Synthesis and pharmacological profiling of analogues of benzyl quinolone carboxylic acid (BQCA) as allosteric modulators of the M1 muscarinic receptor

Article

pubs.acs.org/jmc

Synthesis and Pharmacological Proﬁling of Analogues of Benzyl

Quinolone Carboxylic Acid (BQCA) as Allosteric Modulators of the M₁

Muscarinic Receptor

Shailesh N. Mistry,^†,§Celine Valant,^‡,§Patrick M. Sexton,^‡Ben Capuano,^†Arthur Christopoulos,*^,‡

and Peter J. Scammells*^,†

^†Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia

^‡Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia

ABSTRACT: Established therapy in Alzheimer’s disease involves

potentiation of the endogenous orthosteric ligand, acetylcholine, at the

M₁muscarinic receptors found in higher concentrations in the cortex

and hippocampus. Adverse eﬀects, due to indiscriminate activation of

other muscarinic receptor subtypes, are common. M₁muscarinic

positive allosteric modulators/allosteric agonists such as BQCA oﬀer

an attractive solution, being exquisitely M₁-selective over other

muscarinic subtypes. A common diﬃculty with allosteric ligands is

interpreting SAR, based on composite potency values derived in the

presence of ﬁxed concentration of agonist. In reality these values encompass multiple pharmacological parameters, each

potentially and diﬀerentially sensitive to structural modiﬁcation of the ligand. We report novel BQCA analogues which appear to

augment ligand aﬃnity for the receptor (pK_B), intrinsic eﬃcacy (τ_B), and both binding (α) and functional (β) cooperativity with

acetylcholine. Ultimately, development of such enriched SAR surrounding allosteric modulators will provide insight into their

mode of action.

modulators, PAMs), or activate the receptor in their own right as

well as potentiating agonist activity (allosteric agonists), have

been discovered.⁵PAMs have the potential to oﬀer a more

desirable therapeutic proﬁle than classic orthosteric agonists, as

they would potentiate endogenous ligand activity (via altering

aﬃnity, eﬃcacy, or both) under more physiological conditions

rather than the unselective and continuous agonist activity

conferred by an orthosteric agonist. In addition, the potential for

subtype selectivity is higher due to less evolutionary pressure to

conserve amino acid sequences that are not within the

orthosteric site.⁵

INTRODUCTION

■

Alzheimer’s disease is a progressively debilitating neurodegener-

ative disorder, primarily aﬀecting the aging population. Although

ﬁrst described over 100 years ago, the underlying causative

factors are yet to be identiﬁed. Symptoms include confusion,

memory loss, and dementia, ultimately leading to death.

Progressive degeneration of cholinergic neurons in several

brain areas, including the cerebral cortex and hippocampus, is

known to occur. In particular, the muscarinic M₁receptor has

been found to be more prevalent in these same areas of the

brain.^1,2

The compound 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroqui-

noline-3-carboxylic acid (BQCA) has been shown to exhibit

unprecedented positive allosteric activity for ACh binding as well

as inherent agonist activity at the M₁muscarinic receptor.^6,7

Additionally, BQCA appears to display high subtype selectivity,

with no activity detected at concentrations up to 100 μM at any

of the other four muscarinic receptor subtypes, in several

assays.^6,8

Among the previously reported structure−activity relation-

ships (SARs), one study especially suggests that the activity of

BQCA analogues as M₁muscarinic PAMs is improved by

incorporation of ﬂuorine atoms at either or both the 5- and 8-

positions of the quinolone ring.⁹However, all of these SAR

studies are based on an “allosteric potency” value, determined

Although no disease modifying therapy is currently available,

the majority of pharmacotherapy provides symptomatic relief

through augmentation of cholinergic function via administration

of anticholinesterase inhibitors. The undesired eﬀects (e.g.,

nausea, vomiting, bradycardia) of this type of therapy result from

indiscriminate augmentation of the endogenous orthosteric

ligand, acetylcholine (ACh), and subsequent nonselective

overactivation of other muscarinic receptor subtypes (M₂−

M₅).³A selective orthosteric agonist of the M₁receptor is likely

to oﬀer better therapy, through fewer oﬀ-target eﬀects. However,

subtype selectivity within the muscarinic receptors is notoriously

diﬃcult to achieve, primarily due to the highly conserved amino

acid sequence within the orthosteric site of each muscarinic

receptor subtype.⁴

More recently, compounds binding to topographically distinct

sites on the receptor (allosteric sites), able to potentiate the

agonist activity of an orthosteric ligand (positive allosteric

Received: April 15, 2013

A

dx.doi.org/10.1021/jm400540b | J. Med. Chem. XXXX, XXX, XXX−XXX

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a

Scheme 1. Synthesis of BQCA Analogues with Varied 5- and 8- Substituents

a

Reagents and conditions: (a) diethylethoxymethylene malonate, 100−120 °C, 71−86%; (b) diethylethoxymethylene malonate, Et₃N, 100 °C, 79%;

(c) Eaton’s reagent, 90−100 °C, 35−84%; (d) H₂, 10% Pd/C, EtOH, 100%; (e) alkyl halide, KI, K₂CO₃, DMF, rt, 32−86%; (f) 1,4-dioxane/HCl_(aq)

,

reﬂux, 62−90%; (g) LiOH·H₂O, THF, water, rt, 49−98%; (h) (i) 1 M NaOH_(aq), 1,4-dioxane, 80 °C, (ii) LiOH·H₂O, reﬂux, (iii) microwave 160 °C,

1 h; 53%.

from titration of the modulator in the presence of a ﬁxed (e.g.,

EC₂₀) concentration of the endogenous ligand, ACh. The main

issue with such titration curves is that the estimated “allosteric

potency” reﬂects a composite of at least four operational

properties,¹⁰namely the aﬃnity of the modulator for the free

receptor (K_B), its allosteric eﬀects on the binding and signaling of

the orthosteric ligand (α and β, respectively), and its intrinsic

agonist eﬃcacy (if any) in the system (τ_B). In reality, each of

these parameters may be separately “tuned” through chemical

modiﬁcation, often in opposite directions such that minimal

changes may be observed in the composite allosteric potency

parameter. Consequently, use of an all-encompassing allosteric

potency value can lead to misinterpretation of SAR data. In fact, a

common observation in the ﬁeld, based predominantly on this

approach, is that allosteric ligand SAR is often “ﬂat”.^5,11

Our approach to allosteric ligand SAR is based on the premise

that a more thorough proﬁling of key-selected compounds would

allow us to correlate chemical modiﬁcations to each of the

individual operational parameters of the modulator (aﬃnity,

binding/functional cooperativity, and eﬃcacy). Therefore, using

an operational model of allosterism that we have previously

described,^7,10we sought to quantify the eﬀect of subtle chemical

changes on each of the four pharmacological parameters,

generating an “enriched allosteric ligands SAR” study.

quinolone ring in the 5- and 8-positions, isosteric replacement of

the carboxylic acid moiety or amide derivatives of the acid

function, and ﬁnally, replacement of the N-alkyl group. We

characterized each of these compounds in radioligand binding

experiments to provide aﬃnity and binding cooperativity

estimates, then investigated the agonist properties of 10 of the

most eﬀective modulators by quantifying their eﬃcacy in an

intracellular calcium-mobilization assay. Finally, using the same

functional assay, we assessed ﬁve of the most relevant allosteric

agonist analogues of BQCA in full interaction studies with ACh

to provide functional allosteric modulation estimates. With this

approach, we have been able to better deﬁne the key moieties

triggering the aﬃnity, cooperativity, and eﬃcacy of BQCA and its

analogues for the M₁muscarinic receptor in addition to gathering

some information about the nature of the allosteric binding

pocket implicated. By generating such enriched allosteric SAR,

we believe it is possible to rationally design allosteric ligands for

GPCRs with tailored characteristics (aﬃnity, cooperativity, or

agonism). In addition, such SAR provides valuable insight into

the nature of the target allosteric binding site.

RESULTS AND DISCUSSION

■

Chemistry. The 4-oxo-1,4-dihydroquinoline moiety is

prevalent in a variety of reported compounds, thus its synthesis

is well established. Rapid access to series of analogues substituted

at the 5- and 8-positions of the 4-oxo-1,4-dihydroquinoline ring

system was achieved using the appropriately substituted aniline

With this in mind, we have synthesized over 35 novel

compounds based on the BQCA scaﬀold and introduced four key

structural modiﬁcations, namely alternative substitution of the

B

dx.doi.org/10.1021/jm400540b | J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 2. Synthesis of Amide/N-Acyl-sulfonamide Derivatives of 5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-

a

dihydroquinoline-3-carboxylic Acid

a

Reagents and conditions: (a) (i) DCM, 0 °C, (ii) oxalyl chloride, DMF; (b) amine or amine hydrochloride + TEA, DCM, 18−83%; (c) (i) HCTU,

amine, DMF, (ii) DIPEA, DCM, 62−100%; (d) (i) methanesulfonamide, DCM, 0 °C, (ii) TEA, 0 °C to rt, 39%.

as a starting material, utilizing Gould−Jacobs chemistry.¹²Two

parallel sets of compounds were easily obtained by variation of

the alkyl halide at the N-alkylation step, using literature

precedent to determine the choice of electrophile.⁹

after condensation of the appropriate amine with the

corresponding acid chloride derivative of 5a (not isolated), this

method gave generally poor yields with several side products. In

addition, some N-debenzylation was observed, presumably

during conversion to the acid chloride, under Vilsmeier−Haack

conditions. With the aim of improving yields and using milder

conditions, the coupling reagent HCTU was employed with

DIPEA as a base. This aﬀorded amides (8b, 8e, 8f) in good to

excellent yield, with negligible side product formation.

In the case of amides 10a−b and 11, direct aminolysis of ester

3a proved to be a viable option. Stirring at room temperature in

the presence of MeNH_2(aq), ethanolamine or ammonium

hydroxide respectively gave the desired products in excellent

yields (Scheme 3).

Compounds bearing established acid isosteric groups were

easily accessible via common intermediates employed in the

synthesis of amide derivatives described above. Condensation of

the acid chloride intermediate (Scheme 2) with hydroxylamine

gave the corresponding hydroxamic acid 8i, while using

methanesulfonamide in the presence of TEA gave acylsulfona-

mide 9 following previously reported conditions.¹⁶

Carboxamide 11 was a useful precursor to both the tetrazole

13 and imide 14 (Scheme 3). Initial dehydration of 11 in the

presence of PdCl₂in MeCN¹⁷gave the corresponding nitrile 12.

This was cyclized with NaN₃to form tetrazole 13 using reported

methodology.¹⁸Acylation of 11 was readily achieved with Ac₂O/

pyridine to give imide 14.

Initial thermal condensation of a variety of 2- or 2,5-

disubstituted anilines with commercially available diethylethoxy-

methylene malonate gave substituted (phenylamino)methylene

malonates 1a−d in good yield (Scheme 1). Compounds

generally solidiﬁed from the neat reaction mixture on cooling.

Subsequent cyclization of 1a−d to quinolones 2a−d was carried

out with varied yield by heating in the presence of Eaton’s reagent

(1:10 w/w P₂O₅in methanesulfonic acid). Typically, this type of

cyclization has been achieved using diphenyl ether as the solvent,

requiring high temperatures (over 200 °C).^9,13In addition,

diphenyl ether can prove diﬃcult to remove when used as a

solvent, becoming a viscous liquid or solid on cooling. In

comparison, Eaton’s reagent¹⁴oﬀers lower temperatures, easy

removal (being water miscible), and higher yields.¹⁵Subsequent

selective N-alkylation proceeded in generally good yield, with

crude product precipitating from the reaction mixture after

addition of water to aﬀord the ester precursors 3a−d and 4a−d.

Finally, the desired free acids 5a−d and 6a−d were obtained after

either acidic or basic hydrolysis under standard conditions. It was

found that more aggressive basic hydrolysis of ester 4a resulted in

displacement of the 5-F group, presumably via a S_NAr

mechanism, to give phenolic compound 7.

Amide derivatives of 5,8-diﬂuoro BQCA were obtained

through several synthetic routes, starting with either carboxylic

acid 5a (Scheme 2) or the corresponding ethyl ester 3a (Scheme

3). Although several amides (8a, 8c, 8d, 8g−h) were obtained

Although compounds bearing diﬀerent N-alkyl groups could

be readily accessed through direct N-alkylation of intermediate

2a, as before (Scheme 4), there were several target compounds

C

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Scheme 3. Synthesis of Amide and Acid Isostere Analogues of 5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-

a

carboxylic Acid

a

Reagents and conditions: (a) 40% MeNH_2(aq), EtOH, rt, 83%; (b) ethanolamine, EtOH, rt, 88%; (c) 28−30% NH_3(aq), EtOH, rt, 88%; (d) (i)

PdCl₂, MeCN, water, MeOH, THF, rt, (ii) 50 °C, 35%; (e) NaN₃, TEA·HCl, toluene, reﬂux, 38%; (f) Ac₂O, pyridine, 100 °C, 64%.

that were not accessible through this route. Construction of the

4-oxo-dihydroquinoline core via a tandem addition−elimination

mechanism, where a substituted amine nucleophile ultimately

forms part of the bicyclic core has also been widely reported^9,19

and was employed to synthesize derivatives 26a−b, 28, 29, 31,

and 32 (Scheme 5).

To interrogate the environment surrounding the substituted

N-benzyl group, positional isomers of the literature compound

6a, bearing phenyl substituents in the 2- and 3- position of the

benzyl pendant, were synthesized (16a and 16b, respectively)

(Scheme 4). These were readily accessed in the same manner as

the corresponding 4-phenylbenzyl compound 6a. An additional

analogue, bearing the 4-(3-aminopropynyl)benzyl group was

also made, as this allowed probing of the 4′-position, further

incorporating both a linear extension to the ring and a polar,

charged headgroup.

Initial N-Boc protection of propargyl amine proceeded in

excellent yield using mild aqueous conditions to give carbamate

18. Subsequent Sonogashira coupling²⁰in the presence of CuI

and PdCl₂(PPh₃)₂aﬀorded the arylalkynyl conjugate 19. The

benzylic alcohol group was converted to the corresponding

benzylic bromide 20 in moderate yield, using standard Appel²¹

conditions. Compound 20 was then used to alkylate ethyl 5,8-

diﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (2a) as pre-

viously described, giving compound 21. Under aqueous acidic

conditions, with heating, ester hydrolysis and N-Boc cleavage of

21 occurred in one step, aﬀording amino acid 22 as the

hydrochloride salt.

Although similar compounds are present in the quinolone

class of antibiotic therapeutics, to our knowledge, there are no

reported examples of the N-aryl-4-oxo-1,4-dihydroquinoline-3-

carboxylic acid class possessing allosteric activity at the M₁

muscarinic receptor. These compounds were of interest as they

diﬀer from the core BQCA-type structure, only by the absence of

the methylene group linking the two aromatic moieties. To assess

whether such a structure would be viable as an M₁allosteric

modulator, compounds 26a and 26b were synthesized (Scheme

5).

The ﬁnal group of compounds of interest incorporated a

piperidin-4-ylmethyl group in replacement of the pendant

substituted benzyl group (Scheme 5, compounds 28, 29, 30,

31). Although similar structures have been incorporated into M₁

muscarinic allosteric compounds of the quinolizidinone

class,^5,22−24to our knowledge, no such moieties have been

reported attached to the 4-oxo-1,4-dihydroquinoline-3-carbox-

ylic acid core.

Pharmacology. According to a previous study utilizing

modulator titration curves, the biological activity of BQCA

analogues as M₁muscarinic PAMs of ACh activity in a calcium

mobilization assay was improved by incorporation of ﬂuorine

atoms at the 5- and/or 8-positions of the quinolone ring.⁹To

elucidate the eﬀect engendered by the presence of these

D

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a

Scheme 4. Synthesis of 5,8-Diﬂuoro-1-(substituted-benzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid Analogues

a

Reagents and conditions: (a) alkyl halide, KI, K₂CO₃, DMF, rt, 67−75%; (b) 2 M HCl_(aq), 1,4-dioxane, 80 °C to reﬂux, 91−96%; (c) Boc₂O, water,

rt, 96%; (d) 4-iodobenzyl alcohol, TEA, CuI, PdCl₂(PPh₃)₂, THF, 42%; (e) PPh₃, CBr₄, DCM, 0 °C to rt, 55%; (f) 1 M HCl_(aq), 1,4-dioxane, 80 °C

to reﬂux, 100%.

electronegative atoms on the BQCA scaﬀold, we decided to

investigate the eﬀects of the ﬂuorine incorporation on each of the

four operational parameters describing allosteric modulation by

comparing 5a to BQCA (Figure 1, Table 1). To assess the

binding activity of 5a, we initially performed radioligand binding

experiments, using M₁-expressing CHO cells in the presence of a

K_Dconcentration of the radiolabeled antagonist [³H]-NMS (0.1

nM), increasing concentrations of ACh with or without

increasing concentrations of 5a. Such binding interaction studies

allowed us to estimate the aﬃnity (pK_B) of the modulator for the

allosteric site of the M₁muscarinic receptor as well as its binding

cooperativity (log α) with the endogenous ligand, ACh.

Compared to BQCA, the aﬃnity estimate of 5a (pK_B= 4.96

0.13) was very similar to that of BQCA (pK_B= 4.72 0.07), and

the binding cooperativity exerted by 5a on ACh binding (log α =

2.68 0.16; α = 420) was also not signiﬁcantly diﬀerent from

that exerted by BQCA (log α = 2.60 0.11; α = 400). Next, we

investigated the agonist properties of 5a compared to BQCA in a

calcium mobilization assay. As expected, ACh was a full agonist in

the system, with a potency estimate in the nanomolar range

(pEC₅₀= 8.75 0.12). Interestingly, both allosteric ligands were

full (allosteric) agonists in the M₁-expressing CHO cells, albeit

with lower potency compared to ACh; pEC₅₀= 6.89 0.11 and

7.50 0.20 for BQCA and 5a, respectively. We estimated the

eﬃcacy of both modulators in the system. Interestingly, 5a

appeared to have signiﬁcantly greater eﬃcacy (log τ_B= 2.18

0.06; τ_B= 150) compared to BQCA (log τ_B= 1.45 0.09; τ_B=

28). Finally, we interacted either BQCA or 5a with ACh in a

calcium mobilization assay to estimate the composite coopera-

tivity parameter on binding and function (log αβ) in this assay,

using an operational model of allosteric agonism. Notably, both

BQCA and 5a exhibited comparable binding/function cooper-

ativity estimates, with log αβ = 2.72 0.13 (αβ = 525) and log αβ

= 2.31 0.28 (αβ = 205) for BQCA and 5a, respectively. As

mentioned previously, the “allosteric potency” estimated by

generation of titration curves reﬂects a composite of aﬃnity,

binding/functional cooperativity and eﬃcacy. Therefore, taken

together, these ﬁndings suggest that the observed improved

biological activity of 5a compared to BQCA is predominantly

driven by an improvement in the agonist properties of the

modulator (τ_B) rather than an improvement of its aﬃnity or

allosteric properties (pK_Bor α and β, respectively).

The next phase of this study aimed at investigating the eﬀects

of four key structural modiﬁcations of the BQCA scaﬀold;

alternative substitution of the quinolone ring in the 5- and 8-

positions, isosteric replacement of the carboxylic acid moiety or

amide derivatives of the acid function, and ﬁnally the

replacement of the N-alkyl group. Initially, radioligand binding

experiments were carried out for all ligands, as described above

for BQCA and 5a. This allowed us to determine both aﬃnity and

binding cooperativity estimates for each of the modulators

synthesized in this study. Further functional characterization in a

Ca²⁺mobilization assay was carried out on selected analogues

exhibiting binding cooperativity with ACh (α) of more than 50-

fold. Finally, a small selection of allosteric agonist analogues of

BQCA (where τ_B> 100) were investigated further in order to

estimate the binding/functional cooperativity (log αβ) of each

analogue with ACh, thus allowing estimates of functional

cooperativity (log β) to be extracted.

