ICI 56,780 optimization: Structure-activity relationship studies of 7-(2-phenoxyethoxy)-4(1H)-quinolones with antimalarial activity

Article abstract of DOI:10.1021/acs.jmedchem.6b00759

Though malaria mortality rates are down 48% globally since 2000, reported occurrences of resistance against current therapeutics threaten to reverse that progress. Recently, antimalarials that were once considered unsuitable therapeutic agents have been revisited to improve physicochemical properties and efficacy required for selection as a drug candidate. One such compound is 4(1H)-quinolone ICI 56,780, which is known to be a causal prophylactic that also displays blood schizonticidal activity against P. berghei. Rapid induction of parasite resistance, however, stalled its further development. We have completed a full structure-activity relationship study on 4(1H)-quinolones, focusing on the reduction of cross-resistance with atovaquone for activity against the clinical isolates W2 and TM90-C2B, as well as the improvement of microsomal stability. These studies revealed several frontrunner compounds with superb in vivo antimalarial activity. The best compounds were found to be curative with all mice surviving a Plasmodium berghei infection after 30 days.

Full text of DOI:10.1021/acs.jmedchem.6b00759

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Article
ICI 56,780 Optimization: Structural-Activity and Relationship Studies
of 7-(2-phenoxyethoxy)-4(1H)-quinolones with Antimalarial Activity
Jordany R. Maignan, Cynthia L. Lichorowic, James Giarrusso, Lynn Blake, Deborah Casandra, Tina
S. Mutka, Alexis N. LaCrue, Jeremy N. Burrows, Paul A. Willis, Dennis E Kyle, and Roman Manetsch
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00759 • Publication Date (Web): 11 Jun 2016
Downloaded from http://pubs.acs.org on June 11, 2016
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ICI 56,780 Optimization: StructuralꢀActivity and
Relationship Studies of 7ꢀ(2ꢀphenoxyethoxy)ꢀ4(1H)ꢀ
quinolones with Antimalarial Activity
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§
,#
§,#,†
§
¥
Jordany R. Maignan , Cynthia L. Lichorowic
, James Giarrusso , Lynn Blake , Debora
¥
¥
¥
‡
‡
Casandra , Tina S. Mutka , Alexis N. LaCrue , Jeremy N. Burrows , Paul A. Willis , Dennis E.
¥
,§,†,┴
Kyle , and Roman Manetsch*
.
§
Department of Chemistry, University of South Florida, CHE 205, 4202 E. Fowler Ave, Tampa,
†
Florida 33620, USA, Department of Chemistry and Chemical Biology, Northeastern University,
¥
1
02 Hurtig Hall, 360 Huntington Avenue, Boston, MA 02115, United States. Department of
Global Health, College of Public Health, University of South Florida, 3720 Spectrum Blvd, Suite
‡
304, Tampa, Florida 33612, USA. Medicines for Malaria Venture, 20, Route de PréꢀBois, P.O.
┴
Box 1826, 1215 Geneva 15, Switzerland. Department of Pharmaceutical Sciences, Northeastern
University, 102 Hurtig Hall, 360 Huntington Avenue, Boston, MA 02115, United States.
Abstract
Though malaria mortality rates are down 48% globally since 2000, reported occurrences of
resistance against current therapeutics threaten to reverse that progress. Recently, antimalarials
which were once considered unsuitable therapeutic agents have been revisited to improve
physicochemical properties and efficacy required for selection as a drug candidate. One such
compound is 4(1H)ꢀquinolone ICI 56,780, which is known to be a causal prophylactic that also
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displays blood schizonticidal activity against P. berghei. Rapid induction of parasite resistance
however, stalled its further development. We have completed a full structureꢀactivity relationship
study on 4(1H)ꢀquinolones, focusing on the reduction of crossꢀresistance with atovaquone for
activity against the clinical isolates W2 and TM90ꢀC2B, as well as the improvement of
microsomal stability. These studies revealed several frontrunner compounds with superb in vivo
antimalarial activity. The best compounds were found to be curative with all mice surviving a
Plasmodium berghei infection after 30 days.
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Introduction
Malaria is considered endemic in 97 countries and it is estimated that 3.2 billion people
1–3
are at risk for contracting malaria. Although the number of deaths caused by malaria has
decreased by 48% since the turn of the century, resistance to current treatments is a mounting
3
problem. Therefore, there is an immediate need to develop new antimalarial agents, which
ideally should be active against all developmental stages of the parasite within the host and
within the mosquito vector. In humans, malaria is caused by P. falciparum, P. knowlesi, P.
malariae, P. ovale, and P. vivax, of which P. falciparum is the most common cause for
infections. Malaria begins its life cycle in a host when an infected female Anopheles mosquito
takes a blood meal from a host. Sporozoites are injected from the salivary gland of the mosquito
into the human host and first infect liver cells, which mature into schizonts. Next, schizonts
rupture and release merozoites, which rapidly infect red blood cells causing the clinical
symptoms of the disease. In stark contrast to the most prevalent P. falciparum infections, P.
vivax parasites can infect and stay dormant in liver cells and reemerge weeks, months or even
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–6
years later causing a new infection. Currently, the only effective treatment for the dormant
7,8
liver stage of the parasite is primaquine, which is also used as a causal prophylactic agent.
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1
Effective drug treatment remains the cornerstone of malaria control, nevertheless WHO
states that without new therapeutics, all the strides made in reducing the deaths from the disease
could be reversed owing to resistance of parasite strains to many of the common antimalarials
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and artemisinin combination therapies (ACTs). Due to the limited chemotypes active against
malaria, researchers have begun to optimize old antimalarial agents or drugs, evaluating these in
current preclinical efficacy models and assessing these for proper physicochemical
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,10,11
properties.
This approach has been shown to be effective for endochin, a 3ꢀsubstituted
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4(1H)ꢀquinolone,
floxacrine, a dihydroacridinedione,
chloroquine,
and other
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chemotypes (Figure 1). 4(1H)ꢀquinolone ester ICI 56,780
(6b) is an example of an old
scaffold that is primed for recycling. This analogue was discovered in 1970 by Ryley and Peters
to have blood schizonticidal activity against P. berghei (Pb) and prophylactic activity against P.
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cynomolgi infections.
Compound 6b was found to produce radical cures at 15 mg/kg,
however, resistance was observed after only one passage in Pb infected mice and the compound
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was eventually abandoned. Studies by Manetsch, Kyle, Guy, Ward and O’Neill have shown
that considerable optimization can be done to offset some of the liabilities of the 4(1H)ꢀ
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quinolone compound class.
However, these failed to address the fundamental questions
about why 6b has such broad range antiꢀmalarial activity or how structural modifications of 6b
may reduce the propensity to induce resistance. Given the promise of this scaffold, detailed
structureꢀactivity relationship studies against the blood and liver stages of the parasite were
conducted in order to gain a deeper understanding of this promising scaffold.
Figure 1: Antimalarial structures primed for recycling
Results and Discussion
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Synthetic Chemistry. Previous efforts to optimize 6b using SuzukiꢀMiyaura cross couplings
yielded compounds with reduced antimalarial activities, suboptimal atovaquone crossꢀresistance
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indices, and poor physicochemical properties. In our efforts to improve the bioavailability as
well as to better understand the structural reasons for the broad spectrum activity of compound
6b, initially a focused structureꢀactivity relationship (SAR) study was undertaken with a small
number of analogues (Table 1). The goal was to individually assess the 3ꢀester, the 6ꢀbutyl and
the 7ꢀphenoxyethoxy groups’ contribution to the overall activity of 6b.
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The majority of the compounds have been prepared by a nucleophilic substitution of Nꢀacetylꢀ
protected phenols 1 providing acetamides 2, which under basic conditions were deacetylated to
anilines 3. Treatment of anilines 3 with malonate 4a followed by microwaveꢀassisted cyclization
furnished 4(1H)ꢀquinolone esters 6 (Scheme 1).
Scheme 1: Synthesis of 4(1H)ꢀquinolones 6aꢀv
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Compound 6b was synthesized by an eightꢀstep reaction sequence previously reported.
Analogue 6a was synthesized by the alkylation of acetamide 1a followed by acetyl deprotection
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and GouldꢀJacobs cyclization. Compounds 6c and 6d were both prepared using GouldꢀJacobs
cyclization of the appropriate aniline along with malonate 4a (Scheme 1), while 8aꢀc, were
synthesized by the removal of the ester moiety of their corresponding 4(1H)ꢀquinolones 6aꢀc by
a twoꢀstep reaction sequence of first refluxing in 10% sodium hydroxide then heating the
intermediate acid in toluene at 270 °C for 5 minutes (Scheme 2). Due to the limited commercial
availability of highly substituted anilines, optimization of the 6ꢀ and 7ꢀpositions were conducted
in parallel using two individual compound series focusing either on the 6ꢀposition or 7ꢀposition
(see Tables 2 and 3). Compounds 6kꢀv and 8a were accessed through commercially available
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amines as opposed to 4(1H)ꢀquinolones 6eꢀj, which were synthesized via a reaction sequence
similar to the one of compound 6a.
