpholinomethyl)phenyl)quinoline-4-carboxylic acid (9, X =
H)5 (0.1 g, 0.27 mmol), 2-chloro-4,6-dimethoxy-1,3,5-triazine CDMT
(57 mg, 0.32 mmol, 1.2 equiv), and N-methylmorpholine (0.06 mL,
0.54 mmol, 2 equiv) in DCM (10 mL) was stirred at room
temperature for 1 h. 2-Morpholinoethanamide (0.054 mL, 0.41 mmol,
1.5 equiv) was added, and the reaction mixture was stirred at room
temperature overnight. Reaction was partitioned between DCM (50
mL) and NaHCO3 sat. aq solution (10 mL). Organic phase was dried
over MgSO4, and solvents were removed under reduce pressure.
Product precipitated from acetonitrile (1.5 mL) as a white solid. Yield,
1
63% (83 mg); H NMR (500 MHz, CDCl3) δ 8.22 (dd, J = 5.4, 9.2
Hz, 1H), 8.12 (d, J = 8.2 Hz, 2H), 8.00−7.97 (m, 2H), 7.58−7.52 (m,
3H), 6.67 (t, J = 4.8 Hz, 1H), 3.77−3.70 (m, 10H), 3.61 (s, 2H), 2.70
(t, J = 5.9 Hz, 2H), 2.57 (br s, 4H), 2.51 (t, J = 4.3 Hz, 4H) ppm; 19F
NMR (407.5 MHz; CDCl3) δ −111.23; LC−MS m/z 479 (M + H)+.
6-Fluoro-N-methyl-2-(4-(morpholinomethyl)phenyl)-N-(2-
(pyrrolidin-1-yl)ethyl)quinoline-4-carboxamide (47). To a sus-
pension of 6-fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-car-
boxylic acid (9, X = H)5 (100 mg, 0.27 mmol) in anhydrous DCM (10
mL) were added N-methylmorpholine (0.06 mL, 0.54 mmol, 2 equiv)
and 2-chloro-4,6-dimethoxy-1,3,5-triazine CDMT (57 mg, 0.32 mmol,
1.2 equiv). The reaction mixture was stirred at room temperature for 1
h. N-Methyl-2-pyrrolidin-1-ylethanamine (0.034 g, 0.27 mmol) was
added and the reaction mixture stirred at room temperature overnight.
The mixture was then diluted with DCM (10 mL), water (10 mL) was
added, and the layers were separated. The aqueous portion was
extracted with further DCM (10 mL). The combined DCM extracts
were evaporated in vacuo. The residue was dissolved in DMF and
purified by mass directed autoprep 5−95% MeCN, basic, to afford
impure product. The sample was dissolved in DMF and purified using
mass directed autoprep 25−75% MeCN, basic, and the product
obtained was freeze-dried to afford 47 as a cream colored solid (24 mg,
17% yield); 1H NMR (500 MHz, CDCl3) δ 8.15−8.08 (m, 3H), 8.04−
8.01 (m, 1H), 7.48−7.42 (m, 3H), 7.36 (dd, J = 2.8, 9.2 Hz, 1H),
4.06−4.03 (m, 1H), 3.67 (dd, J = 4.3, 4.3 Hz, 4H), 3.53 (s, 2H), 3.27
(s, 2H), 3.22 (s, 1H), 2.90 (s, 3H), 2.43 (s, 4H), 2.11- 2.06 (m, 4H),
1.81−1.51 (m, 4H) ppm; LC−MS m/z 477 (M + H)+.
Details of other synthetic routes for individual compounds are
described in the Supporting Information.
Biology Materials and Methods. This is included in the
Supporting Information.
Ethical Statements. In vivo antimalarial efficacy studies in P.
berghei carried out at the Swiss Tropical and Public Health Institute
(Basel, Switzerland) adhere to local and national regulations of
laboratory animal welfare in Switzerland (awarded Permission No.
1731). Protocols are regularly reviewed and revised following approval
N - ( 2 - ( C y c l o p r o p y l a m i n o ) e t h y l ) - 6 - fl u o r o - 2 - ( 4 -
(morpholinomethyl)phenyl)quinoline-4-carboxamide (48). To
a suspension of 6-fluoro-2-[4-(morpholinomethyl)phenyl]quinoline-4-
carboxylic acid (9, X = H)5 (0.1 g, 0.27 mmol) in anhydrous DCM (10
mL) were added N-methylmorpholine (0.06 mL, 0.54 mmol, 2 equiv)
and 2-chloro-4,6-dimethoxy-1,3,5-triazine CDMT (57 mg, 0.32 mmol,
1.2 equiv). The reaction mixture was stirred at room temperature for 1
h. N′-Cyclopropylethane-1,2-diamine (0.027 g,0.27 mmol) was added
and the reaction mixture stirred at room temperature for 2.5 days. The
mixture was then diluted with DCM (10 mL), water (10 mL) was
added, and the layers were separated. The aqueous portion was
extracted with further DCM (10 mL). The combined DCM extracts
were evaporated in vacuo. The residue was dissolved in DMF and
purified by mass directed autoprep 5−95% MeCN, basic, method to
by the local authority (Veterinaramt Basel Stadt).