E

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Scheme 5. Synthesis of 4-Oxo-1,4-dihydroquinoline-3-carboxylic Acid Bearing N-aryl or N-(Substituted piperidine-4-methyl)

a

Groups

a

Reagents and conditions: (a) N,N-dimethylformamide, dimethyl acetal, DMF, microwave 100 °C, 30 min, 90%; (b) substituted aniline, dry DMF,

140 °C, 61−71%; (c) LiOH·H₂O, THF, water, rt, 57−74%; (d) N-Boc-(4-aminomethyl)piperidine, dry DMF, 100 °C, 79%; (e) LiOH·H₂O, THF,

water, rt, 22−87%; (f) 4 M HCl/1,4-dioxane, DCM, 100%; (g) BnBr, K₂CO₃, MeCN, rt; (h) phenyl isocyanate, DIPEA, DCM, rt.

First, we investigated the importance of the 5- and 8-ﬂuoro

groups on the BQCA scaﬀold, with compounds, 5b−d, 6b−d,

and 7 as well as the previously reported 5a and 6a. The ester

precursors 3a−d and 4a−d were also incorporated in the study

to determine the role of the carboxylic acid functionality.

Interestingly, when comparing 5a and the corresponding ester

analogue 3a, the replacement of the carboxylic acid function by

an ethyl ester group did not aﬀect the aﬃnity of the analogue for

the allosteric site, however, it had a dramatic eﬀect on the binding

cooperativity with ACh, such that 3a was virtually incapable of

receptor. In contrast, the presence of an 8-OMe group was

signiﬁcantly detrimental for the ligand’s binding cooperativity

with ACh (compare 5b−c with 5a/5d and BQCA), whereas a 5-

OMe group (5d) had very little eﬀect on the binding

cooperativity estimate (compare 5d to BQCA).

Interestingly, replacement of the methoxy group in the R³

position with a phenyl group (6a−d) caused a signiﬁcant

increase in the aﬃnity of each analogue for the allosteric site.

Similarly to the N-(4-methoxy)benzyl series (5a−d), the

presence of an 8-OMe group (6b−c) was detrimental for the

ligand’s binding cooperativity with ACh. As before, a 5-OMe

group (6d) was better tolerated, having less of an eﬀect on the

binding cooperativity estimate compared to that of 6b and 6c.

Finally, replacement of the 5-F group of 6a with a phenol

moiety in compound 7 resulted in around a 0.75 log unit drop in

the binding cooperativity estimate although still being

comparable to other compounds exhibiting high binding

cooperativity estimates (BQCA, 5a, 5d).

modulating ACh binding (log α = 0.18

0.09; α = 1.5).

Additionally, replacement of the ﬂuorine or hydrogen atoms with

methoxy groups in the ester-modiﬁed analogues of BQCA (3b−

d) did not show any improvement in binding cooperativity with

ACh. Similar results were obtained with the N-(4-phenyl)benzyl

analogues (4a−d), with all four analogues exhibiting similar

aﬃnity estimates to BQCA or 5a but considerably lower binding

cooperativity with ACh. In comparison, the corresponding 5- and

8- substituted carboxylic acid derivatives (5b−d, 6b−d)

exhibited a diﬀerent proﬁle of activity. With analogues retaining

the N-(4-methoxy)benzyl group (5b−d), no signiﬁcant eﬀect

was observed on the analogues’ aﬃnity for the M₁muscarinic

These ﬁndings suggest that while the carboxylic acid

functionality is not essential for ligand aﬃnity at the allosteric

site (as masking with an ethyl ester group had no signiﬁcant eﬀect

on pK_B), it appears to be an important determinant of binding

F

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Figure 1. Pharmacological characterization of BQCA and 5a in radioligand binding and calcium mobilization assays. (A,B) Radioligand binding

experiments using M₁-expressing CHO cells in presence of a K_Dconcentration of the radiolabeled antagonist [³H]-NMS (0.1 nM), increasing

concentrations of ACh with or without increasing concentrations of either BQCA (A) or 5a (B). (C−E) Calcium mobilization experiments. (C)

Increasing concentration of ACh, BQCA and 5a alone. (D,E) Increasing concentrations of ACh with or without increasing concentrations of either

BQCA (D) or 5a (E). Values represent the mean SEM from at least three to four experiments performed in duplicate.

Table 1. Binding and Functional Parameters for Analogues of BQCA with Varying 5- and 8- Substituents, Bearing an N-(4-

methoxybenzyl) or N-(4-phenylbenzyl) Pendant

R¹

R²

R³

R⁴

pK_B

log α (α)

log τ_B(τ_B)

log αβ (αβ)

BQCA

3a

H

OMe

Ph

H

Et

H

4.72 0.07

5.12 0.15

4.82 0.28

4.51 0.04

4.22 0.06

3.75 0.10

5.32 0.11

4.08 0.07

5.16 0.15

4.96 0.13

4.86 0.03

4.75 0.05

4.62 0.12

5.36 0.10

5.84 0.04

5.71 0.05

5.67 0.10

5.55 0.17

2.60 0.11 (400)

0.18 0.09 (1.5)

−0.10 0.27 (1)

−0.99 1.04 (0.1)

1.19 0.15 (16)

0.63 0.24 (4)

0.59 0.12 (4)

0.69 0.20 (5)

0.70 0.14 (5)

2.62 0.10(420)

1.31 0.09 (20)

1.68 0.10 (48)

2.20 0.14 (160)

3.16 0.13 (1450)

0.87 0.12 (7)

0.98 0.11 (10)

1.70 0.10 (50)

2.41 0.26 (257)

1.45 0.09 (28)

2.72 0.13 (525)

F

3b

3c

F

OMe

H

3d

4a

OMe

F

4b

4c

F

OMe

H

Ph

H

Ph

4d

5a

OMe

F

Ph

F

OMe

Ph

2.18 0.06 (150)

2.31 0.28 (205)

2.87 0.24 (740)

5b

5c

F

OMe

H

5d

6a

OMe

F

1.75 0.17 (55)

2.37 0.20 (240)

F

6b

6c

F

OMe

H

Ph

H

Ph

6d

7

OMe

OH

Ph

1.92 0.10 (85)

F

Ph

cooperativity with ACh. Additionally, substitution to a methoxy

group in the 8-position is detrimental for the binding

cooperativity of the ligand, while the same substituent at the 5-

position causes relatively less reduction in binding cooperativity.

The 5-position is able to tolerate hydrogen bond donating

(phenol), hydrogen bond accepting (ﬂuoro and methoxy) as well

as non-hydrogen bonding groups (hydrogen atom). It may be of

importance to note that the phenolic group in 7 is ideally situated

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Table 2. Binding and Functional Parameters for Analogues of 5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-

carboxylic Acid Bearing Amide or Acid Isostere Groups

adjacent to the carbonyl moiety in the 4-position, allowing a

possible intramolecular hydrogen bond interaction to take place.

This may impact on the ability of the phenolic group to form

other interactions (so may only be functioning as a hydrogen

bond acceptor).

According to the binding data, apart from BQCA and 5a, the

best modulators were 5d, 6a, 6d, and 7, with binding

cooperativity (α) equal or higher than 50. We therefore

investigated the agonist properties of three of these analogues

(5d, 6a, and 6d).

All three modulators were full agonists, as observed for 5a, in

the calcium mobilization assay. Using an operational model of

agonism, we derived estimates of the eﬃcacy of each modulator

(5d, 6a, and 6d) in the system. Interestingly, all three analogues

exhibited signiﬁcantly higher eﬃcacy estimates than BQCA.

Incidentally, when comparing the three analogues to 5a, both 6a

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Table 3. Binding and Functional Parameters for 5,8-Diﬂuoro-1-(substituted-benzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic

Acid Analogues

and 6d exhibited similar eﬃcacy estimates, while 5d was

signiﬁcantly lower than 5a. These ﬁndings suggest that

modiﬁcations in the 5- and 8-positions of the BQCA scaﬀold

consistently increase the agonistic properties of the modulator

while having very little eﬀect on their ability to modulate ACh

binding. Finally, only one analogue (6a) exhibited an eﬃcacy

estimate (log τ_B= 2.37 0.20; τ_B= 240) higher than 100. We

therefore investigated the combined binding/functional cooper-

ativity of 6a with ACh in a calcium mobilization assay. Notably,

the combined binding/function cooperativity estimate of 6a was

not signiﬁcantly higher than that of BQCA or 5a, with log αβ =

2.87 0.24 (αβ = 740). Overall, these ﬁndings suggest that

modiﬁcation at the 5- and 8-positions of the BQCA scaﬀold

increase the eﬃcacy of the modulators in the system, whereas

replacement of the N-(4-methoxy)benzyl pendant with N-(4-

phenyl)benzyl) increases the aﬃnity of the modulator for the

allosteric site without aﬀecting the binding cooperativity.

We then turned our attention to whether the carboxylic acid

moiety of the 4-oxo-1,4-dihydroquinoline-3-carboxylic acid core

could be replaced by a group other than an ethyl ester. To start, a

series of novel amide derivatives, 8a−h, 10a−b, and 11 were

synthesized, incorporating primary amides to bulky tertiary

amides. The unsubstituted carboxamide 11 was found to retain

aﬃnity but lost an order of magnitude in binding cooperativity

with ACh (log α = 1.51 0.12; α = 32) relative to the parent acid

5a. As mentioned previously, the corresponding ethyl ester 3a

showed a complete loss of binding cooperativity with ACh,

suggesting that the presence of hydrogen bond donor

functionality in the R⁴position may be of importance for

binding cooperativity with the endogenous ligand rather than the

size of the group. Supporting this hypothesis, compounds 8d−f

and 10b, which also possessed this hydrogen bond donor

functionality, retained appreciable binding cooperativity with

ACh. Of particular interest is 8f (log α = 2.22 0.14; α = 170),

which showed only a small drop in binding cooperativity relative

to parent compound 5a, demonstrating that an acidic

functionality is not essential for the transmission of cooperativity

with the endogenous ligand. The secondary alcohol group on this

compound may be able to make further interactions within the

allosteric site. Interestingly, 10b, which possessed a hydroxyethyl

group and should theoretically be able to make a similar type of

interaction to 8f, exhibited signiﬁcantly lower binding cooper-

ativity with ACh (log α = 0.96 0.12; α = 9) compared to that of

8f. This suggests that the cyclohexyl ring in 8f is able to either

make further desirable contacts with the receptor or restrain the

hydroxyl moiety in a manner that is energetically favorable,

holding the compound in a more desirable pose. Notably, the

piperidyl analogue 8h also retained appreciable binding

cooperativity with ACh although lacking the amide proton.

Conversely, methylamide 10a and ethylamide 8a lacked

appreciable positive binding cooperativity with ACh, suggesting

that the presence of a simple hydrogen bond moiety alone is not

suﬃcient to explain the eﬀect on binding cooperativity.

Further investigation of the importance of an acidic moiety at

the 3-position of the 4-oxo-1,4-dihydroquinoline ring was

deemed possible through a series of carboxylic acid isosteres

(Table 2). Many of these isosteres oﬀer advantages in terms of

ADMET properties while also varying in strength of acidity.

Hydroxamic acid 8i (pK_a∼ 8−9),²⁵acylsulfonamide 9 (pK_a∼

4.5),²⁶tetrazole 13 (pK_a∼ 5.4−7.2),²⁶and imide 14 (pK_a∼ 8−

9)²⁷were synthesized and evaluated as they vary in both size and

acidity relative to the parent carboxylic acid 5a (pK_a∼ 6−7).²⁸In

addition, the intermediate nitrile 12 was screened although not

possessing any inherent acidic functionality. All of the acid

isostere analogues retained appreciable binding cooperativity

with ACh (α > 10). Consistent with what we observed in the

previous analogues, no signiﬁcant eﬀect on the analogues aﬃnity

estimates was noted. Interestingly, the nitrile intermediate 12

suﬀered almost complete abolition of positive binding

cooperativity with ACh. When both acid isosteres and amide

analogues are compared, it is evident that replacement of

carboxylic acid is tolerated at the level of the aﬃnity parameter

though causes a reduction in binding cooperativity with ACh.

The pK_aof the heteroatom-bonded hydrogen does not appear to

inﬂuence either the ability of binding or the binding cooperativity

of the modulators for the allosteric site. The possibility to make

such replacements oﬀers potential improvements from a

pharmacokinetic perspective, particularly in terms of intrinsic

permeability and CNS penetration (generally poor for BQCA-

type analogues),^8,9,29−31especially with the analogue 8f that

appears to retain satisfactory binding aﬃnity and binding

cooperativity.

According to the binding data, three analogues of 5a exhibit

binding cooperativity with ACh, higher than 50. These three

analogues, 8d, 8f, and 13, were therefore selected to investigate

their ability to induce agonist activity at the M₁muscarinic

receptor. We performed concentration−response curves with all

three analogues and estimated the eﬃcacy parameter of each

modulator. Interestingly, compared to 5a, the substitution of the

carboxylic acid with either a benzylamide 8d, or a tetrazole 13,

had a deleterious eﬀect on the eﬃcacy of the modulator for the

receptor with log τ_B= 1.25 0.20 (τ_B= 18) and log τ_B= 0.99

0.07 (τ_B= 10) for 8d and 13, respectively. Comparatively, the

substitutions with a 2-hydroxylcyclohexylamide, as in 8f, had no

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signiﬁcant eﬀect on the eﬃcacy of the modulators, with eﬃcacy

estimates similar to the one observed for 5a. With 8f being the

only analogue that exhibited good eﬃcacy, we interacted this

modulator with ACh in a calcium-mobilization assay and

observed that interestingly, 8f exhibited a combined binding/

function cooperativity estimate slightly higher than 5a, with log

αβ = 2.92 0.06 (αβ = 840). Thus far, 8f appears to be the only

compound in this series that appears to exhibit appreciable

cooperativity through binding and function with ACh in the

calcium mobilization assay.

Table 4. Binding and Functional Parameters for 4-Oxo-1,4-

dihydroquinoline-3-carboxylic Acid Derivatives Bearing N-

Aryl or N-(Substituted Piperidine-4-methyl) Groups

Probing of the N-alkyl pendant seemed prudent, given most of

the related compounds reported focused on a 4-substituted-

benzyl group very early in the structural optimization

program.^{9,22−24,30−33}Although compound 6a, which exhibits

over 1000-fold potentiation of ACh binding, has been previously

reported, to our knowledge, the related N-(2-phenyl)benzyl and

N-(3-phenyl)benzyl analogues are unreported. We synthesized

the corresponding analogues 16a−b and an additional

compound, 22, which probes the 4-position of the N-benzylic

pendant in a more linear fashion via an aminopropynyl group

(Table 3). Both 16a and 16b had similar aﬃnity relative to 6a. In

terms of binding cooperativity, both compounds retain

satisfactory cooperativity with ACh, albeit considerably less

than that observed with 6a, such that over a log unit of

cooperativity was lost with 16a and 16b. The amino compound

22 retained both aﬃnity and exhibited only small reduction in

binding cooperativity relative to 5a (to which it is structurally

related). Overall, although the 4-position of the N-benzylic

pendant is still the favored substitution, both the 2- and 3-phenyl

substituted analogues oﬀer good aﬃnity and appreciable binding

cooperativity with ACh. The apparent ability to exchange a

variety of groups at the 4-position of the benzylic pendant, while

retaining good binding cooperativity with ACh, indicates the

presence of a potentially open pocket within the receptor.

Compounds 16a and 22 were selected to assess agonist

properties. Both exhibited greater than 50-fold binding

cooperativity with ACh. In contrast, the 2-phenyl analogue 16a

lost most of its agonist properties compared to the 4-phenyl

analogue 6a (log τ_B= 1.10 0.23; τ_B= 13). Interestingly, the

propargylamine derivative, 22, exhibited just as good eﬃcacy (log

τ_B= 2.29 0.07; τ_B= 195) as 5a, its closest related analogue.

Interacting 22, the only compound with an appreciable eﬃcacy,

with ACh in calcium mobilization assay provided a binding/

function cooperativity estimate nearly identical to the one

established for 5a (log αβ = 2.35 0.09; αβ = 225).

Finally, to further interrogate the N-substituent of the 4-oxo-

1,4-dihydroquinoline core, compounds removing the methylene

linker to the pendant aromatic ring (26a−b) and those replacing

the aromatic ring with an aliphatic heterocycle were synthesized

(28, 29, 31, and 32) (Table 4). Simple removal of the methylene

linker in 26a−b completely abolished the ability of the analogues

to transmit cooperativity with ACh when compared to BQCA.

Similarly, all piperidyl-linked compounds (28, 29, 31, and 32),

which lack aromaticity, exhibited a heavy reduction in binding

cooperativity with ACh relative to BQCA. In the case of amine

salt 29 and N-benzyl amine 31, cooperative binding was

completely abolished. However, the bulky urea 32 displayed

some remaining binding cooperativity with ACh compared to 29

and 31, suggesting that the loss of cooperativity with ACh would

be due to the potential protonation of the amine rather than the

introduction of a bulky group, although a more extensive set of

analogues would need to be investigated to establish this

hypothesis. Taken together, the N-alkyl and N-aryl series suggest

the presence of a fairly open pocket removed from the core 4-

oxo-1,4-dihydroquinoline structure, which is able to tolerate a

variety of groups diﬀering in size and polarity. It would appear,

however, that access to this proposed pocket is via a

conformationally sensitive linker region, preferring planar

aromatic groups, hinged through a methylene unit. Indeed,

literature would suggest that branching or extension of the

methylene linker is not tolerated.⁹Taken with our ﬁndings that

removal of the linker is not tolerated in terms of retaining binding

cooperativity, the methylene linker appears thus far to be

optimal.

CONCLUSIONS

■

The pharmacological activity of the prototypical selective M₁-

muscarinic PAM/allosteric agonist BQCA and related com-

pounds has only previously been described through an all-

encompassing “allosteric potency” estimate. In reality, this

estimate comprises at least four operational parameters (K_B,

aﬃnity for the receptor; α and β, binding and functional

cooperativity factors with the orthosteric ligand, respectively; and

τ_B, intrinsic eﬃcacy). With each of these parameters being

independently tunable through chemical modiﬁcation, we ﬁrst

sought to deﬁne a robust pharmacological proﬁle of BQCA and

key related literature compounds 5a and 6a. Building on this, we

initiated a SAR campaign, synthesizing over 35 novel compounds

with structural modiﬁcation in four areas: alternative substitution

of the 5- and 8- positions of the quinolone ring, isosteric

replacement of the carboxylic acid moiety or amide derivatives,

and replacement of the pendant N-alkyl group.

Evaluation of the literature compounds BQCA, 5a, and 6a

revealed the mechanism through which 5,8-diﬂuoro analogues

display improved biological activity. This was found to be due to

an increased intrinsic eﬃcacy (τ_B) relative to BQCA. Related

analogues bearing varied 5- and 8- substituents (3a−d, 4a−d,

5a−d, 6a−d, and 7) on both the carboxylic acid and ester scaﬀold

revealed that although the presence of the carboxylic acid group

is not essential for aﬃnity, it is important in determining the

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binding cooperativity with ACh. While an 8-methoxy substituent

is generally detrimental toward binding cooperativity with ACh,

the corresponding 5-methoxy substituent is better tolerated.

Furthermore, the 5-position is able to tolerate both hydrogen

bond donating (phenol) and accepting (ﬂuoro and methoxy) as

well as non-hydrogen bonding groups (hydrogen atom) while

still maintaining appreciable aﬃnity for the receptor and binding

cooperativity with ACh. Overall, substituents at the 5- and 8-

positions seem to be more important in tuning the intrinsic

eﬃcacy, whereas changing the N-(4-methoxy)benzyl group to N-

(4-phenyl)benzyl tended to improve aﬃnity for the receptor

without improving cooperative binding with ACh.

Our series of novel amide derivatives (8a−h, 10a−b, and 11)

revealed that the presence of a hydrogen bond donor group,

rather than a carboxylic acid functionality, may be of importance

for binding cooperativity with ACh, although amide derivatives

did suﬀer a reduction in this parameter compared to 5a. Notably,

8f only displayed a small drop in binding cooperativity with ACh

relative to parent compound 5a. The presence of a secondary

alcohol group on the cyclohexylamide substituent may allow for

further hydrogen bond or polar interactions. Furthermore, the

orientation of the amide substituent seems key, as the more

ﬂexible 10b exhibited signiﬁcantly reduced binding cooperativity

with ACh compared to 8f. Further evaluation of 8f revealed it to

be the only compound in our novel series, which also possessed

appreciable functional cooperativity with ACh in the calcium

mobilization assay (i.e., log αβ > log α).

Isosteric replacement of the carboxylic acid group was

tolerated at the level of aﬃnity for the receptor but caused a

reduction in binding cooperativity with ACh. The pK_aof the

acidic hydrogen atom does not seem to correlate with the degree

of cooperative binding with ACh.