Scheme 2: Synthesis of decarboxylated 4(1H)ꢀquinolones 8aꢀc
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To explore the optimal length of the alkyl group in 6ꢀposition of compound 6b, a series
of 4(1H)ꢀquinolones was prepared whose 6ꢀposition was substituted with a variety of groups
ranging from one hydrogen to a pentyl chain. Compounds 6wꢀy were synthesized from 1ꢀbromoꢀ
4ꢀnitroꢀ2ꢀ(2ꢀphenoxyethoxy)benzene (11a) via 1ꢀbromoꢀ2ꢀmethoxyꢀ4ꢀnitrobenzene (9) (Scheme
3
). Phenol 10a was synthesized from anisole 9 via a demethylation with BBr . Compound 10a
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was alkylated with (2ꢀbromoethoxy)ꢀbenzene to give 11a, the needed starting material for the
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next reaction steps. The alkynyl nitrobenzenes 12 were synthesized via Sonogashira coupling
of the available alkynyl starting materials. Nitrobenzene 11a was first subjected to a
Sonogashiraꢀcross coupling using TMSꢀacetylene with PdCl (PPh ) , CuI, and TEA, then finally
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deprotected with TBAF to yield 12a. To access the nitro alkyne 12b, a microwaveꢀassisted
deprotection and coupling reaction using trimethyl(propꢀ1ꢀynꢀ1ꢀyl)silane, Pd(OAc) , and TBAF
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was employed to give the desired product in low yields. Lastly, analogue 12c was obtained in
good yields using 1ꢀpentyne, PdCl (PPh ) , CuI, PPh , and diethylamine in a microwave
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3 2
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3
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reactor. Each alkyne 12 was reduced using hydrogenation conditions in order to reduce both
the alkyne and nitro groups to the corresponding alkylꢀsubstituted anilines 3, which were
subjected to standard GouldꢀJacobs cyclization conditions furnishing the 4(1H)ꢀquinolones 6wꢀy.
Scheme 3: Synthesis of 4(1H)ꢀquinolones 6wꢀy with varying alkyl group in 6ꢀposition
Scheme 4: Synthesis of 4(1H)ꢀquinolones 6zꢀac probing 6ꢀposition electronics
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Next, a small set of 4(1H)ꢀquinolones with electron donating or electron withdrawing
groups were synthesized. This chemistry allowed access to 4(1H)ꢀquinolones 6z, 6aa, 6ab, and
6ac substituted in 6ꢀposition with a bromo, methoxy, chloro, or methyl group. These compounds
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were synthesized starting from the alkylation of corresponding nitrophenols 10 followed by the
reduction of the nitrobenzenes 11aꢀd to give anilines 3zꢀac which were cyclized to give the
desired 4(1H)ꢀquinolones 6zꢀac (Scheme 4).
As the ester group in 3ꢀposition of compound 6b was considered to be a potential
metabolic liability, 4(1H)ꢀquinolone analogues were prepared in which the methyl ester group
was replaced by amides, halides, a nitrile, a keto, an ethyl, or other esters. The amide
functionalities were introduced by the aminolysis of 6b using trimethyl aluminum and the
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appropriate amine in dry benzene to yield compounds 13aꢀe in moderate to low yield after
purification using preparative HPLC (Scheme 5).
Scheme 5: Synthesis of 3ꢀamide substituted 4(1H)ꢀquinolones 13aꢀe
The 3ꢀcyanoꢀ4(1H)ꢀquinolone was synthesized from aniline 3b and the commercially
available ethylꢀ2ꢀcyanoꢀ3ꢀethoxyacrylate (4b). Standard GouldꢀJacobs cyclization of
intermediate Nꢀaryl enamine 5ad afforded the cyanoꢀsubstituted 4(1H)ꢀquinolone 6ad in low
yield (Scheme 6).
Scheme 6: Synthesis of cyanoꢀsubstituted 4(1H)ꢀquinolone 6ad
A threeꢀstep reaction sequence was utilized for the preparation of 3ꢀethylꢀsubstituted
4(1H)ꢀquinolone 6ae. First, ethyl butyrate was reacted with diethyl oxalate (14) to give diester
3
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15, which was used as the starting material along with the aniline 3b to furnish intermediate
5ae followed by cyclization to give 3ꢀethylꢀsubstituted 4(1H)ꢀquinolone 6ae (Scheme 7). 3ꢀethyl
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ester substituted 4(1H)ꢀquinolone 6af was cyclized from enamine 5af following a reaction of
aniline 3b with malonate 4c. (Scheme 8). Ketone 6ag was synthesized through a standard
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ConradꢀLimpach cyclization (Scheme 9). 3ꢀIodoꢀ4(1H)ꢀquinolone 16b was an intermediate for
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the preparation of 3ꢀarylꢀsubstituted 4(1H)ꢀquinolones previously reported. The remaining
halides 16a and 16cꢀ16e were synthesized from 4(1H)ꢀquinolone 8b or 2ꢀmethylꢀ4(1H)ꢀ
quinolone 8d using NBS, NCS or NIS in DMF (Scheme 9).
Scheme 7: Synthesis of 3ꢀethyl substituted 4(1H)ꢀquinolone 6ae
Scheme 8: Synthesis of 3ꢀethyl ester substituted 4(1H)ꢀquinolone 6af
Scheme 9: Synthesis of keto 4(1H)ꢀquinolone 6ag
Scheme 10: Synthesis of 3ꢀhalide substituted 4(1H)ꢀquinolones 16aꢀe
Antimalarial Activity and Cytotoxicity. All synthesized compounds were routinely tested
against the clinically relevant multidrug resistant malarial strains W2 (pyrimethamine and
chloroquine resistant strains) and TM90ꢀC2B (mefloquine, chloroquine, atovaquone,
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2–14
pyrimethamine resistant strains) as previously reported.
Due to the emergence and rapid
3
4
acquisition of crossꢀresistance, each compound was also evaluated on its resistance index (RI),
which is the ratio of the effective concentrations needed to kill 50% of the population of parasites
(
EC ) for TM90ꢀC2B and W2 strains (RI=EC TM90ꢀC2B/EC W2). Ideally, the RI of a
50 50 50
compound should lie between 0.3 and 3 in order to avoid rapidly inducing resistance in the
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parasite.
This range is based upon the natural resistance patterns observed for drugs like
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chloroquine and mefloquine.
The target for 4(1H)ꢀquinolones is cytochrome b of the
mitochondria; TM90ꢀC2B has a Y268S mutation in cytochrome b that confers high grade
resistance to atovaquone. Therefore, the RI values with TM90ꢀC2B reflect potential crossꢀ
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resistance to atovaquone. Selected compounds were also tested for in vitro liver stage activity
using P. berghei sporozoites expressing luciferase, harvested from mosquito salivary glands and
allowed to infect HEPG2 hepatoma cells in order to assess if the compounds possessed causal
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prophylactic activity. Similar to the RI, liver blood indices (LBI) were assessed in order to
relate activity against P. berghei with activity against W2 or TM90ꢀC2B (LBI=EC W2/EC Pb
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or LBI= EC50 TM90ꢀC2B/EC50 Pb). This allowed a simplified method for comparing the
different assays being simultaneously run. Additionally, each compound was also tested for
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cytotoxicity using mammalian J774 cell lines in a 96 well plate format.
Structural Activity Relationships. The original compound 6b was found to have an excellent
EC for W2 at 39.0 pM, however, it suffered from a two orders of magnitude drop in activity for
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the atovaquone resistant strain TM90ꢀC2B with an EC value of 7.89 nM. Compound 6b also
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showed excellent in vitro liver stage activity in P. berghei infected cells with an EC of 52.0
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pM. Initially, each substituent in 3ꢀ, 6ꢀ, and 7ꢀposition was removed sequentially to give
compounds 6a, 6c, and 8b. The removal of the 6ꢀbutyl or 7ꢀ(2ꢀphenoxyethoxy) group provided
slightly less potent compounds 6a and 6c with EC values against W2 of 2.80 and 1.58 nM,
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respectively, whereas both maintained subnanomolar liver stage activity. In contrast, removal of
the ester functionality in 3ꢀposition severely affected the potency, dropping the EC values of
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W2, TM90ꢀC2B and P. berghei to 328 nM, 430 nM, and 5.82 nM, respectively. These data
suggest the importance of the ester in liver stage activity. Despite the severe potency reduction
for compound 8b, it was noted that the resistance index equaled 1.31, which stands in stark
contrast to the large RI values for compounds 6a and 6c which were 26.3 and 141, respectively.