̈
Mouse and rat pharmacokinetics were carried out at the University
of Dundee. All regulated procedures on living animals were carried out
under the authority of a license issued by the Home Office under the
Animals (Scientific Procedures) Act 1986, as amended in 2012 (and in
compliance with EU Directive EU/2010/63). License applications will
have been approved by the University’s Ethical Review Committee
(ERC) before submission to the Home Office. The ERC has a general
remit to develop and oversee policy on all aspects of the use of animals
on University premises and is a subcommittee of the University Court,
its highest governing body.
1
afford 48 as a yellow solid (25 mg, 18% yield); H NMR (500 MHz,
CDCl3) δ 8.18 (dd, J = 5.4, 9.2 Hz, 1H), 8.09 (d, J = 8.2 Hz, 2H),
7.94−7.91 (m, 2H), 7.55−7.49 (m, 3H), 6.74−6.72 (m, 1H), 3.75−
L
DOI: 10.1021/acs.jmedchem.6b00723
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Article
M.; Noedl, H.; Nosten, F.; Kyaw, M. P.; Nhien, N. T.; Imwong, M.;
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Ringwald, P.; Plowe, C. V. Independent emergence of artemisinin
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00723.
■
S
Synthetic details for all compounds, supplementary data
tables, additional information on ADMET and pharma-
cology (PDF)
Molecular formula strings and some data (CSV)
(4) Wells, T. N.; Hooft van Huijsduijnen, R.; Van Voorhis, W. C.
Malaria medicines: a glass half full? Nat. Rev. Drug Discovery 2015, 14,
424−442.
AUTHOR INFORMATION
Corresponding Authors
*K.D.R.: phone, +44 1382 388 688; e-mail, k.read@dundee.ac.
■
(5) Baragana, B.; Hallyburton, I.; Lee, M. C. S.; Norcross, N. R.;
Grimaldi, R.; Otto, T. D.; Proto, W. R.; Blagborough, A. M.; Meister,
S.; Wirjanata, G.; Ruecker, A.; Upton, L. M.; Abraham, T. S.; Almeida,
M. J.; Pradhan, A.; Porzelle, A.; Martinez, M. S.; Bolscher, J. M.;
Woodland, A.; Norval, S.; Zuccotto, F.; Thomas, J.; Simeons, F.;
Stojanovski, L.; Osuna-Cabello, M.; Brock, P. M.; Churcher, T. S.;
Sala, K. A.; Zakutansky, S. E.; Jimenez-Diaz, M. B.; Sanz, L. M.; Riley,
J.; Basak, R.; Campbell, M.; Avery, V. M.; Sauerwein, R. W.;
Dechering, K. J.; Noviyanti, R.; Campo, B.; Frearson, J. A.; Angulo-
Barturen, I.; Ferrer-Bazaga, S.; Gamo, F. J.; Wyatt, P. G.; Leroy, D.;
Siegl, P.; Delves, M. J.; Kyle, D. E.; Wittlin, S.; Marfurt, J.; Price, R. N.;
Sinden, R. E.; Winzeler, E. A.; Charman, S. A.; Bebrevska, L.; Gray, D.
W.; Campbell, S.; Fairlamb, A. H.; Willis, P. A.; Rayner, J. C.; Fidock,
D. A.; Read, K. D.; Gilbert, I. H. A novel multiple-stage antimalarial
agent that inhibits protein synthesis. Nature 2015, 522, 315−320.
(6) Brenk, R.; Schipani, A.; James, D.; Krasowski, A.; Gilbert, I. H.;
Frearson, J.; Wyatt, P. G. Lessons learnt from assembling screening
libraries for drug discovery for neglected diseases. ChemMedChem
2008, 3, 435−444.
(7) Burrows, J. N.; Hooft 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.
(8) (a) Leeson, P. D.; Young, R. J. Molecular property design: does
everyone get it? ACS Med. Chem. Lett. 2015, 6, 722−725. (b) Hann,
M. M. Molecular obesity, potency and other addictions in drug
discovery. MedChemComm 2011, 2, 349−355.
(9) (a) Ritchie, T. J.; Macdonald, S. J. F. The impact of aromatic ring
count on compound developability - are too many aromatic rings a
liability in drug design? Drug Discovery Today 2009, 14, 1011−1020.
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uk.
*I.H.G.: phone, +44 1382 386 240; e-mail, i.h.gilbert@dundee.
ac.uk.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Medicines for Malaria Venture for financial
support. The University of Dundee also acknowledges support
from the Wellcome Trust (Grant 100476 and a Principal
Research Fellowship to A.H.F.). We thank Christian Scheurer
and Jolanda Kamber from the Swiss TPH for assistance in
performing assays against the drug-resistant parasites and the in
vivo (Pb) antimalarial assays.
ABBREVIATIONS USED
■
ACT, artemisinin combination therapy; CDMT, 2-chloro-4,6-
dimethoxy-1,3,5-triazine; Cli, intrinsic clearance; EDC, N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
HOBt, hydroxybenzotriazole; LELP, ligand-efficiency-depend-
ent lipophilicity; LLE, lipophilic ligand efficiency; MMV,
Medicines for Malaria Venture; Pe, permeability; TCP, target
candidate profile; WHO, World Health Organization
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