New Scalable Synthetic Routes to ELQ-300, ELQ-316, and Other Antiparasitic Quinolones

Article abstract of DOI:10.1021/acs.oprd.1c00099

The endochin-like quinolone (ELQ) compound class may yield effective, safe treatments for a range of important human and animal afflictions. However, to access the public health potential of this compound series, a synthetic route needed to be devised, which would lower costs and be amenable to large-scale production. In the new synthetic route described here, a substituted β-keto ester, formed by an Ullmann reaction and subsequent acylation, is reacted with an aniline via a Conrad-Limpach reaction to produce 3-substituted 4(1H)-quinolones such as ELQ-300 and ELQ-316. This synthetic route, the first described to be truly amenable to industrial-scale production, is relatively short (five reaction steps), does not require palladium, chromatographic separation, or protecting group chemistry, and may be performed without high vacuum distillation.

Full text of DOI:10.1021/acs.oprd.1c00099

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Article
New Scalable Synthetic Routes to ELQ-300, ELQ-316, and Other
Antiparasitic Quinolones
Sovitj Pou,*,∇ Rozalia A. Dodean, Lisa Frueh, Katherine M. Liebman, Rory T. Gallagher, Haihong Jin,
Robert T. Jacobs, Aaron Nilsen, David R. Stuart, J. Stone Doggett, Michael K. Riscoe,
and Rolf W. Winter*,∇
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ABSTRACT: The endochin-like quinolone (ELQ) compound class may yield effective, safe treatments for a range of important
human and animal afflictions. However, to access the public health potential of this compound series, a synthetic route needed to be
devised, which would lower costs and be amenable to large-scale production. In the new synthetic route described here, a substituted
β-keto ester, formed by an Ullmann reaction and subsequent acylation, is reacted with an aniline via a Conrad−Limpach reaction to
produce 3-substituted 4(1H)-quinolones such as ELQ-300 and ELQ-316. This synthetic route, the first described to be truly
amenable to industrial-scale production, is relatively short (five reaction steps), does not require palladium, chromatographic
separation, or protecting group chemistry, and may be performed without high vacuum distillation.
KEYWORDS: antimalarial, antiparasitic, ELQ-300, ELQ-316, practical synthesis, process development, Conrad−Limpach reaction,
Ullmann reaction
malaria is a particularly serious and prevalent human disease.
In 2019 alone, it afflicted 229 million people worldwide and
caused an estimated 409,000 deaths.11 ELQ-300, in the form
of a prodrug ELQ-331, has recently been accepted as a
preclinical candidate by the Medicines for Malaria Venture for
potential use in the prevention and treatment of malaria.12
ELQ-316 and its prodrugs, on the other hand, have the
greatest potency against T. gondii and Babesia microti.
Toxoplasmosis may have infected up to one-third of all
humankind; this infection can be serious for immunocompro-
mised individuals and can cause harm to the fetus when
contracted during pregnancy.13,14 Babesiosis affects both
humans and livestock and is, together with neosporosis and
besnoitiosis, a significant problem for livestock husbandry.2−4
If compounds in this series are to become practical drug
candidates, a synthetic route is needed that is amenable to
industrial-scale production. Historically, 3-alkyl-substituted
4(1H)-quinolones, such as endochin, have been synthesized
by the reaction of a substituted β-keto ester with an aniline via
the Conrad−Limpach reaction (Scheme 1).15,16 Endochin
itself is easily synthesized by the Conrad−Limpach reaction, a
sequence of two reactions. In the first stage, 2-n-heptylaceto-
acetic ester is condensed with meta-anisidine to form β-
anilinocrotonate; in the second stage, thermal cyclization
INTRODUCTION
■
When endochin (Figure 1) was first synthesized in 1940 by
Andersag and colleagues, its high activity in the avian malaria
model led to the hope that a new and promising class of
antimalarials had been discovered. Endochin and a series of
derivatives were tested against malaria in humans, but the
results were disappointing and the compounds were not
pursued further as antimalarials.1 This compound class was
reexamined in the early 2000s, when a series of new endochin
derivatives were discovered, which were curative of patent
malaria in mice. The outstanding representatives of this new
generation of endochin-like quinolones (ELQs) bore a methyl
group at position 2, a diphenyl ether substituent at position 3,
and additional substituents in the second ring of the quinolone
system (Figure 1). These ELQs demonstrated high antima-
larial potency in vitro and in vivo, parasite selectivity, chemical
and metabolic stability, desirable pharmacokinetics, and low
mammalian cell toxicity. In addition to their antimalarial
activity, compounds in the series were later found to be highly
active against other Apicomplexa, for which satisfactory
treatments are urgently needed. These include various Babesia
species (affecting humans, cattle, horses, and dogs),2 Theileria
equi (horses),2 Neospora caninum (cattle, sheep, goats, deer,
horses, and dogs),3 Besnoitia besnoiti (cattle),4,5 and
Toxoplasma gondii (humans, sheep, goats, cats, and marine
mammals).6−9 Finally, an ELQ compound has been found to
have a potent, low-dose inhibitory effect on the nematode
Echinococcus multilocularis, a fox-transmitted tapeworm that
may be fatal to its hosts, including humans.10
Received: March 23, 2021
With favorable properties and broad-spectrum activity, the
ELQ compound class may yield effective, safe treatments for a
range of important human and animal afflictions. Of these,
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© XXXX American Chemical Society
A