Although the amide and acid isostere derivatives of 5a

synthesized to date do not improve binding cooperativity with

ACh over that observed with 5a, such groups do allow further

elaboration of the core BQCA scaﬀold while oﬀering potential

improvements in terms of ADMET. Furthermore, our ﬁndings

are in contrast to those recently published,³⁴which report that

functionalization of the carboxylic acid moiety on the quinolone-

based scaﬀold is not tolerated. Indeed, amide 8f while being

comparable to the parent acid 5f in terms of aﬃnity for the

receptor and binding cooperativity with ACh, actually exhibits

improved intrinsic eﬃcacy as well as functional cooperativity

with ACh in the calcium mobilization assay.

demonstrated the subtle tunability of individual ligand

parameters through chemical modiﬁcation, which has previously

only been described as an overall “allosteric potency” estimate.

This allows a more enriched understanding of the SAR

surrounding allosteric ligands, bringing us closer to delineating

some of the “ﬂat” SAR often encountered in this ﬁeld.

EXPERIMENTAL SECTION

■

Chemistry. Chemicals and solvents were purchased from standard

suppliers and used without further puriﬁcation. Davisil silica gel (40−63

μm) for ﬂash column chromatography (FCC) was supplied by Grace

Davison Discovery Sciences (Victoria, Australia), and deuterated

solvents were purchased from Cambridge Isotope Laboratories, Inc.

(USA, distributed by Novachem PTY. Ltd., Victoria, Australia).

Unless otherwise stated, reactions were carried out at ambient

temperature. All microwave reactions took place in a Biotage Initiator

microwave synthesizer. Reactions were monitored by thin layer

chromatography on commercially available precoated aluminum-backed

plates (Merck Kieselgel 60 F₂₅₄). Visualization was by examination

under UV light (254 and 366 nm). General staining was carried out with

KMnO₄or phosphomolybdic acid. A solution of ninhydrin (in ethanol)

was used to visualize primary and secondary amines. All organic extracts

collected after aqueous workup procedures were dried over anhydrous

MgSO₄or Na₂SO₄before gravity ﬁltering and evaporation to dryness.

Organic solvents were evaporated in vacuo at ≤40 °C (water bath

temperature). Puriﬁcation using preparative layer chromatography

(PLC) was carried out on Analtech preparative TLC plates (200 mm ×

200 mm × 2 mm).

¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance

Nanobay III 400 MHz Ultrashield Plus spectrometer at 400.13 and

100.62 MHz, respectively. Chemical shifts (δ) are recorded in parts per

million (ppm) with reference to the chemical shift of the deuterated

solvent. Coupling constants (J) and carbon−ﬂuorine coupling constants

(J_CF) are recorded in Hz, and the signiﬁcant multiplicities described by

singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br), multiplet

(m), doublet of doublets (dd), and doublet of triplets (dt). Spectra were

assigned using appropriate COSY, distortionless enhanced polarization

transfer (DEPT), HSQC, and HMBC sequences.

LC-MS were run to verify reaction outcome and purity using an

Agilent 6100 series Single Quad coupled to an Agilent 1200 series

HPLC. The following buﬀers were used: buﬀer A, 0.1% formic acid in

H₂O; buﬀer B, 0.1% formic acid in MeCN. The following gradient was

used with a Phenomenex Luna 3 μM C8(2) 15 mm × 4.6 mm column,

and a ﬂow rate of 0.5 mL/min and total run time of 12 min; 0−4 min

95% buﬀer A and 5% buﬀer B, 4−7 min 0% buﬀer A and 100% buﬀer B,

7−12 min 95% buﬀer A and 5% buﬀer B. Mass spectra were acquired in

positive and negative ion mode with a scan range of 0−1000 m/z at 5 V.

UV detection was carried out at 254 nm. All retention times (t_R) are

quoted in minutes. All compounds were of >95% purity.

General Procedure A: Condensation of Substituted Anilines

with Diethylethoxymethylene Malonate. Aniline and diethyle-

thoxymethylene malonate (1 equiv) were heated at 100−120 °C (oil

bath) together until TLC analysis (EtOAc/PE 3:7) or LC-MS analysis

indicated conversion was complete (1−7 h). The mixture was cooled to

rt or until precipitation was noted. At this point, the mixture was diluted

with excess PE and the resulting precipitate collected by ﬁltration

(vacuum) before washing with further PE and drying.

General Procedure B: Cyclization of Substituted Diethyl 2-

((phenylamino)methylene)malonates (1a−1d) in Eaton’s Re-

agent, to Give Corresponding Ethyl 4-Oxo-1,4-dihydroquino-

line-3-carboxylates. Substituted diethyl 2-((phenylamino)-

methylene)malonate was dissolved in Eaton’s reagent (a solution of

P₂O₅/methanesulfonic acid 1:10 w/w, 1 mL/mmol) and heated at 90−

100 °C (oil bath) under an atmosphere of nitrogen gas until TLC

analysis (EtOAc) indicated disappearance of starting material (5 h to

overnight). The mixture was cooled to 5 °C before pouring into satd

NaHCO_3(aq)and stirring for 15 min. The resulting precipitate was

isolated by ﬁltration (vacuum) and washed with water, then PE, before

drying.

Finally, our investigations into the N-alkyl pendant showed

that substitution in the 4-position of the benzylic group is the

most favorable, and seemingly a range of groups can be

accommodated. Gratifyingly, propargylamine derivative 22

retained a similar pharmacological proﬁle to 5a, indicating a

potentially open pocket or area able to tolerate a variety of

functional groups and polarities. However, access to this pocket

appears to be tightly controlled, as removal of the methylene

linker in 26a and 26b resulted in complete loss of binding

cooperativity with ACh without major eﬀect on aﬃnity. The

presence of a more bulky aliphatic heterocycle instead of the

planar aromatic ring present in a benzyl group, again caused

severe loss of binding cooperativity with ACh over several

analogues. This again supports the notion that access to a more

open pocket is most optimally achieved via a conformationally

sensitive linker region, preferring planar aromatic groups, hinged

through a methylene unit.

In summary, our detailed pharmacological proﬁling approach

to allosteric ligands for the M₁muscarinic receptor has

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General Procedure C: N-Alkylation of Substituted Ethyl 4-

Oxo-1,4-dihydroquinoline-3-carboxylates (2a−2e). Substituted

ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylate, K₂CO₃(1.1−1.3

equiv), KI (0.1 equiv), and the appropriate alkyl halide (1.1−1.3

equiv) were stirred in DMF (2−4 mL/mmol) at rt overnight. The

reaction was monitored by TLC (EtOAc or EtOAc/DCM 8:2). The

reaction mixture was poured onto ice/water and the resulting precipitate

collected by ﬁltration (vacuum), with further washings of water,

followed by washings of PE, before drying.

J

_CF= 239.4 Hz), 149.78, 145.16 (d, J_CF= 2.3 Hz), 129.85 (d, J_CF= 10.0

Hz), 112.06 (d, J_CF= 9.2 Hz), 110.13 (d, J_CF= 23.0 Hz), 102.04 (d, J_CF

27.9 Hz), 95.09, 60.58, 60.37, 56.58, 14.57, 14.44. LC-MS t_R: 6.15.

=

Diethyl 2-(((2-Methoxyphenyl)amino)methylene)malonate

(1c).³⁶ortho-Anisidine (2.00 g, 16.24 mmol) was reacted according to

general procedure A. After reaction completion, addition of PE on

cooling caused formation of a dark-red oily layer at the bottom of the

ﬂask. This was stirred in PE for 5 min, before allowing full separation to

occur, and decanting the top layer. The red oil was washed with PE in

this manner a further two times before drying under vacuum, causing a

solidiﬁcation to occur. Filtration (vacuum) gave 1.155 g of light-red

solid. The decanted PE washings began to show crystal formation. These

were collected by ﬁltration (vacuum) and washed with PE to give 2.958

g of oﬀ-white crystals. Spectral comparison of the two solids indicated

General Procedure D: Basic Hydrolysis of Esters to Give

Corresponding Carboxylic Acids. The appropriate ester was stirred

in THF/H₂O (1:1, 4−10 mL/mmol), with the vessel atmosphere being

purged with nitrogen gas. To this was added LiOH·H₂O (2−4 equiv)

and the mixture stirred at rt overnight. Once TLC analysis indicated

disappearance of the starting material, the reaction mixture was diluted

with water (to 20 mL) and washed with Et₂O (20 mL). The aqueous

layer was carefully acidiﬁed (∼pH 3) with 2 M HCl_(aq)before extracting

with EtOAc (3 × 20 mL). The combined organic layers were washed

with brine (20 mL) before concentration under reduced pressure.

General Procedure E: Synthesis of Amide Derivatives of 5,8-

Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-

carboxylic Acid (5a), through an Acid Chloride Intermediate.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-car-

boxylic acid (5a) (1.00 g, 2.90 mmol) was dispersed in anhydrous DCM,

under an atmosphere of N₂, and cooled to 0 °C over an ice bath. To this

was added oxalyl chloride (0.278 mL, 3.19 mmol, 1.1 equiv) and DMF

(1 drop). The mixture was stirred over the ice bath for 15 min, before

warming to rt, and stirring for 1 h. Oxalyl chloride (0.5 equiv) was added,

and stirring continued for a further 30 min. One-tenth of this batch

mixture was then added to a vessel containing the appropriate amine

(0.725 mmol, 2.5 equiv) in DCM (3 mL). Where the amine was present

as the hydrochloride salt, TEA (0.754 mmol, 2.6 equiv) was also added.

The mixtures were stirred at rt overnight, before diluting with DCM (20

mL) and washing with 2 M HCl_(aq)(20 mL). After concentration of the

organic layers, the crude products were further puriﬁed by FCC (eluent

EtOAc/PE or EtOAc/DCM).

General Procedure F: HCTU-Mediated Coupling of 5,8-

diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-

carboxylic Acid (5a) with Amines to Give the Corresponding

Amide. 5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquino-

line-3-carboxylic acid (5a) (100 mg, 0.29 mmol), HCTU (144 mg,

0.35 mmol, 1.2 equiv), and amine (1.2−1.5 equiv) were stirred in DMF

(2 mL) at rt. To this mixture was added DIPEA (1.5−3 equiv, depending

on whether the amine was used in the hydrochloride salt form) in DCM

(2 mL) before stirring at rt overnight. DCM was removed from the

reaction mixture under reduced pressure before diluting the residue with

1 M HCl_(aq)(20 mL) and extracting with EtOAc (3 × 20 mL). The

combined organic layers were washed with satd NaHCO_3(aq)(30 mL),

water (30 mL), and brine (30 mL). After concentration of the organic

layers, the crude product was further puriﬁed by FCC.

Diethyl 2-(((2,5-Diﬂuorophenyl)amino)methylene)malonate

(1a).³⁵2,5-Diﬂuoroaniline (11.79 g, 91.32 mmol) was reacted according

to general procedure A to give 22.597 g of oﬀ-white crystalline solid

(83%). ¹H NMR (400 MHz, CDCl₃) δ 11.06 (d, J = 13.1 Hz, 1H), 8.39

(d, J = 13.3 Hz, 1H), 7.11 (ddd, J = 10.1/9.1/4.8 Hz, 1H), 7.00 (ddd, J =

9.2/6.3/2.9 Hz, 1H), 6.76 (dddd, J = 9.0/7.6/3.7/2.9 Hz, 1H), 4.33 (q, J

= 7.1 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H), 1.34 (t, J

= 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.52, 165.40, 159.33

(dd, J_CF= 243.6/2.3 Hz), 150.08, 148.95 (dd, J_CF= 242.1/2.8 Hz),

129.02 (dd, J_CF= 12.9/10.3 Hz), 117.17 (dd, J_CF= 21.4/9.7 Hz), 110.83

(dd, J_CF= 24.2/7.4 Hz), 103.38 (dd, J_CF= 28.4/1.5 Hz), 96.42, 60.90,

60.58, 14.53, 14.39. m/z MS (TOF ES⁻) C₁₄H₁₄F₂NO₄[M − H]⁻calcd

298.1; found 298.0. LC-MS t_R: 6.18.

Diethyl 2-(((5-Fluoro-2-methoxyphenyl)amino)methylene)-

malonate (1b). 5-Fluoro-2-methoxyaniline (2.17 g, 15.37 mmol) was

reacted according to general procedure A to give 3.387 g of oﬀ-white

solid (71%). ¹H NMR (400 MHz, CDCl₃) δ 11.09 (d, J = 13.5 Hz, 1H),

8.44 (d, J = 13.9 Hz, 1H), 6.97 (dd, J = 9.4/2.8 Hz, 1H), 6.85 (dd, J = 9.0,

4.8 Hz, 1H), 6.76 (ddd, J = 8.9/8.0/2.8 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H),

4.26 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H), 1.34 (t, J =

7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.45, 165.82, 157.56 (d,

1

both were the desired product. Total yield: 4.113 g (86%). H NMR

(400 MHz, CDCl₃) δ 11.11 (d, J = 13.9 Hz, 1H), 8.57 (d, J = 14.0 Hz,

1H), 7.29−7.21 (m, 1H), 7.12−7.07 (m, 1H), 6.99 (ddd, J = 7.6/7.6/0.9

Hz, 1H), 6.94 (dd, J = 8.1/1.2 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.25 (q,

J = 7.1 Hz, 2H), 3.94 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H), 1.33 (t, J = 7.1 Hz,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.67, 166.17, 150.55, 149.08,

128.90, 124.94, 121.33, 114.47, 111.38, 93.90, 60.40, 60.19, 56.08, 14.59,

14.48. m/z MS (TOF ES⁺) C₁₅H₁₉NNaO₅[M + Na]⁺calcd 316.3;

found 316.2. LC-MS t_R: 6.08.

Diethyl 2-(((2-Chloro-5-methoxyphenyl)amino)methylene)-

malonate (1d). 6-Chloro-m-anisidine hydrochloride (3.00 g, 15.46

mmol), Et₃N (2.37 mL, 17.00 mmol, 1.1 equiv), and diethylethoxy-

methylene malonate (3.34 g, 15.46 mmol, 1equiv) were stirred together

under a reﬂux condenser at 100 °C for 1 h. TLC analysis (EtOAc/PE

3:7) indicated disappearance of starting aniline. The mixture was cooled

to rt overnight before partitioning between water (100 mL) and Et₂O

(100 mL). The aqueous layer was extracted with EtOAc (100 mL), and

the combined organic layers washed with water (100 mL) then brine (50

mL) before drying over MgSO₄and concentrating to give a crude purple

solid. This was dispersed in PE, sonicated, and then ﬁltered (vacuum),

1

with washings of PE, to give 4.024 g (79%) of purple solid. H NMR

(400 MHz, CDCl₃) δ 11.24 (d, J = 13.1 Hz, 1H), 8.47 (d, J = 13.2 Hz,

1H), 7.31 (d, J = 8.8 Hz, 1H), 6.82 (d, J = 2.7 Hz, 1H), 6.62 (dd, J = 8.9/

2.7 Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H), 3.83 (s,

3H), 1.38 (t, J = 7.1 Hz, 3H), 1.34 (t, J = 7.1 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 168.48, 165.85, 159.62, 150.24, 137.13, 130.80, 115.35,

110.25, 102.05, 95.82, 60.78, 60.49, 55.90, 14.54, 14.46. m/z MS (TOF

ES⁻) C₁₅H₁₇ClNO₅[M − H]⁻calcd 326.8; found 326.0. LC-MS t_R:

6.33.

Ethyl 5,8-Diﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxy-

late (2a).³⁵Diethyl 2-(((2,5-diﬂuorophenyl)amino)methylene)-

malonate (1a) (26.07 g, 87.12 mmol) was cyclized according to general

procedure B to give 18.54 g of yellow solid (84%). ¹H NMR (400 MHz,

DMSO) δ 8.62 (s, 1H), 7.27 (ddd, J = 10.4/8.8/4.2 Hz, 1H), 6.79 (ddd,

J = 12.3/8.7/3.9 Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz,

3H). ¹³C NMR (101 MHz, DMSO) δ 173.24, 166.79, 156.87 (dd, J_CF

256.0/2.8 Hz), 153.47, 152.94 (dd, J_CF= 247.2/3.7 Hz), 140.68 (d, J_CF

=

12.5 Hz), 119.64 (d, J_CF= 4.6 Hz), 114.04 (dd, J_CF= 21.5/11.1 Hz),

108.79, 106.88 (dd, J_CF= 24.5/7.7 Hz), 58.59, 14.46. m/z MS (TOF

ES⁻) C₁₂H₈F₂NO₃[M − H]⁻calcd 252.1; found 252.1. MS (TOF ES⁺)

C₁₂H₁₀F₂NO₃[MH]⁺calcd 254.1; found 254.1. LC-MS t_R: 4.55.

Ethyl 5-Fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-

carboxylate (2b). Diethyl 2-(((5-ﬂuoro-2-methoxyphenyl)amino)-

methylene)malonate (1b) (3.303 g, 10. 61 mmol) was cyclized

according to general procedure B. On pouring the cooled reaction

mixture on to satd NaHCO_3(aq), precipitation did not occur. The

aqueous mixture was extracted with EtOAc (3 × 100 mL), and the

combined organic extracts washed with brine (120 mL). The organic

layers were then concentrated under reduced pressure to give a crude

orange solid. This was redissolved in the minimum amount of EtOAc

before adding enough PE to eﬀect precipitation. The resultant solid was

collected by ﬁltration (vacuum) to give 1.226 g of brown solid (44%). ¹H

NMR (400 MHz, DMSO) δ 11.81 (d, J = 6.0 Hz, 1H), 8.25 (d, J = 6.8

Hz, 1H), 7.27 (dd, J = 8.9/3.8 Hz, 1H), 7.04 (dd, J = 11.9/8.9 Hz, 1H),

4.20 (q, J = 7.1 Hz, 2H), 3.97 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H). ¹³C NMR

(101 MHz, DMSO) δ 172.12, 164.34, 154.32 (d, J_CF= 252.6 Hz),

L

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144.55 (d, J_CF= 3.5 Hz), 143.62, 130.73 (d, J_CF= 3.7 Hz), 117.11 (d, J_CF

= 9.6 Hz), 112.47 (d, J_CF= 9.6 Hz), 111.70, 109.93 (d, J_CF= 22.9 Hz),

59.72, 56.59, 14.26. m/z MS (TOF ES⁺) C₁₃H₁₃FNO₄[MH]⁺calcd

266.1; found 266.1. LC-MS t_R: 4.75.

2H), 5.43 (d, J = 2.8 Hz, 2H), 4.39 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 1.40

(t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 174.20, 165.33,

161.99 (d, J_CF= 233.7 Hz), 159.88, 151.22, 150.53 (d, J_CF= 231.7 Hz),

127.89, 127.88, 127.17 (d, J_CF= 1.0 Hz), 125.16 (d, J_CF= 7.5 Hz),

Ethyl 8-Methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate

(2c). Diethyl 2-(((2-methoxyphenyl)amino)methylene)malonate (1c)

(3.97 g, 13.53 mmol) was cyclized according to general procedure B.

The resulting crude solid was recrystallized from EtOH/water, giving

two crops: 994 mg of pale-brown solid and 944 mg of brown solid. Total

yield = 1.938 g (58%). ¹H NMR (400 MHz, DMSO) δ 11.91 (s, 1H),

8.34 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 6.2 Hz, 1H), 7.44−7.26 (m, 2H),

4.21 (q, J = 7.0 Hz, 2H), 4.00 (s, 3H), 1.27 (t, J = 7.0 Hz, 3H). ¹³C NMR

(101 MHz, DMSO) δ 173.20, 164.66, 148.71, 144.03, 129.34, 128.17,

124.66, 116.78, 112.24, 109.99, 59.61, 56.36, 14.33. m/z MS (TOF ES⁺)

C₁₃H₁₄NO₄[MH]⁺calcd 248.3; found 248.1. LC-MS t_R: 4.79.