Subsequently, compounds 6d, 8a, and 8c were tested, in which two of the 3ꢀ, 6ꢀ and 7ꢀ
substituents were simultaneously removed. Both 6d and 8c retained respectable potency against
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W2, however, both were devoid of any activity against TM90ꢀC2B. Similar to compound 8b,
when the ester group was removed in compounds 8a, inhibition against W2 was lost, however,
the RI value converged towards 1. Importantly, it was also observed that compounds 8a, 8b, and
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c, lacking the ester group in 3ꢀposition, were the least potent analogues against P. berghei of the
entire subseries (Table 1).
Table 1: Sequential removal of original hit compound substituents
Due to the scarcity and costs of commercially available, diꢀ and triꢀsubstituted anilines along
with the awareness that 3ꢀesterꢀsubstituted 4(1H)ꢀquinolones 6a and 6c retained moderate blood
stage activity, a set of compounds were synthesized, in which the 7ꢀ or 6ꢀposition were probed
(Tables 2 and 3). Considering possible metabolic instabilities of the 2ꢀphenoxyethoxy substituent
in 4(1H)ꢀquinolone 6b, analogue 6j was designed in which one of the oxygens in the 7ꢀ(2ꢀ
phenoxyethoxy) moiety was replaced by a methylene unit. Compound 6j, which in comparison
to analogue 6a was 4 times more active against W2 with an EC50 of 0.711 nM, displayed a
greater than tenꢀfold drop in activity against TM90ꢀC2B. This was mirrored in the P. berghei
activities where there was an 18ꢀfold drop in the activity of 6j as compared to the reference 6a,
from EC s of 0.286 nM in 6a to 5.26 nM in 6j. The length of the alkyl chain linker was also
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shortened in hopes of improvement similar to 4(1H)ꢀquinolone 6j, however, analogues 6i, 6h,
and 6k were all less active than the reference 6a. Finally, in order to test electronic and steric
effects, 4(1H)ꢀquinolones substituted in the 7ꢀposition with phenyl, chloro and fluoro
functionalities were tested. 7ꢀChloro and 7ꢀphenyl substituents were not tolerated, as compounds
6
l and 6m were devoid of antimalarial activity, whereas 7ꢀfluoroꢀ4(1H)ꢀquinolone 6n was active
against W2 and TM90ꢀC2B with EC s of 26.2 nM and 395 nM, respectively.
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A similar study was conducted investigating possible moieties in the quinolone’s 6ꢀposition. The
reference compound 6c, which maintains the 6ꢀbutyl substituent, proved to be the most active
compound of this set, with EC s of 1.58 nM for W2, 222 nM for TM90ꢀC2B, and 0.819 nM for
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P. berghei. Among the other compounds tested, substituents derived from the 2ꢀphenoxyethoxy
group were shown to be the most potent analogues. Compound 6e, containing the benzyloxy
substituent in the 6ꢀposition, was the most active analogue against W2, TM90ꢀC2B and P.
berghei with EC values equaling 11.6 nM, 1100 nM, and 127 nM, respectively. Compounds 6f
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and 6g, analogues which increase the alkyl chain linker length in comparison to 6e, were the only
other analogues of this subseries displaying EC50 with doubleꢀdigit nM activity against W2.
Overall, with the exception of 6ꢀphenylꢀsubstituted 4(1H)ꢀquinolone 6u, which was devoid of
activity, probing of the six position gave mediocre compounds leading to the belief that it was
overall less tolerant of structural variation than the 7ꢀposition.
Table 2: Structureꢀactivity relationship of the 7ꢀposition
Table 3: Structureꢀactivity relationship of the 6ꢀposition
In a subseries of close analogues, the optimal length of the 6ꢀalkyl residue was
investigated, leading to the design of compounds 6ac, 6w, 6x and 6y. 7ꢀMethylꢀsubstituted
4
(1H)ꢀquinolone 6ac had modest activity with an EC of 2.01 nM for W2, an EC of 499 nM
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for TM90ꢀC2B, and Pb EC of 152 nM. antimalarial potency increased as the number of
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methylene units were added until the alkyl chain length reached four carbons and decreased as
the chain length grew past four carbons, suggesting that the original 6ꢀbutyl substituent of 6b
was optimal. In general, all compounds in this subseries were potent with most displaying
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subnanomolar EC values for W2, and with the 6ꢀbutylꢀsubstituted 4(1H)ꢀquinolone being the
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most potent.
Table 4: Optimal aliphatic chain length in 6ꢀposition
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In the same vein, analogues in Table 5 were designed to probe the same steric and
electronic effects of the 6ꢀposition. 6ꢀChloro and 6ꢀbromo compounds, 6ab and 6z, possessed
moderate activity with 6ab having an EC of 9.93 nM and 617 nM for W2 and TM90ꢀC2B
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while 6z had EC s of 4.47 nM and 847 nM for W2 and TM90ꢀC2B. Both 6ꢀchloro and 6ꢀbromo
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compounds, 6ab and 6z, also displayed subnanomolar P. berghei EC s of 0.063 nM and 0.336
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nM, respectively. Interestingly, 6ꢀmethoxyꢀ7ꢀphenoxyethoxyꢀsubstituted 4(1H)ꢀquinolone 6aa
had an RI of 1.60 with good activities for both blood and liver stages. It was also observed by the
direct comparison pairs between compound 6aa and its 7ꢀphenoxyethoxyꢀomitted analogue 6o,
and between 6ꢀchloro 4(1H)ꢀquinolone 6ab and its 7ꢀphenoxyethoxyꢀomitted analogue 6r that
the addition of the 7ꢀ(2ꢀphenoxyethoxy) group increases the activity for W2 by >60ꢀfold and
>130ꢀfold for TM90ꢀC2B.
Table 5: Structural activity relationship of 6ꢀposition using electron withdrawing and
donating groups
Following the 6ꢀ and 7ꢀpositions, the 3ꢀposition was probed. A possible metabolite of
ester 6b is acid 7b. Therefore, it was synthesized in order to see whether it could be the active
form of the compound. With activities of 64.0 nM for W2, 1110 nM for TM90ꢀC2B and 0.267
nM for Pb, it was not as active as its parent, however, all activity was not lost. Ethyl ester 6af
showed potent activity, with a W2 EC value of 0.143 nM and was active against P. berghei
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with a subnanomolar EC50. Converting the ester to an amide dropped activity slightly.
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Methylamide 13a was the most active at 13.3 nM for W2 and 3,980 nM for TM90ꢀC2B. As the
alkyl chain increased in size, activity decreased with isopropylamide 13e being completely
inactive with EC values in the µM range. Also, when each amide is compared to its ester
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counterpart, the ester is in all cases significantly more active. The 2ꢀcyanoꢀsubstituted compound
6ad had W2 activity in the high doubleꢀdigit nM range. When the ester was converted to ketone
6ag, the activity dropped by almost 250ꢀfold for W2 moving to 9.67 nM, the change for TM90ꢀ
C2B was not as pronounced, however, with only a 48ꢀfold difference. Lastly, complete removal
of the ketone leaving just an alkyl chain, as with compound 6ae, was potent, having an EC of
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0.440 nM for W2, 1.92 nM for TM90ꢀC2B, and 2.65 for P. berghei. With an RI of 4.36, it
showed once again that removal of the ester left compounds with improved RIs (Table 6).
Unfortunately, compound 6ae displayed cytotoxicity with an EC value of 360 nM against J774.
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Table 6: Structural activity relationship of the 3ꢀposition using ester isosteres
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ꢀIodoꢀsubstituted 4(1H)ꢀquinolone 16b was synthesized with the intention of replacing
the 2ꢀmethylester substituent with a polarizable group. Interestingly, its testing gave an active
compound having EC s of 1.23 nM for W2 and 6.04 nM for TM90ꢀC2B with an RI of 4.91
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Table 7). Other 3ꢀhalo analogues were made, with and without a 2ꢀmethyl group. 3ꢀIodoꢀ2ꢀ
methylꢀ4(1H)ꢀquinolone 16a was approximately 2ꢀfold more potent than its reference 16b. The
ꢀbromo 4(1H)ꢀquinolone 16d and its 2ꢀmethyl analogue 16c were slightly less potent than their
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3ꢀiodo counterparts with W2 activities of 4.64 nM and 2.60 nM, respectively, and TM90ꢀC2B
activities of 48.7 nM and 12.2 nM, respectively. 3ꢀChloroꢀ2ꢀmethyl analogue 16e had EC s of
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6.92 nM for W2 and 67.7 nM for TM90ꢀC2B with an RI of 9.79. Overall, the 3ꢀhaloꢀsubstituted
subseries followed the trend that the 2ꢀmethyl substituted analogues were approximately twoꢀ
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fold more active than the 2ꢀhydrogen analogues and with RIs of 4.69 to 10.5, all five 3ꢀhalo
substituted PEQs 16a– 16e consistently had the best RI values identified thus far.