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Figure 1. Structures of endochin, ELQ-300, ELQ-316, and ELQ-331.
a
Scheme 1. Original Synthesis of Endochin
a
Reaction (a), 1: catalytic H+ and benzene, reflux; 2: Dowtherm A, 250 °C, 40−50% yield.
a
Scheme 2. Optimized Original Synthesis of ELQ-300
a
Reaction (a): 1,4-Dibromobenzene, CuCl, K2CO3, DMG, and DMF, 160 °C, 90 min, 60−80%; (b): Pd(dppf)Cl2, bis(pinacolato)diboron, KOAc,
and DMF, 90 °C, 98%; (c), 1: ethyl acetoacetate, cat. p-TsOH, and PhH, reflux, and 2: Dowtherm A, 250 °C, 68%; (d): I2, NaHCO3, and MeOH,
96%; (e): EtI, K2CO3, and DMF, 81%; (f): Pd(dppf)Cl2, aqueous K2CO3, and DMF, 90 °C, 70%; (g): aqueous HBr and AcOH, 90 °C, 95%; for
synthetic details, see Supporting Information.
produces endochin, usually in Dowtherm A boiling at 250 °C.
Of the two isomers formed in the second step, endochin, the 7-
methoxy isomer, crystallizes out of the reaction mixture upon
cooling, while the 5-methoxy isomer remains dissolved. The
yield of endochin is around 40−50% with this straightforward
procedure.1,17,18 Alternative cyclization methods for 4(1H)-
quinolones have also been explored.19−21
3-Aryl-substituted 4(1H)-quinolones, such as ELQ-300,
have been synthesized by the reaction of 4(1H)-quinolone
with a reactive aryl moiety primarily via a Suzuki−Miyaura
reaction.22−24 However, 4(1H)-quinolones are known to be
sparingly soluble in organic solvents and water and are difficult
to isolate by chromatography. As a result, reactions that
produce a mixture of 3-aryl-4(1H)-quinolones have proven
difficult to separate and generally give poor yields of pure
4(1H)-quinolone products.
a diaryl ether side chain 4 via an Ullmann reaction (reaction a),
was designed to allow for late-stage structural variation at the
3-position using a Suzuki−Miyaura reaction (reaction f). The
synthesis presented in Scheme 2 is a version of the originally
published synthesis that was optimized for a larger scale, the
details of which can be found in the Supporting Information.
We discovered that conversion of 3-halo-4(1H)-quinolone 8 to
the corresponding 4-ethoxy-3-halo-quinoline 9 provided a
protected 4(1H)-quinolone intermediate that performed well
in a Suzuki−Miyaura reaction and provided a product (10)
that could be isolated by chromatography and then readily
converted back to the desired 4(1H)-quinolone, such as ELQ-
300.6,25−27 However, though this route has been used to
prepare hundreds of grams of various ELQ compounds, it is
not ideal for industrial-scale production because it is relatively
long (seven reaction steps), requires the use of a somewhat
expensive palladium catalyst, and involves high vacuum
distillation (compound 4), at least two chromatographic
The original route to synthesize ELQ-300, involving in
parallel the formation of 4(1H)-quinolone 7 via a Conrad−
Limpach reaction (Scheme 2, reaction c) and the formation of
B
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separations (compounds 5 and 10), and multiple recrystalliza-
tions.
GC−MS and was determined to be complete when 11 was
consumed. The product 12 was isolated with a yield of 48−
60% by high vacuum distillation, a method that facilitated the
nearly complete removal of copper. The copper content of 12
using these conditions was <21 ppm, determined by
inductively coupled plasma−atomic emission spectroscopy
(ICP-AES). Other copper couplings were explored, but they
required harsh conditions (e.g., copper metal) or were not
scalable (e.g., copper(II) acetate).33 The copper(II) acetate
reaction (not shown) used in the original ELQ synthetic
method25,26 did not scale well because it was difficult to
maintain a dry, well-oxygenated, homogenous copper(II)
acetate mixture under bulk conditions.
Additional complications arose when performing large-scale
versions of the reactions shown in Scheme 2. The original
iodination reaction (iodine, potassium iodide and n-butyl-
amine in DMF) did not scale well, so it was replaced with
reaction (d) that required at least 72 h and did not progress
much beyond 96% completion. During the formation of the 4-
O-ethyl ether (reaction e), N-ethylation occurred, resulting in a
small amount of an N-ethylated side product that was difficult
to separate. Unwanted reduction of the 3-iodo group during
the Suzuki reaction (f) resulted in a small amount of reduced
side product that was also difficult to separate by
chromatography and was inseparable after the quinolone was
re-formed in reaction (g). Finally, removal of the 4-O-ethyl
ether protecting group (reaction g) required relatively harsh
reaction conditions (HBr in acetic acid (AcOH) at 90 °C) and
long reaction times (>48 h), which can result in demethylation