Ethyl 8-Chloro-5-methoxy-4-oxo-1,4-dihydroquinoline-3-

carboxylate (2d).³⁷Diethyl 2-(((2-chloro-5-methoxyphenyl)amino)-

methylene)malonate (1d) (3.983 g, 12.15 mmol) was cyclized

according to general procedure B. On pouring the cooled reaction

mixture on to satd NaHCO_3(aq), precipitation did not occur. The

aqueous mixture was extracted with EtOAc (3 × 50 mL). TLC analysis

(EtOAc) of the aqueous and organic layers indicated product was still

present in the aqueous layer. This was concentrated and saturated with

NaCl before re-extraction with EtOAc (3 × 50 mL). Concentration of

both organic layers found them to be impure, so they were combined,

concentrated, and further puriﬁed by FCC (eluent MeOH/EtOAc/PE

0:50:50 to 0:100:0, then up to 2:98:0) to give 1.213 g of brown solid

(35%). ¹H NMR (400 MHz, DMSO) δ 11.36 (s, 1H), 8.23 (s, 1H), 7.75

(d, J = 8.9 Hz, 1H), 6.90 (d, J = 8.9 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H),

3.83 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ

172.71, 164.49, 159.09, 149.13, 143.24, 132.62, 121.34, 115.93, 112.29,

106.82, 59.75, 56.09, 14.23. m/z MS (TOF ES⁺) C₁₃H₁₃ClNO₄[MH]⁺

calcd 282.7; found 282.1. LC-MS t_R: 4.88.

Ethyl 5-Methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate

(2e).³⁷Ethyl 8-chloro-5-methoxy-4-oxo-1,4-dihydroquinoline-3-car-

boxylate (2d) (500 mg, 1.78 mmol) was dispersed in EtOH (10 mL)

before sonication to degas the solvent. The vessel was evacuated and

ﬁlled with nitrogen before addition of 10% Pd/C (50 mg), with care.

After resealing, the vessel underwent three cycles of evacuation and

ﬁlling with hydrogen gas (balloon) before stirring at rt overnight. TLC

analysis (MeOH/DCM 1:9) indicated only partial progression, so

EtOH (10 mL) was added to aid dissolution of the starting material, and

hydrogenation continued for a further 2 nights. After this time, 10% Pd/

C (50 mg) was added with care (after evacuation of the vessel and ﬁlling

with nitrogen gas), and hydrogenation continued. After a total of ﬁve

nights of stirring, no starting material was evident by TLC. The mixture

was diluted with DCM and ﬁltered through a bed of Celite, with further

washings of DCM. Concentration of the ﬁltrate gave 457 mg of yellow

solid (quantitative yield). ¹H NMR (400 MHz, DMSO) δ 8.36 (s, 1H),

7.58 (dd, J = 8.2/8.2 Hz, 1H), 7.33 (dd, J = 8.3/0.8 Hz, 1H), 7.15 (dd, J =

8.2/0.6 Hz, 1H), 6.88 (d, J = 8.1 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.82

(s, 3H), 1.27 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 173.26,

165.01, 159.78, 143.15, 141.64, 133.04, 116.86, 111.40, 110.48, 106.35,

59.64, 55.86, 14.28. m/z MS (TOF ES⁺) C₁₃H₁₄NO₄[MH]⁺calcd

248.3; found 248.2. LC-MS t_R: 4.53.

Ethyl 5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydro-

quinoline-3-carboxylate (3a).⁸Ethyl 5,8-diﬂuoro-4-oxo-1,4-dihy-

droquinoline-3-carboxylate (2a) (3.00 g, 11.85 mmol) was alkylated

with 4-methoxybenzyl chloride according to general procedure C. After

the initial period of overnight stirring, further K₂CO₃(0.5 equiv) and 4-

methoxybenzyl chloride (0.5 equiv) were added and stirring continued

for a further overnight period. Because of diﬃculty ﬁltering the product

after pouring onto ice−water, EtOAc (500 mL) was added to redissolve

the wet crude material, and this was washed with water (100 mL) and

brine (100 mL) before concentration under reduced pressure. Crude

residue was redissolved in the minimum amount of EtOAc and

precipitation eﬀected by addition of PE. The desired product was

isolated by ﬁltration (vacuum) to give 3.074 g of brown solid (69%). ¹H

NMR (400 MHz, CDCl₃) δ 8.42 (s, 1H), 7.32−7.15 (m, 1H), 7.08 (d, J

= 8.4 Hz, 2H), 6.96 (ddd, J = 10.4/9.0/3.4 Hz, 1H), 6.86 (d, J = 8.8 Hz,

120.04, 114.64, 113.09, 112.30 (d, J_CF= 25.2 Hz), 61.39, 60.64 (d, J_CF=

17.3 Hz), 55.46, 14.52. m/z MS (TOF ES⁻) C₂₀H₁₇F₂NNaO₄[M +

Na]⁺calcd 396.1; found 396.1. LC-MS t_R: 5.62.

Ethyl 5-Fluoro-8-methoxy-1-(4-methoxybenzyl)-4-oxo-1,4-

dihydroquinoline-3-carboxylate (3b). Ethyl 5-ﬂuoro-8-methoxy-

4-oxo-1,4-dihydroquinoline-3-carboxylate (2b) (200 mg, 0.75 mmol)

was alkylated with 4-methoxybenzyl chloride according to general

procedure C. After the initial period of overnight stirring, further K₂CO₃

(0.3 equiv) and 4-methoxybenzyl chloride (0.3 equiv) were added and

stirring continued for a further overnight period. The desired product

was isolated by ﬁltration as described, to give 160 mg of yellow solid

(58%). ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.06−6.88 (m, 4H),

6.87−6.75 (m, 2H), 5.61 (s, 2H), 4.37 (q, J = 7.1 Hz, 2H), 3.76 (s, 3H),

3.72 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

173.47, 165.68, 159.41, 156.53 (d, J_CF= 258.3 Hz), 151.84, 145.93 (d,

J_CF= 3.9 Hz), 131.71, 128.78, 127.47, 121.11 (d, J_CF= 7.1 Hz), 115.03

(d, J_CF= 10.2 Hz), 114.41, 112.67, 112.13 (d, J_CF= 23.8 Hz), 61.41,

61.15, 56.79, 55.42, 14.54. m/z MS (TOF ES⁺) C₂₁H₂₁FNO₅[MH]⁺

calcd 386.4; found 386.2. LC-MS t_R: 5.62.

Ethyl 8-Methoxy-1-(4-methoxybenzyl)-4-oxo-1,4-dihydro-

quinoline-3-carboxylate (3c). Ethyl 8-methoxy-4-oxo-1,4-dihydro-

quinoline-3-carboxylate (2c) (500 mg, 2.02 mmol) was alkylated with 4-

methoxybenyl chloride, according to general procedure C, to give 578

mg of pale-brown solid. This was further puriﬁed by FCC (eluent

EtOAc/PE 0:100 to 100:0) to give 529 mg of light-brown solid (71%).

¹H NMR (400 MHz, CDCl₃) δ 8.47 (s, 1H), 8.16 (dd, J = 8.1/1.3 Hz,

1H), 7.31 (t, J = 8.0 Hz, 1H), 7.06 (dd, J = 7.9/1.0 Hz, 1H), 6.99 (d, J =

8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.66 (s, 2H), 4.38 (q, J = 7.1 Hz,

2H), 3.75 (s, 6H), 1.40 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 174.06, 165.96, 159.29, 152.08, 150.02, 131.96, 130.27,

129.12, 127.36, 125.67, 120.06, 114.82, 114.33, 110.83, 61.50, 60.99,

56.37, 55.38, 14.55. m/z MS (TOF ES⁺) C₂₁H₂₂NO₅[MH]⁺calcd

368.4; found 368.2. LC-MS t_R: 5.65.

Ethyl 5-Methoxy-1-(4-methoxybenzyl)-4-oxo-1,4-dihydro-

quinoline-3-carboxylate (3d). Ethyl 5-methoxy-4-oxo-1,4-dihydro-

quinoline-3-carboxylate (2e) (100 mg, 0.40 mmol) was alkylated with 4-

methoxybenzyl chloride, according to general procedure C, to give 66

1

mg of oﬀ-white solid (45%). H NMR (400 MHz, CDCl₃) δ 8.42 (s,

1H), 7.41 (dd, J = 8.4/8.4 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 6.93−6.82

(m, 3H), 6.79 (d, J = 8.2 Hz, 1H), 5.24 (s, 2H), 4.38 (q, J = 7.1 Hz, 2H),

3.95 (s, 3H), 3.78 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 174.91, 166.36, 161.75, 159.80, 148.37, 142.00, 132.85,

127.64, 126.42, 119.63, 114.82, 113.30, 108.49, 106.98, 60.98, 57.61,

56.49, 55.47, 14.61. m/z MS (TOF ES⁺) C₂₁H₂₂NO₅[MH]⁺calcd

368.4; found 368.2. LC-MS t_R: 5.46.

Ethyl 1-([1,1′-Biphenyl]-4-ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-

dihydroquinoline-3-carboxylate (4a).⁹Ethyl 5,8-diﬂuoro-4-oxo-

1,4-dihydroquinoline-3-carboxylate (2a) (500 mg, 1.97 mmol) was

alkylated with 4-phenylbenzyl bromide, according to general procedure

C, to give 420 mg of yellow solid (51%). ¹H NMR (400 MHz, CDCl₃) δ

8.46 (s, 1H), 7.64−7.49 (m, 4H), 7.48−7.39 (m, 2H), 7.38−7.30 (m,

1H), 7.29−7.22 (m, 1H), 7.20 (d, J = 8.1 Hz, 2H), 6.97 (ddd, J = 10.3/

9.1/3.4 Hz, 1H), 5.54 (d, J = 2.8 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), 1.41

(t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 165.25, 156.74 (d,

J_CF= 244.7 Hz), 151.39, 147.77 (dd, J_CF= 241.9/4.0 Hz), 141.67,

140.25, 134.33, 130.16, 129.02, 127.96, 127.83, 127.19, 126.65, 120.72

(d, J_CF= 7.8 Hz), 120.06 (dd, J_CF= 25.8/10.6 Hz), 113.31, 112.41 (dd,

J_CF= 24.6, 8.2 Hz), 61.43, 60.83 (d, J_CF= 17.2 Hz), 14.53. m/z MS (TOF

ES⁺) C₂₅H₂₀F₂NO₃[MH]⁺calcd 420.4; found 420.2. LC-MS t_R: 6.08.

Ethyl 1-([1,1′-Biphenyl]-4-ylmethyl)-5-ﬂuoro-8-methoxy-4-

oxo-1,4-dihydroquinoline-3-carboxylate (4b). Ethyl 5-ﬂuoro-8-

methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (2b) (500 mg, 1.89

mmol) was alkylated with 4-phenylbenzyl bromide, according to general

procedure C. After the initial period of overnight stirring, further K₂CO₃

(0.3 equiv) and 4-phenylbenzyl chloride (0.3 equiv) were added and

stirring continued for a further overnight period. The desired product

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was isolated by ﬁltration as described, to give 258 mg of pale-yellow solid

(32%). ¹H NMR (400 MHz, CDCl₃) δ 8.41 (s, 1H), 7.62−7.49 (m, 4H),

7.43 (dd, J = 10.3/4.8 Hz, 2H), 7.34 (ddd, J = 7.3/3.8/1.2 Hz, 1H), 7.12

(d, J = 8.3 Hz, 2H), 7.05−6.91 (m, 2H), 5.72 (s, 2H), 4.39 (q, J = 7.1 Hz,

2H), 3.69 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 173.44, 165.65, 156.58 (d, J_CF= 258.4 Hz), 152.00, 145.89 (d,

J_CF= 3.9 Hz), 140.94, 140.34, 135.92, 131.75 (d, J_CF= 1.6 Hz), 129.00,

product was isolated as 58 mg of pale-brown solid (59%). ¹H NMR (400

MHz, DMSO) δ 15.07 (s, 1H), 9.04 (s, 1H), 7.45 (dd, J = 9.1, 4.5 Hz,

1H), 7.33 (dd, J = 11.2, 9.1 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.86 (t, J =

5.8 Hz, 2H), 5.94 (s, 2H), 3.79 (s, 3H), 3.69 (s, 3H). ¹³C NMR (101

MHz, DMSO) δ 177.48, 165.54, 158.64, 154.52 (d, J_CF= 256.2 Hz),

152.27, 146.40 (d, J_CF= 3.5 Hz), 131.22 (d, J_CF= 1.5 Hz), 128.95,

127.65, 117.48 (d, J_CF= 8.6 Hz), 117.11 (d, J_CF= 9.9 Hz), 113.99, 113.15

(d, J_CF= 22.6 Hz), 108.71, 61.32, 56.89, 55.00;. m/z MS (TOF ES⁻)

C₁₉H₁₅FNO₅[M − H]⁻calcd 356.1; found 356.0. LC-MS t_R: 5.87.

8-Methoxy-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquino-

line-3-carboxylic Acid (5c). Ethyl 8-methoxy-1-(4-methoxybenzyl)-

4-oxo-1,4-dihydroquinoline-3-carboxylate (3c) (105 mg, 0.29 mmol)

was hydrolyzed according to general procedure D. After initial overnight

stirring, further LiOH·H₂O (1.5 equiv) was added and stirring

continued for a further overnight period before workup. In this case,

the acidifed aqueous layer was extracted with DCM (3 × 20 mL) to

obtain the free acid. The product was isolated as 109 mg of pale-brown

solid (98%). ¹H NMR (400 MHz, CDCl₃) δ 14.95 (s, 1H), 8.74 (s, 1H),

8.16 (dd, J = 8.1/1.4 Hz, 1H), 7.47 (dd, J = 8.1 Hz, 1H), 7.21 (dd, J =

8.0/1.3 Hz, 1H), 7.06−6.90 (m, 2H), 6.88−6.72 (m, 2H), 5.80 (s, 2H),

3.83 (s, 3H), 3.77 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 178.35,

167.12, 159.63, 151.30, 150.46, 130.88, 129.22, 128.23, 127.53, 126.98,

119.17, 115.89, 114.55, 108.76, 62.54, 56.55, 55.44/ m/z MS (TOF ES⁺)

C₁₉H₁₈NO₅[MH]⁺calcd 340.1; found 340.2. LC-MS t_R: 5.67.

5-Methoxy-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquino-

line-3-carboxylic Acid (5d). Ethyl 5-methoxy-1-(4-methoxybenzyl)-

4-oxo-1,4-dihydroquinoline-3-carboxylate (3d) (49 mg, 0.13 mmol)

was hydrolyzed according to general procedure D. After initial overnight

stirring, further LiOH·H₂O (1 equiv) was added and stirring continued

for a further hour before workup. The product was isolated as 22 mg of

yellow solid (49%). ¹H NMR (400 MHz, CD₃CN) δ 8.83 (s, 1H), 7.65

(dd, J = 8.5 Hz, 1H), 7.30−7.12 (m, 3H), 7.02 (d, J = 8.3 Hz, 1H), 6.90

(d, J = 8.7 Hz, 2H), 5.48 (s, 2H), 3.93 (s, 3H), 3.75 (s, 3H). ¹³C NMR

(101 MHz, CD₃CN) δ 180.60, 167.96, 162.24, 160.73, 149.65, 143.38,

135.54, 129.30, 127.57, 117.63, 115.37, 110.63, 110.28, 108.69, 58.64,

57.00, 55.99. m/z MS (TOF ES⁺) C₁₉H₁₈NO₅[MH]⁺calcd 340.1;

found 340.2. LC-MS t_R: 5.34.

127.72, 127.69, 127.12, 126.31, 121.08 (d, J_CF= 7.2 Hz), 115.14 (d, J_CF

=

10.1 Hz), 112.83, 112.22 (d, J_CF= 23.7 Hz), 61.74, 61.21, 56.82, 14.55.

m/z MS (TOF ES⁺) C₂₆H₂₃FNO₄[MH]⁺calcd 432.5; found 432.2. LC-

MS t_R: 6.08.

Ethyl 1-([1,1′-Biphenyl]-4-ylmethyl)-8-methoxy-4-oxo-1,4-

dihydroquinoline-3-carboxylate (4c). Ethyl 8-methoxy-4-oxo-1,4-

dihydroquinoline-3-carboxylate (2c) (500 mg, 2.02 mmol) was

alkylated with 4-phenylbenzyl bromide according to general procedure

C. After the initial period of overnight stirring, further K₂CO₃(0.3

equiv) and 4-phenylbenzyl chloride (0.3 equiv) were added and stirring

continued for a further overnight period. The desired product was

isolated by ﬁltration as described, to give 718 mg of yellow solid (86%).

¹H NMR (400 MHz, CDCl₃) δ 8.51 (s, 1H), 8.20 (dd, J = 8.1/1.3 Hz,

1H), 7.62−7.47 (m, 4H), 7.42 (dd, J = 7.5/7.5 Hz, 2H), 7.38−7.29 (m,

2H), 7.12 (d, J = 8.2 Hz, 2H), 7.08 (dd, J = 8.0/1.1 Hz, 1H), 5.77 (s, 2H),

4.41 (q, J = 7.1 Hz, 2H), 3.73 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 174.08, 165.99, 152.30, 150.02, 140.82, 140.40,

136.27, 131.97, 130.36, 128.98, 127.66, 127.64, 127.11, 126.25, 125.79,

120.19, 114.95, 111.05, 61.86, 61.10, 56.43, 14.59. m/z MS (TOF ES⁺)

C₂₆H₂₄NO₄[MH]⁺calcd 414.5; found 414.2. LC-MS t_R: 6.12.

Ethyl 1-([1,1′-Biphenyl]-4-ylmethyl)-5-methoxy-4-oxo-1,4-

dihydroquinoline-3-carboxylate (4d). Ethyl 5-methoxy-4-oxo-1,4-

dihydroquinoline-3-carboxylate (2e) (250 mg, 1.01 mmol) was

alkylated with 4-phenylbenzyl bromide according to general procedure

C. The ﬁltered crude solid was further puriﬁed by FCC (EtOAc/PE

1

0:100 to 100:0) to give 201 mg of pale-yellow solid (48%). H NMR

(400 MHz, CDCl₃) δ 8.47 (s, 1H), 7.66−7.49 (m, 4H), 7.49−7.39 (m,

3H), 7.39−7.29 (m, 1H), 7.22 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.2 Hz,

1H), 6.81 (d, J = 8.2 Hz, 1H), 5.35 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 3.96

(s, 3H), 1.40 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 174.88,

166.28, 161.79, 148.51, 141.99, 141.58, 140.24, 133.59, 132.96, 129.02,

128.09, 127.82, 127.17, 126.58, 119.59, 113.48, 108.50, 107.06, 61.01,

57.81, 56.51, 14.62. m/z MS (TOF ES⁺) C₂₆H₂₄NO₄[MH]⁺calcd

414.5; found 414.2. LC-MS t_R: 5.98.

1-([1,1′-Biphenyl]-4-ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-dihy-

droquinoline-3-carboxylic Acid (6a).³⁸Ethyl 1-([1,1′-biphenyl]-4-

ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (4a)

(100 mg, 0.24 mmol) was dispersed in 1,4-dioxane/1 M HCl_(aq)(1:1,

2 mL) and heated at 80 °C for 3.5 h before stirring at rt overnight. The

mixture was then reﬂuxed for a further 2.5 h before TLC analysis

(MeOH/DCM 2:98) indicated starting material had been consumed.