Table 7: 3ꢀHalo substituted 4(1H)ꢀquinolones with and without 2ꢀmethyl groups
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Correlation Between Blood Stage Activity Liver Stage Activity. In general, 7ꢀphenoxyethoxyꢀ
substituted 4(1H)ꢀquinolones display potent in vitro liver stage activity with lowꢀnanomolar to
subꢀnanomolar EC values. The most potent 4(1H)ꢀquinolone esters 6a, 6b, 6c, 6y, and 6af, as
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well as the medium potent compounds follow an activity order similar to the ranking observed
for W2 and TM90ꢀC2B, whereby the EC values for TM90ꢀC2B are approximately 20ꢀ200ꢀfold
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higher than the EC values for W2 or in vitro liver stage activity. In order to quantify how
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efficiently compounds target the liver stages in comparison to the blood stages, a liver blood
index (LBI) has been calculated, which is the ratio of the effective concentrations needed to kill
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0% of the population of parasites (EC ) for TM90ꢀC2B and W2 strains (LBI =EC TM90ꢀ
50 C2B 50
C2B/EC Pb or LBI =EC TM90ꢀC2B/EC Pb).
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Aqueous Solubility and Lipophilicity. In parallel to testing the compounds for in vitro
antimalarial activity, a limited structureꢀproperty relationship (SPR) study focusing on log D and
aqueous solubility was conducted to assess potential physiochemical liabilities (see Supporting
Information, Table S1). Solubility at pH 6.5 was determined using an inꢀhouse HPLC assay
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based on UV absorption. The log D , the distribution coefficient between octanol and water at
7
.4
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pH 7.4, was experimentally determined via a previously described HPLCꢀbased method.
Overall, solubility continued to be a problem persistent throughout the study, with 13d being the
only compound to reach a solubility of greater than 20 μM at any given pH. Along the same
lines, the majority of the compounds displayed a log D7.4 similar to the starting point 6b. For
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these reasons, physicochemical properties were not strongly considered as selection criteria for
in vivo efficacy studies.
In vivo Efficacy Evaluation of Selected Compounds. The 12 4(1H)ꢀquinolones with potent in
vitro activity against blood stages of P. falciparum were chosen to be screened for in vivo
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efficacy according to a previously reported P. berghei infected mouse model (Table 8). Criteria
such as in vitro blood stage activity, in vitro liver stage activity, compound availability, and to a
limited extent also physicochemical property data, were considered for the selection of the
screening candidates 6b, 6e, 6f, 6g, 6j, 6k, 6i, 6ab, 6ae, 13d, 16a, and 16c. The RI values were
considered a secondary discrimination factor as most of the presented compounds are extremely
potent. The screening was performed by treating two infected mice with a single 50 mg/kg oral
dose of test compound suspended in PEG400 on day 1 postꢀexposure (PE) followed by assessing
parasitemia on days 3 and 6 PE. Compounds with a parasitemia reduction of greater than 50% on
days 3 and 6 PE were considered to be active. The original compound 6b was one of the most
active candidates in the group with 76.9% inhibition on day 3 PE and 49% inhibition on day 6
PE (Table 8). Quinolone ester 6j and 3ꢀbromoꢀ4(1H)ꢀquinolone 16c were the only other two
compounds that displayed a parasitemia reduction similar to the original compound 6b with
47.1% and 49.5% inhibition on day 6 PE. Analogues 6g and 6ae with >65% inhibition on day 3
PE were considered promising, nevertheless, the lack of significant parasitemia reduction on day
6 PE suggested that these compounds may suffer from insufficient bioavailability. A similar
outcome has been observed for the previously reported 3ꢀarylꢀsubstituted 6ꢀchloroꢀ7ꢀmethoxyꢀ
4(1H)ꢀquinolones compound series, for which a long halfꢀlife has been detrimental to the
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development of curative antimalarials.
Table 8: Results of the in vivo efficacy scout screening
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The two frontrunner compounds, 6b and 16c, displaying the best in vivo efficacy were subjected
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to a more rigorous in vivo efficacy testing in a modified Thompson test.
Using mice
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infected with 1 x 10 Plasmodium berghei-GFP parasites, compounds were dosed orally on days
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, 4, and 5 at a 10 mg/kg concentration of compound suspended or dissolved in HEC/Tween. On
days 3, 6, 9, 13, 21, and 30 PE, parasitemia was monitored by Giemsaꢀstained blood smears.
Both compounds were evaluated by the reduction of parasitemia on day 6 PE and the survival up
to day 30 PE. Compound 6b showed no inhibition on day 6 PE and mice were sacrificed on day
13, the same day as the controls. In comparison to the original compound 6b, 3ꢀbromoꢀ4(1H)ꢀ
quinolone 16c is approximately tenfold more soluble, which possibly improved the in vivo
efficacy to 61% inhibition on day 6 PE. Nevertheless, parasitemia rapidly rebounded so that mice
dosed with compound 16c were sacrificed on day 13 PE.
Table 9: Results of Thompson Test
Conclusions
Due to the promising results of past recycling of old antimalarials and the promise of 3ꢀ
4
(1H)ꢀquinolone esters, we synthesized a library of 46 4(1H)ꢀquinolones with a variety of
substituents focusing primarily on the 3ꢀ, 6ꢀ, and 7ꢀpositions. All compounds were tested in vitro
against the two clinically relevant P. falciparum strains W2 and TM90ꢀC2B. Furthermore,
selected compounds with promising activity against W2 and TM90ꢀC2B were evaluated for in
vitro liver stage activity against P. berghei. Overall, this 4(1H)ꢀquinolone series displays potent
erythrocytic and exoerythrocytic activity with many compounds displaying low nanomolar EC50
values. The vast majority of the 4(1H)ꢀquinolones were also shown to be nontoxic in J774
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cytotoxicity assays with EC s >20 µM. However, most compounds were not in vivo efficacious
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most likely due to poor aqueous solubility and low oral bioavailability.
There was, however, invaluable data gained from the analogues synthesized as it was
shown that the 3ꢀester group is very important in addressing atovaquone crossꢀresistance since 3ꢀ
halide compounds 16a – 16e had excellent RI values. We also determined that the six and seven
positions are important for blood stage activity due to the combination of the 6ꢀ and 7ꢀsubstituted
compounds being much more active than the 6ꢀ or 7ꢀsubstituted compounds separately.
Compound 6b and 16c were both tested in vivo using a modified Thompson test. There was a
marked increase in the parasite inhibition for compound 16c over 6b on day 6 PE showing
significant improvements over the original compound 6b. Although bromoꢀsubstituted 4(1H)ꢀ
quinolone 16c showed less in vitro blood and in vitro liver stage activity than the predecessor 6b,
the in vivo activity of 16c on day 6 PE clearly proved its superiority overall. The blood and liver
stage activities of these quinolone esters along with the discovery of the 3ꢀhaloꢀ4(1H)-quinolones
lead us to postulate that there is still much potential in this scaffold and further optimization of
solubility and stability could lead to orally bioavailable, curative agents with activity across the
full spectrum of malaria life cycle stages.
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Experimental Section
General. All reagents and solvents were obtained from Aldrich Chemical Co. and used without
further purification. NMR spectra were recorded at ambient temperature on a 400 MHz or 500
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MHz Varian NMR spectrometer in the solvent indicated. All H NMR experiments are reported
in δ units, parts per million (ppm) downfield of TMS, and were measured relative to the signals
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of chloroform (7.26 ppm) and dimethylsulfoxide (39.5 ppm) with H decoupled observation.
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Data for H NMR are reported as follows: chemicals shift (δ ppm), multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, p = pentet, sext = sextet, sept = septet, oct = octet, m = multiplet),
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integration and coupling constant (Hz) whereas C NMR analyses were reported in terms of
chemical shift. NMR data was analyzed by using MestReNova Software version 6.0.2ꢀ5475. The
purity of the final compounds was determined to be ≥95% by highꢀperformance liquid
chromatography (HPLC) using an Agilent 1100 LC/MSDꢀVL with electrospray ionization. Lowꢀ
resolution mass spectra were performed on an Agilent 1100 LC/MSDꢀVL with electrospray
ionization. Highꢀresolution mass spectra (HRMS) were performed on an Agilent LC/MSD TOF
system G3250AA. Analytical thin layer chromatography (TLC) was performed on silica gel 60
F254 precoated plates (0.25 mm) from EMD Chemical Inc., and components were visualized by
ultraviolet light (254 nm). EMD silica gel 230ꢀ400 (particle size 40ꢀ63 µm) mesh was used for
all flash column chromatography. Microwave heating was performed in a singleꢀmode Anton
Paar Monowave 300 and all microwaveꢀirradiated reactions were conducted in heavyꢀwalled
glass vials sealed with Teflon septa.
All in vivo studies were conducted in compliance with the Guide for the Care and Use of
Laboratory Animals of the National Research Council. The protocol was approved by the
University of South Florida Institutional Animal Care and Use Committee.