The mixture was diluted with water and the resultant precipitate

collected by ﬁltration (vacuum), before washing with further water and

PE, then drying. This gave 82 mg of pale-yellow solid (88%). ¹H NMR

(400 MHz, DMSO) δ 14.79 (s, 1H), 9.19 (s, 1H), 7.79 (ddd, J = 13.8,

9.1, 4.4 Hz, 1H), 7.70−7.52 (m, 4H), 7.50− 7.31 (m, 4H), 7.26 (d, J =

8.2 Hz, 2H), 5.92 (d, J = 3.9 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ

5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquino-

line-3-carboxylic Acid (5a). Ethyl 5,8-diﬂuoro-1-(4-methoxybenzyl)-

4-oxo-1,4-dihydroquinoline-3-carboxylate (3a) (2.37 g, 6.34 mmol) was

dissolved in 1,4-dioxane (15 mL) before adding 2 M HCl_(aq). The

mixture was heated under reﬂux for 2 h. TLC analysis (EtOAc) indicated

complete conversion had taken place. The mixture was cooled to rt,

dilute with water (50 mL), and the precipitate ﬁltered, then washed with

water and petroleum ether 40−60. The crude collected precipitate was

found to contain a mixture of the desired acid as well as some

debenzylated derivative. The desired compound was obtained by

washing the solid with DCM, and concentration of the ﬁltrate. This gave

336 mg (24%) of the debenzylated side-product as a white solid

(precipitate) and 1.417g (65%) of the desired compound as a pale-

yellow solid (DCM washings). ¹H NMR (400 MHz, DMSO) δ 14.76 (s,

1H), 9.11 (s, 1H), 7.77 (ddd, J = 13.8/9.1/4.4 Hz, 1H), 7.39 (ddd, J =

11.1/9.1/3.2 Hz, 1H), 7.12 (d, J = 8.5 Hz, 2H), 6.99−6.78 (m, 2H), 5.78

(d, J = 3.7 Hz, 2H), 3.71 (s, J = 4.7 Hz, 3H). ¹³C NMR (101 MHz,

DMSO) δ 176.96, 165.21, 158.89, 157.12 (dd, J_CF= 261.1/3.1 Hz),

152.20, 147.61 (dd, J_CF= 248.1/4.0 Hz), 130.14 (dd, J_CF= 9.0/2.8 Hz),

127.91 (d, J_CF= 1.9 Hz), 127.59, 121.86 (dd, J_CF= 26.0/10.8 Hz),

117.51 (d, J_CF= 9.1 Hz), 114.17, 113.27 (dd, J_CF= 23.6/8.4 Hz), 109.24,

60.22 (d, J_CF= 16.5 Hz), 55.06. m/z MS (TOF ES⁻) C₁₈H₁₂F₂NO₄[M

− H]⁻calcd 344.3; found 344.1. LC-MS t_R: 5.61.

177.04, 165.23, 157.18 (d, J_CF= 262.1 Hz), 152.57, 147.37 (d, J_CF

=

293.5 Hz), 139.60, 139.47, 135.62 (d, J_CF= 2.1 Hz), 130.29, 128.93,

127.56, 127.06, 126.61, 126.37, 122.04 (dd, J_CF= 22.5, 11.6 Hz), 117.55

(d, J_CF= 9.0 Hz), 113.31 (dd, J_CF= 23.3, 8.5 Hz), 109.40, 60.44 (d, J_CF

=

15.9 Hz). m/z MS (TOF ES⁺) C₂₃H₁₆F₂NO₃[MH]⁺calcd 392.3; found

392.1. LC-MS t_R6.05.

1-([1,1′-Biphenyl]-4-ylmethyl)-5-ﬂuoro-8-methoxy-4-oxo-

1,4-dihydroquinoline-3-carboxylic Acid (6b). Ethyl 1-([1,1′-

biphenyl]-4-ylmethyl)-5-ﬂuoro-8-methoxy-4-oxo-1,4-dihydroquino-

line-3-carboxylate (4b) (214 mg, 0.50 mmol) was hydrolyzed according

to general procedure D. After initial overnight stirring, further

LiOH.H₂O (1 equiv) was added and stirring continued for a further

hour before workup. The isolated product was found to contain a small

amount of starting material, so the crude product was dispersed in 1,4-

dioxane/H₂O (1:1, 2 mL) and heated at 80 °C for 3.5 h, followed by

stirring at rt overnight then reﬂux for 8.5 h. The desired product was

isolated by ﬁltration, after dilution of the reaction mixture with water, to

give 156 mg of pink solid (77%). ¹H NMR (400 MHz, DMSO) δ 15.08

(s, 1H), 9.09 (s, 1H), 7.69−7.55 (m, 4H), 7.51−7.40 (m, 3H), 7.39−

7.28 (m, 2H), 7.18 (d, J = 8.3 Hz, 2H), 6.06 (s, 2H), 3.75 (s, 3H). ¹³C

5-Fluoro-8-methoxy-1-(4-methoxybenzyl)-4-oxo-1,4-dihy-

droquinoline-3-carboxylic Acid (5b). Ethyl 5-ﬂuoro-8-methoxy-1-

(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (3b)

(121 mg, 0.31 mmol) was hydrolyzed according to general procedure

D. After initial overnight stirring, further LiOH.H₂O (1 equiv) was

added and stirring continued for a further hour before workup. The

N

dx.doi.org/10.1021/jm400540b | J. Med. Chem. XXXX, XXX, XXX−XXX

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Article

NMR (101 MHz, DMSO) δ 177.54, 165.55, 154.60 (d, J_CF= 256.2 Hz),

152.58, 146.37 (d, J_CF= 3.4 Hz), 139.46, 139.20, 136.60, 131.36, 128.91,

127.50, 126.85, 126.54, 126.48, 117.53, 117.29 (d, J_CF= 10.1 Hz), 113.20

(d, J_CF= 22.9 Hz), 108.81, 61.67, 56.95. m/z MS (TOF ES⁺)

C₂₄H₁₉FNO₄[MH]⁺calcd 404.1; found 404.2. LC-MS t_R: 6.06.

1-([1,1′-Biphenyl]-4-ylmethyl)-8-methoxy-4-oxo-1,4-dihy-

droquinoline-3-carboxylic Acid (6c). Ethyl 1-([1,1′-biphenyl]-4-

ylmethyl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (4c)

(310 mg, 075 mmol), was hydrolyzed by the same procedure, as

described for the hydrolysis of ethyl 1-([1,1′-biphenyl]-4-ylmethyl)-5-

ﬂuoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (4b) in the

synthesis of 1-([1,1′-biphenyl]-4-ylmethyl)-5-ﬂuoro-8-methoxy-4-oxo-

1,4-dihydroquinoline-3-carboxylic acid (6b) above. The crude product

after acidic hydrolysis required washings of DCM/PE (1:1) to remove

residual starting material. The title compound was isolated as 180 mg of

white solid (62%). ¹H NMR (400 MHz, DMSO) δ 15.16 (s, 1H), 9.14

(s, 1H), 8.01 (dd, J = 8.0, 1.4 Hz, 1H), 7.67−7.53 (m, 5H), 7.48 (dd, J =

8.1, 1.3 Hz, 1H), 7.43 (dd, J = 7.6, 7.6 Hz, 2H), 7.38−7.30 (m, 1H), 7.18

(d, J = 8.3 Hz, 2H), 6.10 (s, 2H), 3.80 (s, 3H). ¹³C NMR (101 MHz,

DMSO) δ 177.50, 165.82, 152.15, 150.35, 139.47, 139.21, 136.78,

130.28, 128.90, 128.06, 127.49, 127.27, 126.86, 126.54, 126.50, 117.59,

116.71, 107.65, 61.54, 56.60. m/z MS (TOF ES⁺) C₂₄H₂₀NO₄[MH]⁺

calcd 386.1; found 386.2. LC-MS t_R: 6.15.

1-([1,1′-Biphenyl]-4-ylmethyl)-5-methoxy-4-oxo-1,4-dihy-

droquinoline-3-carboxylic Acid (6d). Ethyl 1-([1,1′-biphenyl]-4-

ylmethyl)-5-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (4d)

(140 mg, 0.34 mmol) was hydrolyzed by the same procedure, as

described for the hydrolysis of ethyl 1-([1,1′-biphenyl]-4-ylmethyl)-5-

ﬂuoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (4b) in the

synthesis of 1-([1,1′-biphenyl]-4-ylmethyl)-5-ﬂuoro-8-methoxy-4-oxo-

1,4-dihydroquinoline-3-carboxylic acid (6b) above. The title compound

was isolated as 118 mg of pale yellow solid (90%). ¹H NMR (400 MHz,

DMSO) δ 9.22 (s, 1H), 7.74 (dd, J = 8.5 Hz, 1H), 7.68−7.55 (m, 4H),

7.44 (dd, J = 7.6 Hz, 2H), 7.39−7.24 (m, 4H), 7.10 (d, J = 8.3 Hz, 1H),

5.84 (s, 2H), 3.90 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 178.84,

166.37, 160.71, 149.46, 141.88, 139.74, 139.44, 134.89, 134.70, 128.93,

127.59, 127.17, 127.04, 126.61, 115.96, 109.86, 108.68, 107.90, 56.73,

56.29. m/z MS (TOF ES⁺) C₂₄H₂₀NO₄[MH]⁺calcd 386.1; found

386.2. LC-MS t_R: 5.82.

8.80 (s, 1H), 7.35−7.26 (m, 1H), 7.08 (d, J = 8.3 Hz, 2H), 7.00 (ddd, J =

10.8, 9.0, 3.4 Hz, 1H), 6.87−6.79 (m, 2H), 5.52 (d, J = 2.9 Hz, 2H), 3.77

(s, 3H), 3.49 (qd, J = 7.3, 5.6 Hz, 2H), 1.26 (t, J = 7.3 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 175.71, 163.89, 159.90, 158.69 (dd, J_CF= 262.2,

2.7 Hz), 150.44, 147.81 (d, J_CF= 283.9 Hz), 129.35, 127.98, 127.19,

120.14 (d, J_CF= 11.5 Hz), 119.83, 114.60, 113.82, 111.97 (dd, J_CF= 24.4,

8.3 Hz), 61.00 (d, J_CF= 17.1 Hz), 55.44, 34.35, 14.98. m/z MS (TOF

ES⁺) C₂₀H₁₈F₂N₂NaO₃[MH]⁺calcd 395.1; found 395.1. LC-MS t_R:

5.70.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-N,N-dimethyl-4-oxo-1,4-

dihydroquinoline-3-carboxamide (8b). The title compound was

obtained through coupling of dimethylamine hydrochloride with 5,8-

diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic

acid (5a), as described in general procedure F. FCC puriﬁcation (eluent

MeOH/DCM 1:99 to 2:98) gave 112 mg of pale-yellow oil that

1

solidiﬁed on standing to a yellow solid (quantitative yield). H NMR

(400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.23 (ddd, J = 13.6, 6.6, 2.8 Hz, 1H),

7.09 (d, J = 8.4 Hz, 2H), 6.91 (ddd, J = 10.5, 9.0, 3.3 Hz, 1H), 6.87−6.78

(m, 2H), 5.39 (d, J = 2.6 Hz, 2H), 3.75 (s, 3H), 3.08 (s, 3H), 3.03 (s,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.80, 166.31, 159.75, 158.44

(dd, J_CF= 260.5, 1.7 Hz), 147.82 (dd, J_CF= 246.0, 4.4 Hz), 147.18,

130.60 (dd, J_CF= 8.2, 2.8 Hz), 127.98 (d, J_CF= 1.4 Hz), 127.52 (d, J_CF

=

1.0 Hz), 121.17, 119.59 (d, J_CF= 10.7 Hz), 119.33 (d, J_CF= 8.6 Hz),

114.54, 111.08 (dd, J_CF= 24.6, 8.2 Hz), 60.01 (d, J_CF= 17.2 Hz), 55.38,

38.77, 35.61. m/z MS (TOF ES⁺) C₂₀H₁₉F₂N₂O₃[MH]⁺calcd 373.1;

found 373.2. LC-MS t_R: 4.92.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-N-phenyl-1,4-dihy-

droquinoline-3-carboxamide (8c). The title compound was

obtained through condensation of aniline with the acid chloride

intermediate described in general procedure E to give 47 mg of white

solid (39%). ¹H NMR (400 MHz, CDCl₃) δ 11.97 (s, 1H), 8.89 (s, 1H),

7.77 (dd, J = 8.6, 1.0 Hz, 2H), 7.41−7.28 (m, 3H), 7.16−7.00 (m, 4H),

6.90−6.80 (m, 2H), 5.56 (d, J = 2.8 Hz, 2H), 3.77 (s, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 175.78, 162.07, 160.01, 158.73 (dd, J_CF= 266.7,

4.2 Hz), 150.79, 148.03 (dd, J_CF= 247.2, 3.6 Hz), 138.61, 130.27,

129.08, 128.09, 126.95, 124.22, 120.68, 120.36 (dd, J_CF= 26.2, 10.7 Hz),

119.71 (d, J_CF= 7.4 Hz), 114.69, 113.79, 112.37 (dd, J_CF= 24.9, 8.2 Hz),

61.24 (d, J_CF= 17.5 Hz), 55.45. m/z MS (TOF ES⁺) C₂₄H₁₉F₂N₂O₃

[MH]⁺calcd 421.1; found 421.1. LC-MS t_R: 6.12.

1-([1,1′-Biphenyl]-4-ylmethyl)-8-ﬂuoro-5-hydroxy-4-oxo-

1,4-dihydroquinoline-3-carboxylic Acid (7). Ethyl 1-([1,1′-biphen-

yl]-4-ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxylate

(4a) (102 mg, 0.24 mmol) was dispersed in 1 M NaOH_(aq)/1,4-dioxane

(1:1, 2 mL) and heated at 80 °C for 3.5 h, followed by stirring at rt

overnight. After this time, complete conversion to the acid had been

achieved. The mixture was heated under reﬂux for a further 30 h, with no

appreciable progress noted. LiOH·H₂O (41 mg, 1.22 mmol, 5.1 equiv)

were added and reﬂux continued overnight, followed by stirring at rt for

a further week. After this time, the entire mixture was transferred to a

microwave vial and LiOH·H₂O (5 equiv) added. The vessel was sealed

and heated at 160 °C for 1 h. LC-MS analysis after this time indicated

complete conversion to the title compound. The mixture was diluted

with water (20 mL) and washed with Et₂O (20 mL) before acidiﬁcation

with 2 M HCl_(aq). The resultant precipitate was collected by ﬁltration

(vacuum) to give 75 mg of yellow/brown solid. This was recrystallized

N-Benzyl-5,8-diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihy-

droquinoline-3-carboxamide (8d). The title compound was

obtained through condensation of benzylamine with the acid chloride

intermediate described in general procedure E to give 49 mg of white

solid (39%). ¹H NMR (400 MHz, CDCl₃) δ 10.18 (t, J = 5.4 Hz, 1H),

8.83 (s, 1H), 7.47−7.19 (m, 7H), 7.08 (d, J = 8.4 Hz, 2H), 7.00 (ddd, J =

10.8, 9.0, 3.4 Hz, 1H), 6.91−6.76 (m, 2H), 5.52 (d, J = 2.9 Hz, 2H), 4.65

(d, J = 5.8 Hz, 2H), 3.77 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

175.69, 164.13, 159.93, 158.76 (dd, J_CF= 244.7, 2.6 Hz), 150.63, 147.88

(d, J_CF= 263.1 Hz), 138.62, 129.50 (d, J_CF= 10.1 Hz), 128.75, 128.00,

127.99, 127.34, 127.12 (d, J_CF= 1.2 Hz), 120.12 (dd, J_CF= 25.8, 10.7

Hz), 119.84 (d, J_CF= 7.4 Hz), 114.62, 113.64, 112.08 (dd, J_CF= 24.6, 8.6

Hz), 61.06 (d, J_CF= 17.3 Hz), 55.44, 43.61. m/z MS (TOF ES⁺)

C₂₅H₂₁F₂N₂O₃[MH]⁺calcd 435.2; found 435.1. LC-MS t_R: 5.99.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-N-(piperidin-1-yl)-

1,4-dihydroquinoline-3-carboxamide (8e). The title compound

was obtained through coupling of 1-aminopiperidine with 5,8-diﬂuoro-

1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

(5a), as described in general procedure F. FCC puriﬁcation (eluent

MeOH/DCM 0:100 to 5:95), followed by PLC puriﬁcation (MeOH/

DCM 2:98, plate run 3 times), gave 77 mg of oﬀ-white solid (62%). ¹H

NMR (400 MHz, CDCl₃) δ 10.58 (s, 1H), 8.81 (s, 1H), 7.36−7.26 (m,

1H), 7.14−6.94 (m, 3H), 6.89−6.76 (m, 2H), 5.53 (d, J = 2.9 Hz, 2H),

3.77 (s, 3H), 3.09−2.68 (m, 4H), 1.88−1.65 (m, 4H), 1.47 (d, J = 5.2

Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 175.34, 161.54, 159.92, 158.73

1

from EtOAc, to give 49 mg of oﬀ-white solid (53%). H NMR (400

MHz, DMSO) δ 13.37 (s, 1H), 13.29 (s, 1H), 9.17 (s, 1H), 7.78−7.54

(m, 5H), 7.45 (dd, J = 7.6 Hz, 2H), 7.35 (dd, J = 7.3 Hz, 1H), 7.25 (d, J =

8.1 Hz, 2H), 6.87 (dd, J = 9.0, 3.2 Hz, 1H), 5.89 (d, J = 3.0 Hz, 2H). ¹³C

NMR (101 MHz, DMSO) δ 180.38, 164.38, 157.74 (d, J_CF= 1.8 Hz),

153.29, 143.26 (d, J_CF= 240.5 Hz), 139.62, 139.47, 135.58 (d, J_CF= 1.6

Hz), 128.92, 128.47, 127.55, 127.05, 126.61, 126.42, 123.44 (d, J_CF

=

24.7 Hz), 114.04, 111.99 (d, J_CF= 7.7 Hz), 107.97 (d, J_CF= 1.9 Hz),

60.38 (d, J_CF= 14.9 Hz). m/z MS (TOF ES⁺) C₂₃H₁₇FNO₄[MH]⁺

calcd 390.1; found 390.1. LC-MS t_R: 5.93.

N-Ethyl-5,8-diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihy-

droquinoline-3-carboxamide (8a). The title compound was

obtained through condensation of 2 M ethylamine in THF with the

acid chloride intermediate described in general procedure E to give 40

mg of white solid (37%). ¹H NMR (400 MHz, CDCl₃) δ 9.77 (s, 1H),

(d, J_CF= 257.5 Hz), 150.65, 147.95 (d, J_CF= 242.2 Hz), 128.66 (d, J_CF

=

5.6 Hz), 127.97, 127.13, 120.05 (d, J_CF= 5.2 Hz), 119.76 (d, J_CF= 6.7

Hz), 114.61, 113.78, 112.07 (dd, J_CF= 24.7, 8.3 Hz), 61.08 (d, J_CF= 16.9

Hz), 57.18, 55.44, 25.25, 23.64. m/z MS (TOF ES⁺) C₂₃H₂₄F₂N₃O₃

[MH]⁺calcd 428.2; found 428.2. LC-MS t_R: 5.21.

O

dx.doi.org/10.1021/jm400540b | J. Med. Chem. XXXX, XXX, XXX−XXX

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Article

5,8-Diﬂuoro-N-((1S,2S)-2-hydroxycyclohexyl)-1-(4-methoxy-

benzyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (8f). The

title compound was obtained through coupling of (1S,2S)-2-amino-

cyclohexanol hydrochloride with 5,8-diﬂuoro-1-(4-methoxybenzyl)-4-

oxo-1,4-dihydroquinoline-3-carboxylic acid (5a), as described in general

procedure F. FCC puriﬁcation (eluent EtOAc/PE 0:100 to 100:0) gave

Hz), 59.55 (d, J_CF= 16.2 Hz), 55.05. m/z MS (TOF ES⁺)

C₁₈H₁₅F₂N₂O₄[MH]⁺calcd 361.1; found 361.1. LC-MS t_R: 5.51.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-N-(methylsulfonyl)-4-

oxo-1,4-dihydroquinoline-3-carboxamide (9). 5,8-Diﬂuoro-1-(4-

methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5a)

(100 mg, 0.29 mmol) was dispersed in dry DCM (1 mL) under an

atmosphere of N₂and cooled to 0 °C over an ice bath. To this were

added oxalyl chloride (0.028 mL, 0.32 mmol, 1.1 equiv) and DMF (1

drop) and the mixture stirred on the ice bath for 10 min before stirring at

rt for 30 min. Oxalyl chloride (0.2 equiv) was added, and stirring

continued at rt for 10 min. The mixture was concentrated under reduced

pressure before redissolving in dry DCM (1 mL) and methanesulfo-

namide (28 mg, 0.29 mmol, 1 equiv). The mixture was cooled over an

ice bath, under an atmosphere of N₂, before adding TEA (0.121 mL,

0.87 mmol, 3 equiv). The mixture was warmed to rt, and left to stir

overnight, before diluting with DCM (20 mL) and washing with 1 M

HCl_(aq)(20 mL). The organic layer was concentrated and the residue

puriﬁed by FCC (eluent EtOAc/PE 0:100 to 100:0) to give 48 mg of

white crystalline solid (39%). ¹H NMR (400 MHz, CDCl₃) δ 12.61 (s,

1H), 8.72 (s, 1H), 7.39 (ddd, J = 13.5, 9.0, 4.3 Hz, 1H), 7.18−7.01 (m,

3H), 6.95−6.77 (m, 2H), 5.56 (d, J = 2.8 Hz, 2H), 3.79 (s, 3H), 3.36 (s,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 175.36, 163.08, 160.28, 159.30 (d,

J_CF= 240.6 Hz), 151.09, 147.11 (d, J_CF= 278.8 Hz), 129.65, 128.35,

1

148 mg of oﬀ-white solid (quantitative yield). H NMR (400 MHz,

CDCl₃) δ 9.99 (d, J = 6.9 Hz, 1H), 8.80 (s, 1H), 7.39−7.17 (m, 1H),

7.14−6.94 (m, 3H), 6.93−6.73 (m, 2H), 5.52 (d, J = 2.7 Hz, 2H), 4.07

(s, 1H), 3.91−3.67 (m, 4H), 3.58−3.37 (m, 1H), 2.19−1.95 (m, 2H),

1.83−1.68 (m, 2H), 1.53−1.27 (m, 4H). ¹³C NMR (101 MHz, CDCl₃)

δ 175.63, 165.92, 159.94, 156.41 (d, J_CF= 238.8 Hz), 150.80, 145.47 (d,

J_CF= 255.7 Hz), 130.24 (dd, J_CF= 8.7, 1.9 Hz), 127.97, 127.03, 120.26

(dd, J_CF= 25.4, 10.5 Hz), 119.71 (d, J_CF= 7.5 Hz), 114.64, 113.06,

112.25 (dd, J_CF= 24.9, 8.2 Hz), 76.11, 61.14 (d, J_CF= 17.1 Hz), 55.97,

55.44, 38.75, 34.48, 31.49, 24.83, 24.10. m/z MS (TOF ES⁺)

C₂₄H₂₅F₂N₂O₄[MH]⁺calcd 443.2; found 443.2. LC-MS t_R: 5.32;

[α]²_D⁴= +498° (0.0105, DCM).