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General Procedure A: Aniline (5 mmol) and dimethyl methoxy methylene malonate (7.6
mmol) were added to a flask and heated to 110 °C for 30 minutes (monitored by TLC). After
cooling, diethyl ether was added at which point precipitation occurred. This solid was collected
by filtration and used in the next step without further purification.
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General Procedure B: Enamine intermediate (5.8 mmol) was added to toluene (11.6 mL) and
heated in a microwave reactor for 5 minutes at 270 °C. The precipitated product was filtered and
washed with cold methanol. The solid residue was in most cases recrystallized in 4:1
MeOH:DMF to afford the final product.
General Procedure C: An ester (4 mmol) was refluxed for 2 hours in 10% NaOH (10 mL)
solution. It was then cooled and acidified to pH 5 where the precipitate was filtered and washed
with water. It was dried and used in the next step without further purification.
General Procedure D: A carboxylic acid (5 mmol) and toluene (10 mL) were refluxed in a
microwave for 5 minutes at 280 °C. Diethyl ether was added to the reaction and the resulting
precipitate was collected and washed with cold methanol.
General Procedure E: To Nꢀ(3ꢀhydroxyphenyl)acetamide (15 mmol) was added (2ꢀ
bromoethoxy)benzene (19.5 mmol) along with Cs CO (45 mmol) and DMF (30 mL). The
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reaction was heated at 40 °C overnight. The reaction was extracted with DCM, dried over
Na SO , filtered, and concentrated under reduced pressure. The crude product was recrystallized
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in hexanes/ethyl acetate.
General Procedure F: An acetamide (4 mmol) along with KOH (40 mmol) and 9:1 DI
water:ethanol (50 mL) was refluxed overnight. After the reaction was complete, the ethanol was
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removed under reduced pressure. The residue was then dissolved in DCM washed with DI water,
dried over Na SO , filtered and concentrated under reduced pressure. The resulting oil could then
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be used without further purification in most cases. If not, aqueous HCl was added and the
solution was washed with EA. The aqueous layer was basified then washed with EA and the
organic layer was dried over Na SO , filtered and concentrated under reduced pressure.
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General Procedure G: An amine or amine hydrochloride (0.76 mmol) and trimethyl aluminum
0.8 mL) were added dropwise at 0 °C. The reaction was then stirred at room temperature for 30
(
minutes. At that time, 6b (0.38 mmol) was added as a solid and the reaction was allowed to
reflux overnight. The reaction was purified using preparative HPLC to yield the title compound.
General Procedure H: Quinolone (1 mmol) and Nꢀhalo succinimide (2.2 mmol) were refluxed
in DMF (5 mL) for 2 hours. The reaction was concentrated and water was added. The resulting
precipitate was filtered and recrystallized in acetone.
General Procedure I: An alkyne substituted nitrobenzene was added with palladium on carbon
and ethyl acetate to a hydrogenation flask. The flask was shaken under 60 psi hydrogen gas for
24 hours. The reaction mixture was filtered over celite and concentrated under reduced pressure.
The title compound was isolated via flash chromatography.
Nꢀ(3ꢀ(2ꢀPhenoxyethoxy)phenyl)acetamide 2a was synthesized following general procedure E
1
in 91% yield. H NMR (400 MHz, DMSO) δ 9.94 (s, 1H), 7.37 (s, 1H), 7.35 – 7.26 (m, 2H), 7.26
–
2
7.10 (m, 2H), 7.02 – 6.91 (m, 3H), 6.66 (dd, J = 8.1, 1.5 Hz, 1H), 4.27 (td, J = 5.2, 3.5 Hz, 4H),
13
.05 (s, 3H). C NMR (101 MHz, DMSO) δ 168.44, 158.60, 158.33, 140.56, 129.56, 120.78,
+
114.50, 111.63, 108.85, 105.61, 66.27, 66.17, 24.11. HRMS (ESIꢀTOF) m/z: [M + H] calcd for
C H NO 272.1281; found 272.1284.
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Nꢀ(4ꢀ(Benzyloxy)phenyl)acetamide 2c was synthesized following general procedure E in 98%
1
yield. H NMR (400 MHz, DMSO) δ 9.80 (s, 1H), 7.53 – 7.47 (m, 2H), 7.45 – 7.41 (m, 2H),
1
3
7
.41 – 7.35 (m, 2H), 7.34 – 7.29 (m, 1H), 6.97 – 6.92 (m, 2H), 5.05 (s, 2H), 2.02 (s, 3H). C
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NMR (101 MHz, DMSO) δ 167.73, 154.07, 137.19, 132.76, 128.36, 127.73, 127.63, 120.50,
+
114.76, 69.35, 23.80. HRMS (ESIꢀTOF) m/z: [M + H] calcd for C H NO 242.1176; found
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242.1200.
Nꢀ(4ꢀ(3ꢀPhenylpropoxy)phenyl)acetamide 2e was synthesized following general procedure E
1
in 94% yield. H NMR (400 MHz, DMSO) δ 9.79 (s, 1H), 7.51 – 7.45 (m, 2H), 7.31 – 7.14 (m,
13
5
H), 6.88 – 6.82 (m, 2H), 3.90 (t, J = 6.4 Hz, 2H), 2.78 – 2.68 (m, 2H), 2.05 – 1.94 (m, 5H). C
NMR (101 MHz, DMSO) δ 167.62, 154.33, 141.39, 132.50, 128.31, 128.30, 125.79, 120.44,
+
1
14.39, 66.75, 31.48, 30.44, 23.76. HRMS (ESIꢀTOF) m/z: [M + H] calcd for C H NO
1
7
19
2
270.1489; found 270.1471.
Nꢀ(3ꢀ(Benzyloxy)phenyl)acetamide 2f was synthesized following general procedure E in 98%
1
yield. H NMR (400 MHz, DMSO) δ 9.91 (s, 1H), 7.46 – 7.42 (m, 2H), 7.42 – 7.36 (m, 3H),
7.35 – 7.29 (m, 1H), 7.18 (t, J = 8.1 Hz, 1H), 7.11 (dt, J = 8.1, 1.3 Hz, 1H), 6.71 – 6.66 (m, 1H),
1
3
5.06 (s, 2H), 2.03 (s, 3H). C NMR (101 MHz, DMSO) δ 168.76, 159.00, 140.95, 137.50,
129.88, 128.85, 128.22, 128.07, 111.97, 109.55, 106.23, 69.53, 24.52. HRMS (ESIꢀTOF) m/z:
+
[
M + H] calcd for C H NO 242.1176; found 242.1162.
1
5
15
2
Nꢀ(3ꢀ(3ꢀPhenylpropoxy)phenyl)acetamide 2h was synthesized following general procedure E
1
in 99% yield. H NMR (400 MHz, CDCl ) δ 9.24 (s, 1H), 7.37 (d, J = 1.9 Hz, 1H), 7.26 – 7.21
3
(m, 2H), 7.16 – 7.11 (m, 5H), 6.59 (dt, J = 7.4, 2.1 Hz, 1H), 3.86 (t, J = 6.3 Hz, 2H), 2.76 – 2.68
1
3
(
m, 2H), 2.12 (s, 3H), 2.00 (tt, J = 12.9, 6.3 Hz, 2H). C NMR (101 MHz, CDCl ) δ 169.18,
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59.14, 141.22, 139.59, 129.15, 128.17, 128.08, 125.58, 111.96, 109.85, 106.19, 66.54, 31.79,
+
30.52, 24.01. HRMS (ESIꢀTOF) m/z: [M + H] calcd for C H NO 270.1489; found 270.1464.
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4ꢀ(Benzyloxy)aniline 3e was synthesized following general procedure F in 93% yield. H NMR
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(
400 MHz, CDCl ) δ 7.46 – 7.36 (m, 4H), 7.35 – 7.30 (m, 1H), 6.86 – 6.81 (m, 2H), 6.67 – 6.62
3
1
3
(m, 2H), 5.00 (s, 2H), 3.31 (s, 2H). C NMR (101 MHz, CDCl ) δ 152.04, 140.31, 137.58,
3
1
28.57, 127.86, 127.57, 116.46, 116.14, 70.85.
1
4
ꢀ(3ꢀPhenylpropoxy)aniline 3g was synthesized following general procedure F in 98% yield. H
NMR (400 MHz, CDCl ): δ 7.29 –7.16 (m, 5H,), 6.75 –6.60 (m, 4H), 3.88 (t, J = 6.4 Hz 2H),
3
13
3
.40 (s, 2H), 2.79 (t, J = 7.4 Hz 2H), 2.06 (dt, J = 7.4/6.4 Hz 2H). C NMR (100 MHz, CDCl ):
3
δ 152.2, 141.6, 139.9, 128.5, 128.3, 125.8, 116.4, 115.7, 67.6, 32.1, 30.9. HRMS (ESIꢀTOF) m/z:
+
[
M + H] calcd for C H NO Calcd.: 227.1310; Found 227.1310.