5,8-Diﬂuoro-1-(4-methoxybenzyl)-3-(morpholine-4-

carbonyl)quinolin-4(1H)-one (8g). 5,8-Diﬂuoro-1-(4-methoxyben-

zyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5a) (100 mg, 0.29

mmol) was dispersed in dry DCM (1 mL) under an atmosphere of N₂

and cooled to 0 °C over an ice bath. To this was added oxalyl chloride

(0.028 mL, 0.32 mmol, 1.1 equiv) and DMF (1 drop) and the mixture

stirred on the ice bath for 10 min before warming to rt and stirring for a

further 20 min. TLC analysis (EtOAc) indicated starting material had

been consumed. Morpholine (0.053 mL, 0.61 mmol, 2.1 equiv) was

added, and the mixture stirred for 10 min at rt before diluting with DCM

(20 mL) and washing with 0.5 M HCl_(aq)(20 mL) and satd NaHCO_3(aq)

(20 mL). After concentration of the organic layer under reduced

pressure, the crude was further puriﬁed by FCC (eluent EtOAc 10:90 to

126.04, 121.39 (dd, J_CF= 13.6, 10.8 Hz), 121.15, 114.89, 113.63 (d, J_CF

=

12.6 Hz), 111.19, 61.62 (d, J_CF= 17.6 Hz), 55.49, 41.97. m/z MS (TOF

ES⁺) C₁₉H₁₆F₂N₂NaO₅S [M + Na]⁺calcd 445.1; found 445.1. LC-MS

t_R: 5.32.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-N-methyl-4-oxo-1,4-dihy-

droquinoline-3-carboxamide (10a). Ethyl 5,8-diﬂuoro-1-(4-me-

thoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (3a) (100 mg,

0.27 mmol) was stirred in a mixture of EtOH/40% MeNH_2(aq)(1:1, 2

mL) at rt overnight. TLC analysis (EtOAc/DCM 1:1) indicated

complete conversion had taken place. The mixture was poured on to ice

water, and the resulting precipitate collected by ﬁltration (vacuum),

before drying to give 80 mg of pale-yellow solid (83%). ¹H NMR (400

MHz, CDCl₃) δ 9.72 (d, J = 3.7 Hz, 1H), 8.80 (s, 1H), 7.38−7.19 (m,

1H), 7.07 (d, J = 8.4 Hz, 2H), 7.00 (ddd, J = 10.8, 9.1, 3.3 Hz, 1H), 6.83

(d, J = 8.7 Hz, 2H), 5.52 (d, J = 2.7 Hz, 2H), 3.77 (s, 3H), 2.99 (d, J = 4.9

Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 175.64, 164.76, 159.91, 158.68

(dd, J_CF= 262.7, 2.8 Hz), 150.35, 147.88 (d, J_CF= 259.7 Hz), 130.33 (dd,

J_CF= 8.0, 2.4 Hz), 127.98 (d, J_CF= 1.5 Hz), 127.18 (d, J_CF= 1.0 Hz),

120.03 (dd, J_CF= 25.7, 11.0 Hz), 119.86 (d, J_CF= 7.4 Hz), 114.61,

113.79, 111.98 (dd, J_CF= 24.8, 8.2 Hz), 60.98 (d, J_CF= 17.2 Hz), 55.43,

26.10. m/z MS (TOF ES⁺) C₁₉H₁₆F₂N₂NaO₃[M + Na]⁺calcd 381.1;

found 381.2. LC-MS t_R: 5.16.

5,8-Diﬂuoro-N-(2-hydroxyethyl)-1-(4-methoxybenzyl)-4-

oxo-1,4-dihydroquinoline-3-carboxamide (10b). Ethyl 5,8-di-

ﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate

(3a) (100 mg, 0.27 mmol) was stirred in a mixture of EtOH/

ethanolamine (2:1, 3 mL) at rt for 3.75 h. TLC analysis (EtOAc)

indicated only partial progression, so EtOH (2 mL) was added to aid

further dissolution. After overnight stirring, complete conversion had

taken place. The mixture was poured on to ice/2 M HCl_(aq)and the

resulting precipitate collected by ﬁltration (vacuum) with washings of

water. After drying, 91 mg of yellow solid was obtained (88%). ¹H NMR

(400 MHz, CDCl₃) δ 10.17 (s, 1H), 8.79 (s, 1H), 7.31 (ddd, J = 13.6,

9.0, 4.3 Hz, 1H), 7.14−6.96 (m, 3H), 6.91−6.78 (m, 2H), 5.52 (d, J = 2.8

Hz, 2H), 3.83 (s, 2H), 3.77 (s, 3H), 3.63 (dd, J = 10.0, 5.6 Hz, 2H), 3.37

(d, J = 4.5 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 175.60, 165.93,

159.96, 157.29 (d, J_CF= 273.1 Hz), 150.58, 147.96 (d, J_CF= 245.2 Hz),

130.28 (d, J_CF= 8.5 Hz), 127.98 (d, J_CF= 1.5 Hz), 127.02 (d, J_CF= 1.1

Hz), 120.25 (dd, J_CF= 25.6, 11.0 Hz), 119.74 (d, J_CF= 7.7 Hz), 114.65,

113.21, 112.25 (dd, J_CF= 24.7, 8.1 Hz), 63.43, 61.11 (d, J_CF= 17.1 Hz),

55.44, 43.16. m/z MS (TOF ES⁺) C₂₀H₁₉F₂N₂O₄[MH]⁺calcd 389.1;

found 389.2. LC-MS t_R: 4.90.

1

100:0) to give 100 mg of colorless glassy solid (83%). H NMR (400

MHz, DMSO) δ 8.35 (s, 1H), 7.66−7.49 (m, 1H), 7.22−7.00 (m, 3H),

6.95−6.80 (m, 2H), 5.52 (d, J = 3.2 Hz, 2H), 3.70 (s, 3H), 3.67−3.53

(m, 6H), 3.40−3.19 (m, 2H). ¹³C NMR (101 MHz, DMSO) δ 170.90,

164.13, 158.74, 158.71 (d, J_CF= 235.1 Hz), 146.88, 146.86 (d, J_CF

=

260.9 Hz), 130.37 (d, J_CF= 3.4 Hz), 128.82, 127.30, 120.50 (d, J_CF= 6.2

Hz), 119.91 (d, J_CF= 2.4 Hz), 114.15, 111.51, 110.76 (dd, J_CF= 22.5/9.0

Hz), 66.47, 66.05, 58.74 (d, J_CF= 15.3 Hz), 55.04, 47.10, 41.99. m/z MS

(TOF ES⁻) C₂₂H₂₁F₂N₂O₄[M + H]⁺calcd 415.2; found 415.2. LC-MS

t_R: 5.27.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-3-(piperidine-1-

carbonyl)quinolin-4(1H)-one (8h). The title compound was

obtained through condensation of piperidine with the acid chloride

intermediate described in general procedure E to give 45 mg of oﬀ-white

semisolid (38%). ¹H NMR (400 MHz, CDCl₃) δ 7.88 (s, 1H), 7.26−

7.16 (m, 1H), 7.09 (d, J = 8.5 Hz, 2H), 6.91 (ddd, J = 10.5, 9.0, 3.3 Hz,

1H), 6.87−6.80 (m, 2H), 5.39 (d, J = 2.5 Hz, 2H), 3.77 (s, 3H), 3.69 (s,

2H), 3.36 (s, 2H), 1.83−1.43 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ

171.91, 164.51, 159.77, 158.51 (dd, J_CF= 261.8, 2.9 Hz), 147.84 (dd, J_CF

= 246.0, 4.4 Hz), 146.82, 130.66 (dd, J_CF= 8.1, 2.8 Hz), 128.00, 127.99,

127.62, 121.38, 119.41 (dd, J_CF= 25.6, 10.7 Hz), 114.56, 111.24, 111.00

(dd, J_CF= 24.6, 8.2 Hz), 60.01 (d, J_CF= 17.2 Hz), 55.41, 48.75, 43.45,

26.64, 25.69, 24.62. m/z MS (TOF ES⁺) C₂₃H₂₃F₂N₂O₃[MH]⁺calcd

413.2; found 413.2. LC-MS t_R: 5.55.

5,8-Diﬂuoro-N-hydroxy-1-(4-methoxybenzyl)-4-oxo-1,4-di-

hydroquinoline-3-carboxamide (8i). The title compound was

obtained through condensation of hydroxylamine hydrochloride with

the acid chloride intermediate described in general procedure E. The

crude product was dissolved in the minimum amount of MeOH, before

addition of EtOAc, causing precipitation of a pink solid to occur. This

was collected by ﬁltration (vacuum) to give 19 mg of the desired

compound (18%, > 90% pure). ¹H NMR (400 MHz, DMSO) δ 11.46 (s,

1H), 9.28 (s, 1H), 8.89 (s, 1H), 7.75−7.54 (m, 1H), 7.33−7.18 (m, 1H),

7.07 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 5.71 (d, J = 3.1 Hz,

2H), 3.70 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 173.59, 161.53,

160.37 (d, J_CF= 250.8 Hz), 158.80, 150.21, 147.49 (d, J_CF= 244.2 Hz),

129.96 (dd, J_CF= 6.6, 1.7 Hz), 128.48, 127.45, 120.54 (dd, J_CF= 26.3, 9.9

Hz), 118.58 (d, J_CF= 7.9 Hz), 114.19, 112.56, 112.04 (dd, J_CF= 23.3, 8.1

5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquino-

line-3-carboxamide (11). Ethyl 5,8-diﬂuoro-1-(4-methoxybenzyl)-4-

oxo-1,4-dihydroquinoline-3-carboxylate (3a) (520 mg, 1.39 mmol) was

dispersed in 28−30% NH_3(aq)/EtOH (1:1, 8 mL) and stirred at rt in a

closed vessel for 60 h. TLC analysis (EtOAc) indicated complete

P

dx.doi.org/10.1021/jm400540b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

conversion had taken place. The mixture was diluted with water and

washed with water and PE. The crude solid was further puriﬁed by FCC

(eluent EtOAc/PE 0:100 to 100:0) to give 37 mg of oﬀ-white solid

(64%). ¹H NMR (400 MHz, CDCl₃) δ 12.27 (s, 1H), 8.77 (s, 1H), 7.36

(ddd, J = 13.5, 9.0, 4.3 Hz, 1H), 7.16−7.01 (m, 3H), 6.95−6.80 (m, 2H),

5.55 (d, J = 2.8 Hz, 2H), 3.78 (s, 3H), 2.46 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 175.41, 171.69, 162.89, 160.16, 158.71 (dd, J_CF= 264.1, 3.1

Hz), 151.64, 147.98 (d, J_CF= 247.4 Hz), 130.10 (dd, J_CF= 9.3, 2.5 Hz),

128.18, 126.45, 120.95 (dd, J_CF= 26.0, 10.5 Hz), 119.72 (d, J_CF= 7.7

Hz), 114.80, 113.14 (dd, J_CF= 25.0, 8.4 Hz), 112.28, 61.45 (d, J_CF= 17.5

Hz), 55.47, 26.39. m/z MS (TOF ES⁺) C₂₀H₁₆F₂N₂NaO₄[M + Na]⁺

calcd 409.1; found 409.1. LC-MS t_R: 5.26.

Ethyl 1-([1,1′-Biphenyl]-2-ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-

dihydroquinoline-3-carboxylate (15a). Ethyl 5,8-diﬂuoro-4-oxo-

1,4-dihydroquinoline-3-carboxylate (2a) (500 mg, 1.97 mmol) was

alkylated with 2-(bromomethyl)biphenyl, according to general

procedure C. The resulting crude product was further puriﬁed by

FCC (eluent EtOAc/PE 0:100 to 20:80) to give 480 mg (73%) of pale-

yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.58−7.21 (m,

8H), 7.16 (ddd, J = 13.5/9.0/4.3 Hz, 1H), 7.02−6.83 (m, 2H), 5.42 (d, J

= 2.7 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H). ¹³C

NMR (101 MHz, CDCl₃) δ 172.35, 164.99, 158.52 (dd, J_CF= 263.1/2.7

Hz), 151.17, 147.66 (dd, J_CF= 246.9/4.3 Hz), 141.06, 139.89, 133.07 (d,

J_CF= 2.1 Hz), 130.75, 130.24 (d, J_CF= 5.7 Hz), 128.82, 128.78, 128.32,

128.23, 127.90, 125.61, 120.52 (d, J_CF= 7.7 Hz), 119.83 (dd, J_CF= 25.6/

stirred for 10 min before collecting the resulting precipitate by ﬁltration

1

(vacuum) and drying to give 423 mg (88%) of white solid. H NMR

(400 MHz, CDCl₃) δ 9.50 (s, 1H), 8.79 (s, 1H), 7.30 (ddd, J = 13.6/9.0/

4.3 Hz, 1H), 7.08 (d, J = 8.2 Hz, 2H), 7.02 (ddd, J = 10.7/9.0/3.4 Hz,

1H), 6.88−6.80 (m, 2H), 5.75 (s, 1H), 5.51 (d, J = 2.9 Hz, 2H), 3.77 (s,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 175.41, 165.80, 159.92, 158.69 (d,

J_CF= 262.4 Hz), 151.03, 147.98 (d, J_CF= 246.8 Hz), 130.37 (dd, J_CF

=

8.6/2.9 Hz), 127.92, 127.90, 127.08, 120.26 (dd, J_CF= 25.8/10.7 Hz),

119.87 (d, J_CF= 7.2 Hz), 114.62, 113.42, 112.30 (dd, J_CF= 24.8/8.4 Hz),

61.10 (d, J_CF= 16.9 Hz), 55.44. m/z MS (TOF ES⁺) C₁₈H₁₄F₂N₂NaO₃

[M + Na]⁺calcd 367.3; found 367.1. LC-MS t_R: 5.38.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquino-

line-3-carbonitrile (12). 5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-

1,4-dihydroquinoline-3-carboxamide (11) (104 mg, 0.30 mmol) was

stirred in MeCN/H₂O/MeOH (3:3:1, 7 mL) at rt before adding PdCl₂

(20 mg, 0.12 mmol, 0.4 equiv). The mixture was stirred at rt for 2 h

before adding THF (2 mL) and continuing stirring at rt overnight. TLC

analysis (EtOAc) indicated partial reaction progression, so stirring was

continued at 50 °C for a further overnight period before adding in PdCl₂

(0.1 equiv) and continuing stirring at 50 °C for a ﬁnal overnight period.

After this, organic solvents were removed under reduced pressure and

the residue diluted with water (20 mL) before extraction with DCM (3

× 20 mL). The combined organic extracts were washed with brine (30

mL) before concentration under reduced pressure. The crude product

was further puriﬁed by FCC (eluent EtOAc/PE 0:100 to 100:0) to give

34 mg of oﬀ-white solid (35%). ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s,

1H), 7.35 (ddd, J = 13.7, 9.1, 4.4 Hz, 1H), 7.12 (d, J = 8.4 Hz, 2H), 7.05

(ddd, J = 10.0, 9.1, 3.4 Hz, 1H), 6.96−6.82 (m, 2H), 5.44 (d, J = 2.9 Hz,

2H), 3.81 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.03, 160.35,

160.02 (dd, J_CF= 257.5, 6.9 Hz), 150.26, 147.99 (dd, J_CF= 247.6, 4.3

10.5 Hz), 113.04, 112.13 (dd, J_CF= 24.7/8.1 Hz), 61.22, 59.55 (d, J_CF

=

16.9 Hz), 14.48. m/z MS (TOF ES⁺) C₂₅H₂₀F₂NO₃[M + H]⁺calcd

420.1; found 420.2. LC-MS t_R: 6.06.

Ethyl 1-([1,1′-Biphenyl]-3-ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-

dihydroquinoline-3-carboxylate (15b). Ethyl 5,8-diﬂuoro-4-oxo-

1,4-dihydroquinoline-3-carboxylate (2a) (500 mg, 1.97 mmol) was

alkylated with 3-phenylbenzyl bromide according to general procedure

C. The resulting crude product was further puriﬁed by FCC (eluent

EtOAc/PE 0:100 to 20:80) to give 619 mg (75%) of pale-yellow solid.

¹H NMR (400 MHz, CDCl₃) δ 8.46 (s, 1H), 7.58−7.47 (m, 3H), 7.47−

7.31 (m, 5H), 7.29−7.17 (m, 1H), 7.07 (d, J = 7.7 Hz, 1H), 6.96 (ddd, J

= 10.0, 9.3, 3.4 Hz, 1H), 5.56 (d, J = 2.9 Hz, 2H), 4.39 (q, J = 7.1 Hz,

2H), 1.40 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.48,

Hz), 130.12 (dd, J_CF= 6.7, 3.5 Hz), 128.58, 125.91, 121.20 (dd, J_CF

=

26.0, 10.4 Hz), 118.47 (d, J_CF= 8.5 Hz), 115.01, 114.68, 113.24 (dd, J_CF

= 24.0, 8.4 Hz), 98.65, 60.87 (d, J_CF= 18.3 Hz), 55.52. m/z MS (TOF

ES⁺) C₁₈H₁₂F₂N₂NaO₂[M + Na]⁺calcd 349.1; found 349.1. LC-MS t_R:

5.20.

5,8-Diﬂuoro-1-(4-methoxybenzyl)-3-(1H-tetrazol-5-yl)-

quinolin-4(1H)-one (13). 5,8-Diﬂuoro-1-(4-methoxybenzyl)-4-oxo-

1,4-dihydroquinoline-3-carbonitrile (12) (34 mg, 0.20 mmol), NaN₃

(20 mg, 0.31 mmol, 3 equiv), and triethylammonium chloride (43 mg,

0.31 mmol, 3 equiv) were heated with stirring in toluene (5 mL) at 100

°C overnight. TLC analysis (EtOAc) indicated no progress, so further

triethylammonium chloride (143 mg, 10 equiv) and NaN₃(67 mg, 10

equiv) were added, and heating continued under reﬂux for a further

overnight period. TLC analysis indicated almost complete conversion,

so heating under reﬂux was continued for a further 7 h, then reduced to

70 °C over the weekend. The mixture was cooled, then diluted with

Et₂O (10 mL) and extracted with water (2 × 10 mL). The aqueous layer

was washed with Et₂O (10 mL) before acidifying with 2 M HCl_(aq)and

extracting with EtOAc (2 × 10 mL). Concentration of the EtOAc layers

followed by PLC puriﬁcation (MeOH/DCM 5:95, plate run twice) gave

14 mg of yellow solid (38%). ¹H NMR (400 MHz, CDCl₃) δ 14.22 (s,

1H), 9.00 (s, 1H), 7.40 (ddd, J = 13.6, 9.0, 4.3 Hz, 1H), 7.20−7.01 (m,

3H), 6.96−6.72 (m, 2H), 5.61 (d, J = 2.4 Hz, 2H), 3.78 (s, 3H). ¹³C

NMR (101 MHz, CDCl₃) δ 176.71, 160.18, 158.50 (dd, J_CF= 266.5, 3.1

Hz), 150.66, 148.30 (d, J_CF= 271.9 Hz), 146.61, 130.12 (d, J_CF= 11.1

165.17, 158.68 (dd, J_CF= 263.2/2.6 Hz), 151.38, 147.76 (dd, J_CF

246.5/4.5 Hz), 142.40, 140.39, 136.04 (d, J_CF= 1.4 Hz), 130.11 (d, J_CF

=

10.7 Hz), 129.74, 129.06, 127.91, 127.48, 127.26, 124.92, 124.80, 120.65

(d, J_CF= 7.5 Hz), 120.07 (dd, J_CF= 25.5/10.7 Hz), 113.26, 112.40 (dd,

J_CF= 24.7/8.3 Hz), 61.39, 61.05 (d, J_CF= 16.9 Hz), 14.50. m/z MS

(TOF ES⁺) C₂₅H₂₀F₂NO₃[MH]⁺calcd 420.1; found 420.2. LC-MS t_R:

6.09.

1-([1,1′-Biphenyl]-2-ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-dihy-

droquinoline-3-carboxylic Acid (16a). Ethyl 1-([1,1′-biphenyl]-2-

ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (15a)

(100 mg, 0.24 mmol) was dispersed in 1,4-dioxane/2 M HCl_(aq)(1:1, 2

mL) and heated at 80 °C for 5 h. TLC analysis (EtOAc) indicated

complete hydrolysis had taken place. The mixture was cooled to rt

before diluting with water (20 mL) and extracting with DCM (3 × 20

mL). Concentration of the combined organic layers gave 90 mg of pale-

yellow solid (96%). ¹H NMR (400 MHz, CDCl₃) δ 14.32 (s, 1H), 8.48

(s, 1H), 7.50−7.27 (m, 7H), 7.23−7.16 (m, 2H), 7.09 (ddd, J = 10.2/

9.1/3.4 Hz, 1H), 6.94 (d, J = 7.7 Hz, 1H), 5.58 (d, J = 3.0 Hz, 2H). ¹³C

NMR (101 MHz, CDCl₃) δ 177.71, 165.83, 158.24 (dd, J_CF= 265.1/2.7

Hz), 151.52, 147.94 (dd, J_CF= 248.7/4.5 Hz), 141.25, 139.69, 132.10 (d,

J_CF= 2.1 Hz), 131.07, 131.03, 129.02, 128.87, 128.59, 128.33, 128.13,

126.25 (d, J_CF= 1.6 Hz), 121.35 (dd, J_CF= 25.6/10.8 Hz), 118.33 (d, J_CF

= 9.2 Hz), 113.19 (dd, J_CF= 23.98.0 Hz), 110.22, 60.87 (d, J_CF= 16.9

Hz). m/z MS (TOF ES⁺) C₂₃H₁₆F₂NO₃[MH]⁺calcd 392.1; found

392.1. LC-MS t_R: 6.00.

1-([1,1′-Biphenyl]-3-ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-dihy-

droquinoline-3-carboxylic Acid (16b). Ethyl 1-([1,1′-biphenyl]-3-

ylmethyl)-5,8-diﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (15b)

(101 mg, 0.24 mmol) was dispersed in 1,4-dioxane/0.2 M HCl_(aq)(1:1, 2

mL) and heated at 60 °C for 3 h. TLC analysis (EtOAc/DCM 1:1)

indicated no progress. Heating was continued at 80 °C for a further 1.25

h before adding in 2 M HCl_(aq)(0.15 mL). Heating was continued at 80

°C for a further 1.75 h, followed by rt stirring overnight. After a ﬁnal 6 h

Hz), 128.13, 126.55, 120.80 (dd, J_CF= 26.1, 10.9 Hz), 118.73 (d, J_CF

8.7 Hz), 114.83, 113.01 (dd, J_CF= 23.9, 7.6 Hz), 106.86, 61.34 (d, J_CF

=

17.8 Hz), 55.48. m/z MS (TOF ES⁻) C₁₈H₁₂F₂N₅O₂[M − H]⁻calcd

368.1; found 368.0. LC-MS t_R: 5.10.

N-Acetyl-5,8-diﬂuoro-1-(4-methoxybenzyl)-4-oxo-1,4-dihy-

droquinoline-3-carboxamide (14). 5,8-Diﬂuoro-1-(4-methoxyben-

zyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (11) (50 mg, 0.15

mmol) was dissolved in pyridine (1 mL) before adding Ac₂O (0.021

mL, 0.22 mmol, 1.5 equiv) and stirring at rt overnight. TLC analysis

(EtOAc) indicated no progress, so Ac₂O (1 mL) and DCM (1 mL) were

added and stirring continued at rt overnight. The temperature was then

increased to 100 °C, and after 2 d the starting material had disappeared.

The mixture was cooled, diluted with water, and stirred for 30 min at rt.

The resulting precipitate was collected by ﬁltration (vacuum) and

Q

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Article

of heating under reﬂux, the mixture was cooled, diluted with water, and

the resulting precipitate collected by ﬁltration (vacuum), with washings

of water and PE. After drying, 85 mg of pale-yellow solid was obtained

(91%). ¹H NMR (400 MHz, DMSO) δ 14.78 (s, 1H), 9.18 (s, 1H), 7.78

(ddd, J = 13.8/9.1/4.4 Hz, 1H), 7.68−7.56 (m, 3H), 7.54 (s, 1H), 7.50−

7.31 (m, 5H), 7.09 (d, J = 7.7 Hz, 1H), 5.94 (d, J = 3.9 Hz, 2H). ¹³C

NMR (101 MHz, DMSO) δ 177.06, 165.24, 157.16 (dd, J_CF= 261.6/2.3

Hz), 152.57, 147.26 (dd, J_CF= 232.5/7.8 Hz), 140.62, 139.60, 137.16 (d,

J_CF= 2.4 Hz), 130.32 (dd, J_CF= 12.0/2.8 Hz), 129.46, 128.95, 127.67,

126.73, 126.18, 124.61, 124.33, 121.88 (dd, J_CF= 25.7/10.3 Hz), 117.55

(d, J_CF= 10.0 Hz), 113.26 (dd, J_CF= 23.2/9.0 Hz), 109.40, 60.70 (d, J_CF

= 15.3 Hz). m/z MS (TOF ES⁺) C₂₃H₁₆F₂NO₃[MH]⁺calcd 392.1;

found 392.1. LC-MS t_R: 6.05.

tert-Butyl Prop-2-yn-1-ylcarbamate (18). Propargylamine (17)

(5.126 g, 93.06 mmol) was dissolved in water (100 mL) with stirring. To

this was added Boc₂O (21.325 g, 97.71 mmol, 1.05 equiv), and the

mixture was stirred at rt overnight. TLC analysis (MeOH/DCM 1:9)

indicated complete conversion. The mixture was extracted with DCM (3

× 30 mL) and the combined organic extracts washed with water (50

mL). Concentration of the organic layers gave 13.902 g of pale-yellow

oil, which rapidly crystallized to oﬀ-white crystals (96%). ¹H NMR (400

MHz, CDCl₃) δ 4.70 (s, 1H), 3.92 (d, J = 3.0 Hz, 2H), 2.21 (t, J = 2.5 Hz,

1H), 1.45 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 146.90, 85.32, 80.21,

71.38, 28.48, 27.57.

158.68 (d, J_CF= 265.5 Hz), 155.40, 151.31, 147.68 (d, J_CF= 243.2 Hz),

135.53 (d, J_CF= 1.3 Hz), 132.55, 129.99, 126.07, 123.28, 120.66 (d, J_CF

=

7.8 Hz), 120.06 (dd, J_CF= 25.5/10.5 Hz), 113.40, 112.49 (d, J_CF= 33.3

Hz), 86.70, 82.33, 80.26, 61.46, 60.80 (d, J_CF= 17.0 Hz), 31.30, 28.50,

14.51. m/z MS (TOF ES⁺) C₂₇H₂₇F₂N₂O₅[MH]⁺calcd 497.2; found

497.2. LC-MS t_R: 5.90.

1-(4-(3-Aminoprop-1-yn-1-yl)benzyl)-5,8-diﬂuoro-4-oxo-1,4-

dihydroquinoline-3-carboxylic Acid Hydrochloride (22). Ethyl 1-

(4-(3-((tert-butoxycarbonyl)amino)prop-1-yn-1-yl)benzyl)-5,8-di-

ﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (21) (230 mg, 0.46

mmol) was dispersed in a mixture of 1,4-dioxane/1 M HCl_(aq)(1:1, 2

mL) and heated at 80 °C for 3.5 h, then at rt overnight (TLC analysis

(MeOH/DCM 1:9) indicated complete N-Boc deprotection after 1.5

h). Heating was continued under reﬂux for a further 5 h, before cooling

and concentration under reduced pressure, to give 205 mg of pale-

yellow solid (quantitative yield). ¹H NMR (400 MHz, DMSO) δ 14.75

(s, 1H), 9.14 (s, 1H), 8.45 (s, 3H), 7.77 (ddd, J = 13.7, 9.1, 4.3 Hz, 1H),

7.56−7.31 (m, 3H), 7.21 (d, J = 8.3 Hz, 2H), 5.90 (d, J = 4.0 Hz, 2H),

3.97 (s, 2H).; ¹³C NMR (101 MHz, DMSO) δ 177.04, 165.18, 158.46

(d, J_CF= 261.7 Hz), 152.63, 147.97 (d, J_CF= 262.4 Hz), 137.80, 131.87,

130.23, 126.15, 120.50, 119.70 (d, J_CF= 23.1 Hz), 119.04, 113.35 (d, J_CF

= 13.3 Hz), 109.46, 85.09, 83.04, 60.31 (d, J_CF= 4.2 Hz), 28.93. m/z MS

(TOF ES⁺) C₂₀H₁₅F₂N₂O₃[MH]⁺calcd 369.1; found 369.1. LC-MS t_R:

4.36.

Ethyl 3-(Dimethylamino)-2-(2-ﬂuorobenzoyl)acrylate (24).⁴⁰

Ethyl-(2-ﬂuorobenzyl)acetate (23) (1.04 g, 4.95 mmol) was dissolved in

DMF (1 mL) and N,N-dimethylformamide and dimethyl acetal (627

mg, 5.26 mmol, 1 equiv) added. The mixture was heated at 100 °C in the

microwave reactor for 30 min before pouring onto ice water (200 mL)

and stirring for 5 min. The aqueous slurry was then extracted with Et₂O

(20 mL) before saturating the aqueous layer with NaCl and extracting

again with Et₂O (2 × 20 mL). The combined organic layers were washed

with brine (30 mL) before concentration to give 1.185 g of yellow oil

(90%). ¹H NMR (400 MHz, CDCl₃) δ 7.75 (s, 1H), 7.59 (s, 1H), 7.45−

7.31 (m, 1H), 7.16 (ddd, J = 7.6, 0.9 Hz, 1H), 7.01 (ddd, J = 10.3, 8.3, 0.8

Hz, 1H), 3.95 (q, J = 7.1 Hz, 2H), 3.28 (s, 3H), 2.90 (s, 3H), 0.88 (t, J =

7.1 Hz, 3H). m/z MS (TOF ES⁺) C₁₄H₁₇FNO₃[MH]⁺calcd 266.1;

found 266.2.1. LC-MS t_R: 5.66.

Ethyl 1-(4-Methoxyphenyl)-4-oxo-1,4-dihydroquinoline-3-

carboxylate (25a).⁴⁰Ethyl 3-(dimethylamino)-2-(2-ﬂuorobenzoyl)-

acrylate (24) (100 mg, 0.38 mmol) and p-anisidine (47 mg, 0.38 mmol,

1 equiv) were dissolved in dry DMF (2 mL) and heated at 140 °C in the

microwave reactor for 1 h. TLC analysis (EtOAc/PE 1:1) indicated

partial progression, so the mixture was heated at 140 °C (oil bath) over

the weekend. LC-MS analysis after this time indicated only 1 major peak,

the mixture was poured on to ice water, and the resulting precipitate

ﬁltered, washed with water and PE, and dried to give 87 mg of pale-

brown solid (71%). ¹H NMR (400 MHz, CDCl₃) δ 8.55 (dd, J = 8.0, 1.4

Hz, 1H), 8.50 (s, 1H), 7.51 (ddd, J = 8.6, 7.1, 1.6 Hz, 1H), 7.45−7.37 (m,

1H), 7.37−7.28 (m, 2H), 7.16−7.04 (m, 2H), 6.99 (d, J = 8.4 Hz, 1H),

4.40 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 174.59, 165.95, 160.61, 149.25, 141.14, 133.52,

132.40, 128.70, 128.62, 127.65, 125.33, 117.85, 115.55, 111.39, 61.08,

55.88, 14.59. m/z MS (TOF ES⁺) C₁₉H₁₈NO₄[MH]⁺calcd 324.1;

found 324.2. LC-MS t_R: 5.27.

Ethyl 1-([1,1′-Biphenyl]-4-yl)-4-oxo-1,4-dihydroquinoline-3-

carboxylate (25b). Ethyl 3-(dimethylamino)-2-(2-ﬂuorobenzoyl)-

acrylate (24) (100 mg, 0.38 mmol) and 4-aminobiphenyl (64 mg,

0.38 mmol, 1 equiv) were dissolved in dry DMF (2 mL) and heated at

140 °C (oil bath) over the weekend. LC-MS analysis after this time

indicated only one major peak, the mixture was poured on to ice water,

and the resulting precipitate was ﬁltered, washed with water and PE, and

dried to give 85 mg of yellow solid (61%). ¹H NMR (400 MHz, CDCl₃)

δ 8.64−8.50 (m, 2H), 7.88−7.77 (m, 2H), 7.72−7.64 (m, 2H), 7.64−

7.48 (m, 5H), 7.48−7.36 (m, 2H), 7.08 (dd, J = 13.6, 4.9 Hz, 1H), 4.41

(q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 174.58, 165.83, 148.84, 143.27, 140.75, 139.83, 139.48,

132.50, 129.27, 129.16, 128.63, 128.46, 127.87, 127.74, 127.39, 125.47,

117.81, 111.63, 61.13, 14.60. m/z MS (TOF ES⁺) C₂₄H₂₀NO₃[MH]⁺

calcd 370.1; found 370.2. LC-MS t_R: 5.77.

tert-Butyl (3-(4-(Hydroxymethyl)phenyl)prop-2-yn-1-yl)-

carbamate (19).³⁹4-Iodobenzyl alcohol (2.02 g, 8.55 mmol), tert-

butyl prop-2-yn-1-ylcarbamate (18) (1.724 g, 11.11 mmol, 1.3 equiv),

and TEA (3.573 mL, 25.64 mmol, 3 equiv) were placed in a ﬂask in dry

THF (20 mL) and degassed by sonication (10 min). CuI (82 mg, 0.43

mmol, 0.05 equiv) and PdCl₂(PPh₃)₂(302 mg, 0.43 mmol, 0.05 equiv)

were added and the mixture stirred at rt overnight. TLC analysis

(EtOAc/PE 3:7) indicated complete depletion of the alkyne. The

mixture was diluted with EtOAc (100 mL) before ﬁltering through a bed

of Celite (vacuum). The ﬁltrate was washed with water (2 × 50 mL) and

brine (50 mL) before concentration and further puriﬁcation by FCC

(eluent EtOAc/PE 0:100 to 30:70) to give 933 mg of light-brown solid

(42%). ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J = 8.2 Hz, 2H), 7.30 (d,

J = 8.4 Hz, 2H), 4.77 (s, 1H), 4.69 (d, J = 5.5 Hz, 2H), 4.15 (d, J = 4.7 Hz,

2H), 1.76 (t, J = 5.8 Hz, 1H), 1.47 (s, 9H). ¹³C NMR (101 MHz,

CDCl₃) δ 150.57, 141.26, 132.02, 126.89, 122.08, 85.55, 83.07, 71.61,

65.05, 31.35, 28.52. m/z MS (TOF ES⁺) C₁₁H₁₂NO₃[M − ^tBu + 2H]⁺

calcd 206.1; found 206.2. LC-MS t_R: 5.54.

tert-Butyl (3-(4-(Bromomethyl)phenyl)prop-2-yn-1-yl)-

carbamate (20). tert-Butyl (3-(4-(hydroxymethyl)phenyl)prop-2-yn-

1-yl)carbamate (19) (880 mg, 3.37 mmol) and CBr₄(1.227 g, 3.70

mmol, 1.1 equiv) were dissolved in DCM (20 mL) before cooling over

an ice bath. PPh₃(970 mg, 3.70 mmol, 1.1 equiv) was added and the

mixture allowed to warm to rt with stirring. After 1.5 h of stirring at rt,

TLC analysis (EtOAc/PE 3:7) indicated good progression. PPh₃(0.5

equiv) was added, and stirring continued overnight. The mixture was

concentrated under reduced pressure and the crude residue further

puriﬁed by FCC (eluent EtOAc/PE 0:100 to 30:70) to give 595 mg of

shiny oﬀ-white solid (55%). ¹H NMR (400 MHz, CDCl₃) δ 7.40−7.34

(m, 2H), 7.34−7.27 (m, 2H), 4.75 (s, 1H), 4.46 (s, 2H), 4.14 (d, J = 4.7

Hz, 2H), 1.46 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 155.40, 137.97,

132.16, 129.08, 122.98, 86.40, 82.68, 79.62, 32.96, 31.31, 28.46. m/z MS

(TOF ES⁺) C₁₁H₁₁BrNO₂[M − ^tBu + 2H]⁺calcd 268.0; found 268.0.

LC-MS t_R: 6.29.

Ethyl 1-(4-(3-((tert-Butoxycarbonyl)amino)prop-1-yn-1-yl)-

benzyl)-5,8-diﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxy-

late (21). Ethyl 5,8-diﬂuoro-4-oxo-1,4-dihydroquinoline-3-carboxylate

(2a) (199 mg, 0.79 mmol) was alkylated with tert-butyl (3-(4-

(bromomethyl)phenyl)prop-2-yn-1-yl)carbamate (20) according to

general procedure C. The resulting crude product was further puriﬁed

by FCC (eluent EtOAc/PE 5:95 to 100:0) to give 392 mg (67%) of

yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.39 (s, 1H), 7.38 (d, J = 8.3

Hz, 2H), 7.22 (ddd, J = 13.5/9.0/4.3 Hz, 1H), 7.05 (d, J = 8.1 Hz, 2H),

6.96 (ddd, J = 10.3/9.1/3.4 Hz, 1H), 5.47 (d, J = 2.8 Hz, 2H), 4.74 (s,

1H), 4.39 (q, J = 7.1 Hz, 2H), 4.13 (d, J = 4.5 Hz, 2H), 1.46 (s, 9H), 1.40

(t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.42, 165.20,

R

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1-(4-Methoxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carbox-

ylic Acid (26a). Ethyl 1-(4-methoxyphenyl)-4-oxo-1,4-dihydroquino-

line-3-carboxylate (25a) (68 mg, 0.21 mmol) was hydrolyzed according

to general procedure D. On acidiﬁcation of the aqueous layer during

workup, a pink precipitate formed. This was collected by ﬁltration

(vacuum) and washed with water and PE before drying to give 46 mg of

pink solid (74%). ¹H NMR (400 MHz, CDCl₃) δ 14.92 (s, 1H), 8.80 (s,

1H), 8.56 (dd, J = 8.1, 1.3 Hz, 1H), 7.68 (ddd, J = 8.7, 7.1, 1.6 Hz, 1H),

7.57 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H), 7.41−7.28 (m, 2H), 7.17 (d, J = 8.5

Hz, 1H), 7.15−7.05 (m, 2H), 3.93 (s, 4H). ¹³C NMR (101 MHz,

CDCl₃) δ 178.92, 167.11, 161.03, 149.04, 141.64, 133.90, 132.77,

128.36, 126.90, 126.49, 126.07, 118.72, 115.71, 109.02, 55.94. m/z MS

(TOF ES⁺) C₁₇H₁₄NO₄[MH]⁺calcd 296.1; found 296.2. LC-MS t_R:

5.25.