1
5
17
1
3
ꢀ(Benzyloxy)aniline 3h was synthesized following general procedure E in 91% yield. H NMR
(500 MHz, CDCl ) δ 7.47 (d, J = 7.1 Hz, 2H), 7.45 – 7.39 (m, 2H), 7.36 (dd, J = 8.3, 6.1 Hz,
3
1
H), 7.10 (t, J = 8.0 Hz, 1H), 6.48 – 6.42 (m, 1H), 6.38 – 6.31 (m, 2H), 5.05 (s, 2H), 3.67 (s,
1
3
2
H). C NMR (126 MHz, CDCl ) δ 160.05, 147.89, 137.29, 130.19, 128.61, 127.93, 127.52,
3
108.24, 104.88, 102.05, 69.86. HRMS (ESI) calcd for C H NO [M+H]+ : 200.1070,
1
3
13
Found:200.1094.
1
3
ꢀ(3ꢀphenylpropoxy)aniline 3j was synthesized following general procedure E in 89% yield. H
NMR (500 MHz, CDCl ) δ 7.30 (t, J = 7.5 Hz, 2H), 7.22 (dd, J = 12.6, 7.1 Hz, 3H), 7.06 (t, J =
3
8
.0 Hz, 1H), 6.33 (dd, J = 8.2, 2.2 Hz, 1H), 6.29 (dd, J = 7.9, 1.9 Hz, 1H), 6.25 (t, J = 2.1 Hz,
1H), 3.93 (t, J = 6.3 Hz, 2H), 3.65 (s, 2H), 2.89 – 2.75 (m, 2H), 2.10 (dd, J = 14.8, 6.6 Hz, 2H).
1
3
C NMR (126 MHz, CDCl ) δ 160.28, 147.84, 141.69, 130.16, 128.63, 128.49, 125.99, 107.93,
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Found:228.1356.
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ꢀEthylꢀ3ꢀ(2ꢀphenoxyethoxy)aniline 3w was synthesized from 12a following general procedure
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I in 77% yield. H NMR (400 MHz, CDCl ) δ 7.30 (t, J = 7.1 Hz, 2H), 7.01 – 6.89 (m, 4H), 6.31
3
– 6.23 (m, 2H), 4.36 – 4.30 (m, 2H), 4.30 – 4.24 (m, 2H), 2.53 (q, J = 7.4 Hz, 2H), 1.13 (t, J =
7
.5 Hz, 3H).
3
ꢀ(2ꢀPhenoxyethoxy)ꢀ4ꢀpropylaniline 3x was synthesized from 12b following general
procedure I and the crude product was used without further purification.
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ꢀPentylꢀ3ꢀ(2ꢀphenoxyethoxy)aniline 3y was synthesized from 12c following general procedure
I and the crude product was used without further purification.
4
ꢀBromoꢀ3ꢀ(2ꢀphenoxyethoxy)aniline 3z. To
a
solution of 1ꢀbromoꢀ4ꢀnitroꢀ2ꢀ(2ꢀ
phenoxyethoxy)benzene (572 mg, 1.6916 mmol) in ethanol (5 mL) was added zinc powder (1.1
g, 16.916 mmol) and acetic acid (0.97 mL, 16.916 mmol) at 0 °C. The solution was allowed to
warm to room temperature and stirred until the disappearance of the starting material was
observed as indicated by TLC (4 hours). The reaction solution was filtered through celite then
concentrated under reduced pressure. The residue was dissolved in DCM (10 mL), washed with
sodium bicarbonate, DI water and brine, dried over Na SO , filtered and concentrated under
2
4
reduced pressure. The crude product was purified by flash chromatography to give the 3z (486
1
mg, 93% yield). H NMR (400 MHz, DMSO) δ 7.33 – 7.26 (m, 2H), 7.11 (d, J = 8.5 Hz, 1H),
7
.02 – 6.98 (m, 2H), 6.97 ꢀ 6.92 (m, 1H), 6.37 (d, J = 2.4 Hz, 1H), 6.13(dd, J = 8.5, 2.4 Hz, 1H),
13
5
.28 (s, 2H), 4.35 – 4.28 (m, 2H), 4.27 – 4.21 (m, 2H). C NMR (101 MHz, DMSO) δ 158.33,
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54.94, 149.78, 132.68, 129.51, 120.76, 114.62, 108.01, 99.83, 95.55, 67.11, 66.20. HRMS
(ESI) calcd for C H BrNO [M+H]+ : 308.0281, Found: 308.0268.
14 14 2
4ꢀMethoxyꢀ3ꢀ(2ꢀphenoxyethoxy)aniline 3aa. To a solution of 1ꢀmethoxyꢀ4ꢀnitroꢀ2ꢀ(2ꢀ
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phenoxyethoxy)benzene (3.2 mmol) in ethanol (10 mL) was added zinc powder (32 mmol) and
acetic acid (32 mmol) at 0 °C. The solution was allowed to warm to room temperature and stirred
until the disappearance of the starting material was observed as indicated by TLC (4 hours). The
reaction solution was filtered through celite then concentrated under reduced pressure. The
resulting residue was dissolved in DCM, washed with sodium bicarbonate, DI water and brine,
dried over Na SO , filtered and concentrated under reduced pressure. The residue was purified
2
4
1
by flash chromatography to give 3aa in 62% yield. H NMR (399 MHz, CDCl ) δ 7.33 – 7.26
3
(m, 2H), 6.95 (d, J = 9.0 Hz, 3H), 6.73 (d, J = 8.5 Hz, 1H), 6.40 (d, J = 2.6 Hz, 1H), 6.28 (dd, J =
13
8
1
5
.4, 2.6 Hz, 1H), 4.34 (s, 4H), 3.79 (s, 3H), 3.44 (bs, 2H). C NMR (100 MHz, CDCl ) δ
3
58.72, 142.98, 140.68, 133.86, 129.58, 121.10, 114.77, 114.34, 107.77, 103.41, 67.69, 66.38,
7.08.
4
8
6
ꢀChloroꢀ3ꢀ(2ꢀphenoxyethoxy)aniline 3ab was synthesized following general procedure F in
1
7% yield. H NMR (500 MHz, CDCl ) δ 7.36 – 7.28 (m, 2H), 7.11 (d, J = 8.4 Hz, 1H), 7.03 –
3
.95 (m, 3H), 6.33 (d, J = 2.5 Hz, 1H), 6.25 (dd, J = 8.4, 2.5 Hz, 1H), 4.39 – 4.27 (m, 4H), 3.68
13
(
s, 2H). C NMR (126 MHz, CDCl ) δ 158.58, 154.77, 146.39, 130.59, 129.53, 121.16, 114.81,
3
112.30, 108.61, 101.72, 67.74, 66.33.
4ꢀMethylꢀ3ꢀ(2ꢀphenoxyethoxy)aniline 3ac To a flame dried flask was added 1ꢀmethylꢀ4ꢀnitroꢀ
2
ꢀ(2ꢀphenoxyethoxy)benzene (10 mmol) and SnCl (40 mmol) and 300 mL of ethanol. The
2
reaction was refluxed for 3 hours. The EtOH was removed and 4M KOH was added until basic
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and the reaction was extracted with EA. The reaction was then dried and concentrated to give the
1
title compound in 75% yield. H NMR (500 MHz, CDCl ) δ 7.33 (t, J = 8.0 Hz, 2H), 7.01 (t, J =
3
7.7 Hz, 3H), 6.94 (d, J = 7.6 Hz, 1H), 6.30 – 6.23 (m, 2H), 4.37 – 4.32 (m, 2H), 4.30 – 4.26 (m,
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13
2
H), 3.54 (s, 2H), 2.15 (s, 3H). C NMR (126 MHz, CDCl ) δ 158.82, 157.54, 145.56, 131.23,
3
129.58, 121.10, 117.13, 114.83, 107.55, 100.01, 66.81, 66.64, 15.48. HRMS (ESI) calcd for
C H NO [M+H]+ : 244.1332, Found:244.1320.
15
17
2
Dimethyl 2ꢀ(((3ꢀ(2ꢀphenoxyethoxy)phenyl)amino)methylene)malonate 5a was synthesized
1
following general procedure A in 86% yield. H NMR (400 MHz, DMSO) δ 10.69 (d, J = 13.9
Hz, 1H), 8.44 (d, J = 13.9 Hz, 1H), 7.30 (tt, J = 8.1, 2.5 Hz, 3H), 7.06 (t, J = 2.2 Hz, 1H), 7.02 –
6
3
1
5
.91 (m, 4H), 6.78 (dd, J = 8.2, 2.2 Hz, 1H), 4.37 – 4.33 (m, 2H), 4.32 – 4.28 (m, 2H), 3.73 (s,
1
3
H), 3.66 (s, 3H). C NMR (101 MHz, DMSO) δ 167.57, 165.25, 159.40, 158.26, 151.40,
40.63, 130.57, 129.52, 120.74, 114.45, 111.07, 109.85, 104.15, 92.72, 66.53, 66.08, 51.12,
+
1.09. HRMS (ESIꢀTOF) m/z: [M + H] calcd for C H NO 372.1442; found 372.1429.
20 21
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Dimethyl 2ꢀ((phenylamino)methylene)malonate 5d was synthesized following general
1
procedure A in quantitative yield. H NMR (399 MHz, DMSO) δ 10.75 (d, J = 14.0 Hz, 1H),
8.43 (d, J = 14.0 Hz, 1H), 7.41 – 7.30 (m, 4H), 7.15 (t, J = 6.8 Hz, 1H), 3.72 (s, 3H), 3.66 (s,
13
3
H). C NMR (100 MHz, DMSO) δ 167.74, 165.30, 151.60, 139.37, 129.63, 124.76, 117.66,
2.55, 51.10, 51.07.