4-Oxo-1-(piperidin-4-ylmethyl)-1,4-dihydroquinoline-3-car-

boxylic Acid Hydrochloride (29). 1-((1-(tert-Butoxycarbonyl)-

piperidin-4-yl)methyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

(28) (155 mg, 0.40 mmol) was dissolved in DCM (2 mL) with stirring

at rt before addition of 4 M HCl in 1,4-dioxane (2 mL). The mixture was

stirred at rt for 1 h, before addition of PE, followed by collection of the

resulting precipitate by ﬁltration (vacuum). The collected solid was

further washed with PE before drying to give 149 mg of oﬀ-white solid

(quantitative yield). ¹H NMR (400 MHz, DMSO) δ 9.18−8.95 (m, J =

16.9 Hz, 2H), 8.89−8.62 (m, J = 10.1 Hz, 1H), 8.41 (dd, J = 8.1, 1.5 Hz,

1H), 8.11 (d, J = 8.7 Hz, 1H), 7.99 (ddd, J = 8.7, 7.1, 1.6 Hz, 1H), 7.69

(dd, J = 11.2, 3.9 Hz, 1H), 4.56 (d, J = 7.4 Hz, 2H), 3.23 (d, J = 12.5 Hz,

2H), 2.75 (q, J = 12.3 Hz, 2H), 2.16 (ddd, J = 11.2, 7.5, 3.6 Hz, 1H), 1.71

(d, J = 12.8 Hz, 2H), 1.60−1.38 (m, 2H). ¹³C NMR (101 MHz, DMSO)

δ 177.83, 166.08, 149.81, 139.52, 134.30, 126.43, 125.97, 125.61, 118.45,

107.40, 57.13, 42.36, 32.69, 25.50. m/z MS (TOF ES⁺) C₁₆H₁₉N₂O₃

[MH]⁺calcd 287.1; found 287.2. LC-MS t_R: 3.70.

Ethyl 4-Oxo-1-(piperidin-4-ylmethyl)-1,4-dihydroquinoline-

3-carboxylate hydrochloride (30). Ethyl 1-((1-(tert-

butoxycarbonyl)piperidin-4-yl)methyl)-4-oxo-1,4-dihydroquinoline-3-

carboxylate (27) (342 mg, 0.83 mmol) was dissolved in DCM (2 mL)

and stirred at rt before addition of 4 M HCl in 1,4-dioxane (2 mL). The

mixture was stirred at rt for 1 h before addition of PE and attempted

collection of the resulting precipitate by ﬁltration (vacuum). However,

the product was found to be hygroscopic, so the solid was redissolved in

MeOH/DCM and combined with the ﬁltrate, before concentration

under reduced pressure, to give 325 mg of oﬀ-white solid (quantitative

yield). ¹H NMR (400 MHz, DMSO) δ 9.11 (d, J = 10.1 Hz, 1H), 8.90

(d, J = 9.9 Hz, 1H), 8.68 (s, 1H), 8.26 (dd, J = 8.0, 1.5 Hz, 1H), 7.86 (d, J

= 8.5 Hz, 1H), 7.83−7.74 (m, 1H), 7.49 (t, J = 7.5 Hz, 1H), 4.35 (d, J =

7.3 Hz, 2H), 4.23 (q, J = 7.1 Hz, 2H), 3.23 (d, J = 12.3 Hz, 2H), 2.86−

2.64 (m, 2H), 2.13 (dd, J = 9.1, 5.8 Hz, 1H), 1.70 (d, J = 12.8 Hz, 2H),

1.50 (td, J = 15.6, 3.6 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H). ¹³C NMR (101

MHz, DMSO) δ 172.79, 164.78, 149.74, 139.00, 132.72, 128.35, 126.46,

124.96, 117.54, 109.70, 59.82, 56.47, 42.38, 32.53, 25.58, 14.36. m/z MS

(TOF ES⁺) C₁₈H₂₃N₂O₃[MH]⁺calcd 315.2; found 315.2. LC-MS t_R:

3.67.

1-((1-Benzylpiperidin-4-yl)methyl)-4-oxo-1,4-dihydroquino-

line-3-carboxylic Acid (31). Ethyl 4-oxo-1-(piperidin-4-ylmethyl)-

1,4-dihydroquinoline-3-carboxylate hydrochloride (30) (80 mg, 0.23

mmol), K₂CO₃(79 mg, 0.57 mmol, 2.5 equiv), and BnBr (47 mg, 0.28

mmol, 1.2 equiv) were dispersed in MeCN (2 mL) and stirred at rt

overnight. MeCN was removed under reduced pressure, and the residue

dispersed in water, causing a precipitate to form. This was collected by

ﬁltration (vacuum) and washed with PE to give 60 mg of oﬀ-white solid.

This was puriﬁed by FCC (MeOH/DCM 0:100 to 10:90) to give 48 mg

of glassy solid. This was taken up in water/THF (1:1, 4 mL), and the

vessel purged with nitrogen gas, before addition of LiOH·H₂O (39 mg,

0.92 mmol, 4 equiv). The mixture was stirred at rt overnight before

diluting with water (20 mL) and washing with Et₂O (20 mL). The

aqueous layer was then neutralized with careful addition of 2 M HCl_(aq)

before extraction with EtOAc (3 × 20 mL). Finally, concentration of the

EtOAc layers gave 35 mg of oﬀ-white solid (40%). ¹H NMR (400 MHz,

DMSO) δ 15.23 (s, 1H), 9.01 (s, 1H), 8.40 (dd, J = 8.1, 1.5 Hz, 1H), 8.10

(d, J = 8.7 Hz, 1H), 7.97 (ddd, J = 8.7, 7.0, 1.6 Hz, 1H), 7.67 (t, J = 7.2

Hz, 1H), 7.39−7.15 (m, 5H), 4.50 (d, J = 7.4 Hz, 2H), 3.41 (s, 2H), 2.78

(d, J = 11.4 Hz, 2H), 1.95−1.62 (m, 3H), 1.56−1.27 (m, 4H). ¹³C NMR

(101 MHz, DMSO) δ 177.73, 166.07, 149.64, 139.54, 137.41, 134.27,

128.62, 128.10, 126.79, 126.36, 125.91, 125.51, 118.45, 107.26, 62.13,

58.12, 52.44, 34.95, 28.70. m/z MS (TOF ES⁺) C₂₃H₂₅N₂O₃[MH]⁺

calcd 377.2; found 377.2. LC-MS t_R: 4.16.

1-([1,1′-Biphenyl]-4-yl)-4-oxo-1,4-dihydroquinoline-3-car-

boxylic Acid (26b). Ethyl 1-([1,1′-biphenyl]-4-yl)-4-oxo-1,4-dihydro-

quinoline-3-carboxylate (25b) (68 mg, 0.18 mmol) was hydrolyzed

according to general procedure D. On extracting the reaction mixture

with Et₂O, the lithium salt of the product was found to suspend in the

organic layer. This was collected by ﬁltration (vacuum) before

suspending in 2 M HCl_(aq)(10 mL) and extracting the acidic suspension

with DCM (2 × 10 mL). Concentration of the DCM layers gave 35 mg

1

of pale-yellow solid (57%). H NMR (400 MHz, CDCl₃) δ 14.88 (s,

1H), 8.86 (s, 1H), 8.59 (dt, J = 5.1, 2.5 Hz, 1H), 7.93−7.79 (m, 2H),

7.76−7.64 (m, 3H), 7.60 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H), 7.57−7.39 (m,

5H), 7.27 (d, J = 8.1 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 178.96,

166.99, 148.68, 143.94, 141.27, 139.21, 139.10, 134.01, 129.32, 129.31,

128.65, 127.54, 127.42, 127.01, 126.61, 126.11, 118.68, 109.23. m/z MS

(TOF ES⁺) C₂₂H₁₆NO₃[MH]⁺calcd 342.1; found 342.2. LC-MS t_R:

5.72.

Ethyl 1-((1-(tert-Butoxycarbonyl)piperidin-4-yl)methyl)-4-

oxo-1,4-dihydroquinoline-3-carboxylate (27). Ethyl 3-(dimethy-

lamino)-2-(2-ﬂuorobenzoyl)acrylate (24) (500 mg, 1.88 mmol), N-

Boc-(4-aminomethyl)piperidine (404 mg, 1.88 mmol, 1 equiv), and

Cs₂CO₃(735 mg, 2.26 mmol, 1.2 equiv) were dissolved in dry DMF (10

mL) under an atmosphere of nitrogen gas. The mixture was heated at

100 °C (oil bath) for 6.5 h, after which time TLC analysis (EtOAc/PE

6:4) indicated disappearance of starting material. The mixture was

cooled to rt before diluting with ice water and stirring for 30 min. The

resulting precipitate was collected by ﬁltration (vacuum) to give 619 mg

of oﬀ-white solid (79%). ¹H NMR (400 MHz, CDCl₃) δ 8.56 (dd, J =

8.1, 1.5 Hz, 1H), 8.40 (s, 1H), 7.70 (ddd, J = 8.6, 7.1, 1.6 Hz, 1H), 7.46

(td, J = 7.6, 0.8 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 4.41 (q, J = 7.1 Hz,

2H), 4.34−3.84 (m, 4H), 2.63 (t, J = 12.2 Hz, 2H), 2.09 (ddt, J = 15.4,

7.6, 3.8 Hz, 1H), 1.60 (d, J = 17.3 Hz, 2H), 1.46 (s, 9H), 1.42 (t, J = 7.1

Hz, 3H), 1.37−1.10 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 174.32,

166.09, 154.70, 149.49, 139.07, 132.76, 129.51, 128.49, 125.34, 115.67,

110.94, 79.99, 61.17, 59.33, 43.40, 35.69, 29.83, 28.56, 14.60. m/z MS

(TOF ES⁺) C₂₃H₃₁N₂O₅[MH]⁺calcd 415.2; found 415.3. LC-MS t_R:

5.42.

1-((1-(tert-Butoxycarbonyl)piperidin-4-yl)methyl)-4-oxo-1,4-

dihydroquinoline-3-carboxylic Acid (28). Ethyl 1-((1-(tert-

butoxycarbonyl)piperidin-4-yl)methyl)-4-oxo-1,4-dihydroquinoline-3-

carboxylate (27) (250 mg, 0.60 mmol) was dispersed in THF/water

(1:1, 6 mL) and the ﬂask atmosphere purged with nitrogen gas.

LiOH·H₂O (101 mg, 2.41 mmol, 4 equiv) was added, and the mixture

stirred at rt overnight. TLC analysis (MeOH/DCM 5:95) indicated

conversion was complete. The mixture was diluted with water (20 mL)

and washed with Et₂O (30 mL), before careful acidiﬁcation (∼pH 4−5)

by dropwise addition of 2 M HCl_(aq), causing a precipitate to form. This

was collected by ﬁltration (vacuum) to give 202 mg of oﬀ-white solid

(87%). ¹H NMR (400 MHz, CDCl₃) δ 14.85 (s, 1H), 8.69 (s, 1H), 8.58

(dd, J = 8.1, 1.5 Hz, 1H), 7.87 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H), 7.68−7.54

(m, 2H), 4.19 (s, 4H), 2.63 (t, J = 12.0 Hz, 2H), 2.11 (dtd, J = 15.4, 7.7,

3.9 Hz, 1H), 1.83−1.52 (m, J = 13.5 Hz, 2H), 1.45 (d, J = 7.0 Hz, 9H),

1.32 (dt, J = 23.1, 13.5 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 178.62,

167.03, 154.66, 148.79, 139.62, 134.26, 127.79, 126.85, 126.48, 116.46,

108.82, 80.13, 59.94, 43.06, 36.02, 29.82, 28.54. m/z MS (TOF ES⁺)

C₂₁H₂₇N₂O₅[MH]⁺calcd 387.2; found 387.2. LC-MS t_R: 5.45.

4-Oxo-1-((1-(phenylcarbamoyl)piperidin-4-yl)methyl)-1,4-

dihydroquinoline-3-carboxylic Acid (32). Ethyl 4-oxo-1-(piperidin-

4-ylmethyl)-1,4-dihydroquinoline-3-carboxylate hydrochloride (30)

(80 mg, 0.23 mmol) was dispersed in DCM (2 mL), with addition of

DIPEA (0.060 mL, 0.35 mmol, 1.5 equiv), and stirred at rt before

addition of phenyl isocyanate (0.027 mL, 0.25 mmol, 1.1 equiv). The

mixture was stirred at rt overnight. Further phenyl isocyanate (0.2

equiv) with stirring at rt for a further 3 h before diluting the reaction

mixture with EtOAc (30 mL) and washing with 2 M NaOH_(aq)(20 mL),

S

dx.doi.org/10.1021/jm400540b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2 M HCl_(aq)(20 mL), and brine (20 mL). The organic layer was

concentrated and further puriﬁed by FCC (eluent MeOH/DCM 0:100

to 10:90) to give 88 mg of yellow solid. This was taken up in water/THF

(1:1, 4 mL) and the vessel purged with nitrogen gas, before addition of

LiOH·H₂O (39 mg, 0.92 mmol, 4 equiv). The mixture was stirred at rt

overnight before diluting with water (20 mL) and washing with Et₂O

(20 mL). The aqueous layer was then acidiﬁed (∼pH 4) with 2 M

HCl_(aq), causing a precipitate to form. This was collected by ﬁltration

(vacuum) to give 20 mg of oﬀ-white solid (22%). ¹H NMR (400 MHz,

DMSO) δ 15.24 (s, 1H), 9.05 (s, 1H), 8.46 (s, 1H), 8.42 (dd, J = 8.1, 1.4

Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H), 7.99 (ddd, J = 8.6, 7.1, 1.4 Hz, 1H),

7.69 (dd, J = 7.5 Hz, 1H), 7.44 (d, J = 7.6 Hz, 2H), 7.21 (dd, J = 7.9 Hz,

2H), 6.91 (dd, J = 7.3 Hz, 1H), 4.53 (d, J = 7.5 Hz, 2H), 4.13 (d, J = 13.4

Hz, 2H), 2.68 (t, J = 12.1 Hz, 2H), 2.24−1.97 (m, 1H), 1.54 (d, J = 10.9

Hz, 2H), 1.28 (ddd, J = 19.5, 9.8, 6.2 Hz, 2H). ¹³C NMR (101 MHz,

DMSO) δ 177.78, 166.09, 154.73, 149.68, 140.68, 139.58, 134.28,

128.23, 126.38, 125.94, 125.56, 121.54, 119.50, 118.47, 107.32, 57.81,

43.47, 35.12, 28.63. m/z MS (TOF ES⁺) C₂₃H₂₄N₃O₄[MH]⁺calcd

406.2; found 406.2. LC-MS t_R: 5.13.

Pharmacology. Radioligand Binding Assays. FlpInCHO cells

stably expressing the human M₁muscarinic acetylcholine receptor

(FlpInCHO-hM₁mAChR) were generated as described previously⁴¹

and maintained in DMEM containing 5% FBS under humidiﬁed

conditions at 37 °C and 5% CO₂. Radioligand binding experiments were

performed on whole cells an dseeded at 20000 cells per well in complete

DMEM into 96-well ISOPLATE TC plates. Next, cells were allowed to

grow for 8 h at 37 °C, then serum starved overnight. Following a wash

with phosphate-buﬀered saline (100 μL), the cells were then

resuspended in binding buﬀer (10 mM HEPES, 100 mM NaCl, 10

mM MgCl₂, pH 7.4). Assays were performed in a total volume of 200 μL

with a 1/10 dilution of drug for a duration of 4 h at 4 °C. Assays were

terminated by buﬀer removal followed by rapid washing, twice, with ice-

cold 0.9% NaCl (100 μL). Plates were allowed to dry inverted for 30

min; OptiPhase Supermix scintillation cocktail (100 μL) was added,

plates were sealed (TopSeal), and radioactivity was measured in a

MicroBeta²LumiJET microplate counter. All inhibition binding

experiments were performed with 0.1 nM [³H]-NMS (K_Dconcen-

tration) in the presence of increasing concentrations of ACh with or

without increasing concentrations of BQCA analogues.

where Y is percentage (vehicle control) binding, [A], [B], and [I] are the

concentrations of [³H]NMS, BQCA (or analogues), and ACh,

respectively, K_Aand K_Bare the equilibrium dissociation constants of

[³H]NMS and BQCA, respectively, K_Bis the equilibrium dissociation

constant of CCh, and α′ and α are the cooperativities (or analogues)

between BQCA and [³H]NMS or CCh, respectively. Values of α (or α′)

> 1 denote positive cooperativity, values <1 (but >0) denote negative

cooperativity, and values = 1 denote neutral cooperativity.

Functional data were analyzed using an operational model of

allosterism and agonism according to eq 2:^10,42

(

_A)ⁿ)

E = E τ A (K + αβ[B]) + τ [B]K

_A[ ]

m

B

( A K + K K + [B]K + α[A][B])ⁿ

(

[ ]

B

A

B

A

_A)ⁿ)

(2)

+ τ A (K + αβ[B]) + τ [B]K

(

_A[ ]

B

where E_mis the maximum possible cellular response, [A] and [B] are the

concentrations of orthosteric and allosteric ligands, respectively, K_Aand

K_Bare the equilibrium dissociation constant of the orthosteric and

allosteric ligands, respectively, τ_Aand τ_Bare operational measures of

orthosteric and allosteric ligand eﬃcacy (which incorporate both signal

eﬃciency and receptor density), respectively, α is the binding

cooperativity parameter between the orthosteric and allosteric ligand,

and β denotes the magnitude of the allosteric eﬀect of the modulator on

the eﬃcacy of the orthosteric agonist. In all instances, the equilibrium

dissociation constant of each agonist and BQCA (or analogues) was

ﬁxed to that determined from the binding assays.

AUTHOR INFORMATION

Corresponding Author

■

*For P.J.S.: phone, +61 0 3 9903 9542; E-mail, Peter.

Scammells@monash.edu. For A.C.: phone, +61 0 3 9903 9067;

E-mail, Arthur.Christopoulos@monash.edu.

Author Contributions

^§These authors contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

Intracellular Calcium Mobilization Assays. On the day prior to the

experiment, FlpInCHO-hM₁mAChR cells were seeded at 20000 cells

per well (100 μL ﬁnal volume per well) into clear 96-well plates in

DMEM containing 5% FBS and incubated 8 h under humidiﬁed

conditions at 37 °C and 5% CO₂prior serum starving the cells overnight.

On the day of the experiment, the assay plates were washed once with

100 μL per well of phosphate buﬀered saline. The cells were then

incubated with Ca²⁺assay buﬀer (150 mM NaCl, 2.6 mM KCl, 1.18 mM

MgCl₂·6H₂O, 10 mM D-glucose, 10 mM HEPES, 2.2 mM CaCl₂·2H₂O,

0.5% (w/v) bovine serum albumin (BSA), and 4 mM Probenecid, pH =

7.4) containing 1 μM Fluo-4-AM for 1 h under humidiﬁed conditions at

37 °C and 0% CO₂. Each assay plate was then loaded into a FLEXstation

(Molecular Devices Inc., Sunnyvale, CA) with its stock compound plate.

The FLEXstation measured ﬂuorescence over a 100 s time period using

485 nm excitation and 520 nm emission wavelengths and performed the

addition of drugs (1/10 dilution) at the 20 s time point. For all

interaction studies, ACh and BQCA (or its analogue) were coadded

(simultaneously) at 20 s. For each, the peak of maximum ﬂorescence

between 20 and 100 s was chosen and corrected to the baseline

(ﬂuorescence from 0 to 19 s), then normalized to 100 μM ATP as

internal control.

ACKNOWLEDGMENTS

■

This research was supported by Discovery grant DP110100687

of the Australian Research Council and Program grant 519461 of

the National Health and Medical Research Council (NHMRC)

of Australia. A.C. and P.M.S. are Principal Research Fellows of

the NHMRC.

ABBREVIATIONS USED

■

BQCA, benzyl quinolone carboxylic acid (1-(4-methoxybenzyl)-

4-oxo-1,4-dihydroquinoline-3-carboxylic acid); CHO, Chinese

hamster ovary; DIPEA, N,N-diisopropylethylamine; FCC, ﬂash-

column chromatography; PE, petroleum ether 40−60; PLC,

preparative layer chromatography; TEA, triethylamine

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Synthesis and pharmacological profiling of analogues of benzyl quinolone carboxylic acid (BQCA) as allosteric modulators of the M1 muscarinic receptor

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