9
Dimethyl 2ꢀ(((4ꢀ(benzyloxy)phenyl)amino)methylene)malonate 5e was synthesized following
1
general procedure A in 89% yield. H NMR (400 MHz, CDCl ) δ 11.00 (d, J = 13.9 Hz, 1H),
3
8.43 (d, J = 13.9 Hz, 1H), 7.45 – 7.29 (m, 5H), 7.09 – 7.03 (m, 2H), 6.99 – 6.93 (m, 2H), 5.03 (s,
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H), 3.84 (s, 3H), 3.76 (s, 3H). C NMR (101 MHz, CDCl ) δ 169.51, 166.05, 156.44, 152.87,
3
1
36.64, 132.84, 128.64, 128.11, 127.45, 118.89, 116.07, 91.94, 70.38, 51.52, 51.37.
Dimethyl 2ꢀ(((4ꢀphenethoxyphenyl)amino)methylene)malonate 5f was synthesized following
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general procedure A in 96% yield. H NMR (500 MHz, CDCl ) H NMR (500 MHz, CDCl ) δ
3
3
11.02 (d, J = 13.8 Hz, 1H), 8.46 (d, J = 13.9 Hz, 1H), 7.37 – 7.23 (m, 5H), 7.08 (d, J = 8.9 Hz,
H), 6.91 (d, J = 8.9 Hz, 2H), 4.17 (t, J = 7.1 Hz, 2H), 3.86 (s, 3H), 3.78 (s, 3H), 3.11 (t, J = 7.1
2
1
3
Hz, 2H). C NMR (126 MHz, CDCl ) δ 169.67, 166.23, 156.65, 153.06, 138.12, 132.76, 129.10,
3
128.65, 126.71, 119.02, 115.83, 91.94, 69.21, 51.66, 51.51, 35.88.
Dimethyl 2ꢀ(((3ꢀ(benzyloxy)phenyl)amino)methylene)malonate 5h was synthesized following
1
general procedure A in 97% yield. H NMR (400 MHz, CDCl ) δ 10.97 (d, J = 13.7 Hz, 1H),
3
8
.47 (d, J = 13.7 Hz, 1H), 7.42 – 7.28 (m, 5H), 7.25 – 7.20 (m, 1H), 6.72 (d, J = 1.4 Hz, 1H),
13
6
.75 – 6.68 (m, 2H), 5.02 (s, 2H), 3.82 (s, 3H), 3.75 (s, 3H). C NMR (101 MHz, CDCl ) δ
3
169.22, 165.76, 159.97, 151.98, 140.28, 136.40, 130.67, 128.60, 128.09, 127.42, 111.08, 109.84,
+
1
04.18, 92.95, 70.09, 51.53, 51.38. HRMS (ESI) [M+H] calcd for C H NO 342.1336; found
1
9
19
5
342.1319.
Dimethyl 2ꢀ(((3ꢀphenethoxyphenyl)amino)methylene)malonate 5i was synthesized following
1
general procedure A in 97% yield. H NMR (400 MHz, CDCl ) δ 10.78 (d, J = 13.7 Hz, 1H),
3
8
.29 (d, J = 13.8 Hz, 1H), 7.13 – 7.01 (m, 6H), 6.53 – 6.42 (m, 3H), 3.96 (t, J = 7.0 Hz, 2H), 3.63
13
(s, 3H), 3.56 (s, 3H), 2.88 (t, J = 7.0 Hz, 2H). C NMR (101 MHz, CDCl ) δ 169.39, 165.99,
3
1
60.17, 152.19, 140.35, 138.02, 130.75, 129.04, 128.61, 126.68, 110.96, 109.72, 104.05, 92.98,
8.93, 51.67, 51.54, 35.77.
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Dimethyl 2ꢀ(((3ꢀ(3ꢀphenylpropoxy)phenyl)amino)methylene)malonate 5j was synthesized
1
following general procedure A in 96% yield. H NMR (500 MHz, CDCl ) δ 11.01 (d, J = 13.7
3
Hz, 1H), 8.54 (d, J = 13.8 Hz, 1H), 7.37 – 7.16 (m, 6H), 6.78 – 6.65 (m, 3H), 3.98 (t, J = 6.2 Hz,
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2H), 3.87 (s, J = 0.5 Hz, 3H), 3.80 (s, J = 0.6 Hz, 3H), 2.83 (t, J = 7.6 Hz, 2H), 2.17 – 2.09 (m,
1
3
2H). C NMR (126 MHz, CDCl ) δ 169.47, 166.04, 160.41, 152.29, 141.38, 140.38, 130.79,
3
128.60, 128.57, 126.12, 111.07, 109.63, 104.01, 92.97, 67.19, 51.73, 51.59, 32.19, 30.82.
Dimethyl 2ꢀ(((3ꢀchlorophenyl)amino)methylene)malonate 5l was synthesized following
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general procedure A in 75% yield. H NMR (500 MHz, CDCl ) δ 11.01 (d, J = 13.2 Hz, 1H),
3
8.48 (d, J = 13.6 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.20 – 7.09 (m, 2H), 7.02 (dd, J = 8.1, 1.5 Hz,
13
1
H), 3.86 (s, 3H), 3.79 (s, 3H). C NMR (126 MHz, CDCl ) δ 169.35, 165.81, 151.81, 140.46,
3
135.84, 131.03, 125.12, 117.39, 115.61, 94.10, 51.89, 51.74.
Dimethyl 2ꢀ(([1,1'ꢀbiphenyl]ꢀ3ꢀylamino)methylene)malonate 5m was synthesized from [1,1'ꢀ
1
biphenyl]ꢀ3ꢀamine following general procedure A in quantitative yield. H NMR (399 MHz,
CDCl ) δ 11.14 (d, J = 14.2 Hz, 1H), 8.61 (d, J = 13.7 Hz, 1H), 7.58 (d, J = 7.3 Hz, 2H), 7.46 (q,
3
J = 14.7, 7.3 Hz, 3H), 7.39 (t, J = 6.4 Hz, 2H), 7.34 (bs, 1H), 7.14 (d, J = 7.6 Hz, 1H), 3.88 (s,
1
3
3
H), 3.79 (s, 3H). C NMR (100 MHz, CDCl ) δ 169.56, 166.12, 152.44, 143.38, 140.24,
3
139.70, 130.39, 129.06, 128.09, 127.30, 124.12, 116.30, 116.07, 93.17, 51.81, 51.67.
Dimethyl 2ꢀ(((3ꢀfluorophenyl)amino)methylene)malonate 5n was synthesized following
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general procedure A in 39% yield. H NMR (400 MHz, CDCl ) δ 11.01 (d, J = 13.3 Hz, 1H),
3
8.46 (d, J = 13.6 Hz, 1H), 7.32 (dd, J = 14.3, 8.0 Hz, 1H), 6.93 – 6.80 (m, 3H), 3.84 (s, 3H), 3.77
1
3
(
s, 3H). C NMR (101 MHz, CDCl ) δ 169.31, 165.76, 163.71 (d, J = 247.4 Hz), 151.81, 140.88
3
(d, J = 9.8 Hz), 131.33 (d, J = 9.5 Hz), 113.06, 111.85 (d, J = 21.2 Hz), 104.58 (d, J = 25.5 Hz),
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3.99, 51.83 (s, J = 15.0 Hz), 51.68. C NMR (101 MHz, CDCl ) δ 169.31, 165.76, 163.71 (d, J
3
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247.4 Hz), 151.81, 140.88 (d, J = 9.8 Hz), 131.33 (d, J = 9.5 Hz), 113.06, 111.85 (d, J = 21.2
Hz), 104.58 (d, J = 25.5 Hz), 93.99, 51.83 (s, J = 15.0 Hz), 51.68.
Dimethyl 2ꢀ(((4ꢀmethoxyphenyl)amino)methylene)malonate 5o was synthesized following
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general procedure A in 85% yield. H NMR (399 MHz, CDCl ) δ 10.99 (d, J = 13.7 Hz, 1H),
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8
3
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.42 (d, J = 13.9 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H), 6.87 (d, J = 8.9 Hz, 2H), 3.82 (s, 3H), 3.77 (s,
1
3
H), 3.74 (s, 3H). C NMR (100 MHz, CDCl ) δ 169.56, 166.10, 157.31, 152.95, 132.61,
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18.94, 115.02, 91.80, 55.59, 51.57, 51.41.
Dimethyl 2ꢀ(((4ꢀethoxyphenyl)amino)methylene)malonate 5p was synthesized following
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general procedure A in 97% yield. H NMR (400 MHz, DMSO) δ 10.72 (d, J = 14.1 Hz, 1H),
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.33 (d, J = 14.1 Hz, 1H), 7.32 – 7.24 (m, 2H), 6.97 – 6.88 (m, 2H), 4.00 – 3.95 (m, 2H), 3.71 (s,
1
3
3H), 3.64 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H). C NMR (101 MHz, DMSO) δ 167.78, 165.37,
56.02, 152.22, 132.67, 119.38, 115.22, 91.32, 63.29, 50.93, 50.91, 14.58.
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Dimethyl 2ꢀ(((4ꢀisopropylphenyl)amino)methylene)malonate 5q was synthesized following
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general procedure A in 96% yield. H NMR (400 MHz, DMSO) δ 10.74 (d, J = 14.1 Hz, 1H),
8
1
1
.41 (d, J = 14.1 Hz, 1H), 7.32 – 7.21 (m, 4H), 3.72 (s, 3H), 3.65 (s, 3H), 2.87 (hept, J = 6.9 Hz,
1
3
H), 1.19 (s, 3H), 1.17 (s, 3H). C NMR (101 MHz, DMSO) δ 167.70, 165.31, 151.78, 145.12,
37.26, 127.40, 117.80, 91.96, 54.90, 51.03, 32.83, 23.82.
Dimethyl 2ꢀ(((4ꢀchlorophenyl)amino)methylene)malonate 5r was synthesized following
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general procedure A in 56% yield H NMR (400 MHz, DMSO) δ 10.68 (d, J = 11.3 Hz, 1H),
1
3
8
.38 (d, J = 11.9 Hz, 1H), 7.42 (s, 4H), 3.73 (s, 3H), 3.66 (s, 3H). C NMR (101 MHz, DMSO)
δ 167.75, 165.63, 151.66, 138.93, 129.80, 128.99, 119.94, 93.63, 51.61, 51.55.
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Dimethyl 2ꢀ(((4ꢀ(4ꢀfluorophenoxy)phenyl)amino)methylene)malonate 5s was synthesized
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following general procedure A in 86% yield H NMR (400 MHz, CDCl ) δ 11.02 (d, J = 13.7
3
Hz, 1H), 8.44 (d, J = 13.8 Hz, 1H), 7.11 – 7.06 (m, 2H), 7.05 – 6.98 (m, 2H), 6.98 – 6.91 (m,
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H), 3.83 (s, 3H), 3.75 (s, 3H). C NMR (101 MHz, CDCl ) δ 169.58, 166.07, 159.05 (d, J =
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242.1 Hz), 155.18, 152.87 (d, J = 2.6 Hz), 152.77, 134.81, 120.49 (d, J = 8.3 Hz), 119.72,
19.04, 116.55 (d, J = 23.4 Hz), 92.72, 51.72, 51.58.
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Dimethyl 2ꢀ(((4ꢀethylphenyl)amino)methylene)malonate 5t was synthesized following general
1
procedure A in 50% yield. H NMR (400 MHz, DMSO) δ 10.72 (d, J = 14.1 Hz, 1H), 8.41 (d, J
=
14.1 Hz, 1H), 7.28 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 3.72 (s, 3H), 3.65 (s, 3H), 2.58
13
(
q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). C NMR (101 MHz, DMSO) δ 167.66, 165.31,
151.71, 140.49, 137.18, 128.87, 117.78, 91.97, 51.05, 51.03, 27.48, 15.57.
Dimethyl 2ꢀ(([1,1'ꢀbiphenyl]ꢀ4ꢀylamino)methylene)malonate 5u was synthesized following
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general procedure A in 79% yield. H NMR (399 MHz, CDCl ) δ 11.11 (d, J = 13.8 Hz, 1H),
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8.57 (d, J = 13.8 Hz, 1H), 7.57 (dd, J = 12.9, 8.0 Hz, 4H), 7.43 (t, J = 7.6 Hz, 2H), 7.34 (t, J =
1
3
7
.3 Hz, 1H), 7.19 (d, J = 8.5 Hz, 2H), 3.87 (s, 3H), 3.79 (s, 3H). C NMR (100 MHz, CDCl ) δ
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169.41, 165.94, 151.96, 139.89, 138.27, 138.01, 128.92, 128.44, 127.47, 126.79, 117.54, 93.03,
1.70, 51.55.
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Dimethyl 2ꢀ(((4ꢀphenoxyphenyl)amino)methylene)malonate 5v was synthesized following
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general procedure A in 93% yield. H NMR (399 MHz, DMSO) δ 10.75 (d, J = 13.9 Hz, 1H),
8.38 (d, J = 14.0 Hz, 1H), 7.47 – 7.35 (m, 4H), 7.13 (t, J = 7.4 Hz, 1H), 7.08 – 6.96 (m, 4H), 3.72
1
3
(
s, 3H), 3.65 (s, 3H). C NMR (100 MHz, DMSO) δ 167.59, 165.35, 156.88, 153.59, 135.37,
130.09, 123.43, 119.91, 119.77, 118.33, 92.16, 51.11, 51.09.
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Dimethyl 2ꢀ(((4ꢀbromoꢀ3ꢀ(2ꢀphenoxyethoxy)phenyl)amino)methylene)malonate 5z was
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synthesized following general procedure A in 91% yield. H NMR (400 MHz, DMSO) δ 10.70
(
d, J = 13.8 Hz, 1H), 8.44 (d, J = 13.8 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.35 – 7.26 (m, 3H),
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.05 – 6.91 (m, 4H), 4.49 – 4.42 (m, 2H), 4.38 – 4.33 (m, 2H), 3.74 (s, 3H), 3.67 (s, 3H). C
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3
NMR (100 MHz, DMSO) C NMR (100 MHz, DMSO) δ 167.48, 165.29, 158.31, 155.32,
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51.18, 140.25, 133.52, 129.55, 120.83, 114.62, 110.82, 106.06, 104.42, 93.28, 67.84, 66.09,
1.23.
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Dimethyl 2ꢀ(((4ꢀmethoxyꢀ3ꢀ(2ꢀphenoxyethoxy)phenyl)amino)methylene)malonate 5aa was
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synthesized from 3aa following general procedure A in 78% yield. H NMR (500 MHz, CDCl )
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δ 11.03 (d, J = 13.9 Hz, 1H), 8.45 (d, J = 13.8 Hz, 1H), 7.29 (dd, J = 8.7, 7.4 Hz, 2H), 6.99 –
6.93 (m, 3H), 6.87 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H), 6.74 (dd, J = 8.6, 2.6 Hz, 1H),
1
3
4
.42 – 4.38 (m, 2H), 4.38 – 4.35 (m, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.77 (s, 3H). C NMR (100
MHz, CDCl ) δ 169.68, 166.22, 158.60, 152.96, 149.25, 147.65, 132.96, 129.63, 121.26, 114.77,
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112.92, 110.43, 104.91, 92.12, 68.21, 66.44, 56.46, 51.72, 51.58.
Methyl 4ꢀoxoꢀ7ꢀ(2ꢀphenoxyethoxy)ꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylate 6a was synthesized
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following general procedure B in 48% yield. H NMR (400 MHz, DMSO) δ 12.15 (s, 1H), 8.51
(
s, 1H), 8.07 (s, 1H), 7.30 (s, 2H), 7.03 (d, J = 27.9 Hz, 5H), 4.39 (d, J = 20.9 Hz, 4H), 3.73 (s,
+
3
H). HRMS (ESIꢀTOF) m/z: [M + H] calcd for C H NO 340.1180; found 340.1153.
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Methyl 6ꢀbutylꢀ4ꢀoxoꢀ7ꢀ(2ꢀphenoxyethoxy)ꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylate 6b was
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synthesized following general procedure A and B as a white powder in 22% yield. H NMR (500
MHz, CDCl and 3 drops TFAꢀd) δ 9.29 (s, 1H), 8.18 (s, 1H), 7.77 (s, 1H), 7.31 (t, J = 8.0 Hz,
3
2H), 6.99 (t, J = 7.4 Hz, 1H), 6.96 (d, J = 7.9 Hz, 2H), 4.59 (dd, J = 5.3, 3.1 Hz, 2H), 4.45 (dd, J
=
5.2, 3.1 Hz, 2H), 4.12 (s, 3H), 2.83 – 2.78 (m, 2H), 1.66 (t, J = 7.6 Hz, 2H), 1.37 (dd, J = 14.9,
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