of the 7-OMe ether.
As part of our effort to establish a synthetic route to
sythesize ELQ-300 which would be amenable to industrial-
scale preparation, we returned to Andersag’s original method
by which 3-substituted 4(1H)-quinolones were prepared from
substituted β-keto esters using the Conrad−Limpach reaction
as the final step. Recently, the synthesis of 3-diaryl ether
4(1H)-quinolones via substituted β-keto esters has been
revisited.28,29 However, these routes were not shown to be
amenable to industrial-scale production. Our optimized
approach, presented here, is relatively short (five reaction
steps), does not require palladium, involves no chromato-
graphic separation, and avoids high vacuum distillation.
Additionally, it requires no protecting group chemistry because
the poorly soluble 4(1H)-quinolone is not formed until the
final reaction step.
In order to avoid high vacuum distillation, which may be
problematic on an industrial scale, we attempted to find an
alternative method for the preparation of diaryl ether 12. By
substituting the corresponding carboxylic acid 13 for the ester
11 in the Ullmann reaction, we obtained a solid intermediate,
diaryl ether 14, which could be purified without distillation
(Scheme 3, reaction b): 4-bromophenylacetic acid 13 was
allowed to react with 4-trifluoromethoxyphenol 3 in the
presence of a catalytic amount of CuCl, DMG, and K2CO3 in
DMF at 160 °C. After 3.5 h, 13 was completely consumed as
determined by GC−MS. During the workup, the copper
catalyst was removed by the addition of ammonium
pyrrolidinedithiocarbamate (APDTC) according to the
procedure described by Gallagher and Vo.34 The solid
carboxylic acid 14 was easily purified by treatment with hot
water to remove most of the primary impurity (starting
material, phenol 3) and water-soluble residues, and was
1
obtained in 78% yield with >95% purity by GC−MS and H-
NMR. Esterification of 14 with ethanol in the presence of
catalytic hydrochloric acid over 18 h afforded the desired diaryl
ether 12 in 91% yield after removal of ethanol in vacuo and
passage of the crude product through a silica gel plug, which
was rinsed with 3:1 hexanes/ethyl acetate. The product 12 was
RESULTS AND DISCUSSION
■
1
at least 95% pure as observed by GC−MS and H-NMR and
The first step of the synthesis of diaryl ether 12 is an Ullmann
reaction.30 The copper-mediated coupling of 4-trifluorome-
thoxyphenol 3 and ethyl 2-(4-bromophenyl)acetate 11 with
copper(I) chloride (CuCl), N,N-dimethylglycine (DMG) as a
copper(I) chloride chelator, and potassium carbonate
(K2CO3) in DMF at 160 °C for 90 min afforded the diaryl
ether 12 (Scheme 3).31,32 The reaction was monitored by
was suitable for use in the next step without further
purification. The overall yield of this two-step process was
71%. The successful preparation of the solid intermediate 14
and its conversion to the high-purity key intermediate 12 show
that this ELQ compound synthesis can be performed without
high vacuum distillation.
It was important to verify that the copper content of 12
obtained via the carboxylic acid route (from 13) was as low as
the copper content of 12 obtained via the ester route (from
11). By an ICP-AES analysis, it was determined that the
copper content of 14 after treatment with APDTC was 73
ppm. After esterification and passage through a silica gel plug,
the copper content of 12 was <12 ppm. Thus, the copper
content of 12 obtained from 13 was no higher than the copper
content of 12 (<21 ppm) obtained from 11 after high vacuum
distillation.
Scheme 3. Two Alternate routes for the Ullmann Synthesis
of Ethyl 2-(4-(4-(trifluoromethoxy)phenoxy)phenyl)acetate
a
12
The synthesis of diaryl ether 12 using hypervalent iodine
was also explored as a possible alternative approach to the
synthesis of ELQ-300. This approach would avoid the use of
copper in the synthetic sequence, as iodine(III) mediates the
formation of the diaryl ether C−O bond. A similar strategy has
been used in previous syntheses of ELQ-300 with symmetrical
diaryliodonium salts.28,29 In our case, we considered the use of
an unsymmetrical diaryliodonium salt in which a relatively
inexpensive aryl auxiliary replaces half of the desired aryl
group, which would otherwise go to the waste stream, in the
a
Reaction (a): CuCl, K2CO3, DMG, and DMF, 160 °C, 90 min, 48−
60% yield; (b), 1: CuCl, K2CO3, DMG, and DMF, 160 °C, 3.5 h, and
2: APDTC, 78%; (c) catalytic HCl and EtOH, 18 h, 91%.
C
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a
Scheme 4. Hypervalent Iodine Synthesis of Ethyl 2-(4-(4-(trifluoromethoxy)phenoxy)phenyl)acetate 12
a
Reaction (a), 1: meta-Chloroperoxybenzoic acid, para-toluenesulfonic acid, and acetonitrile, 65 °C, 40 min, and 2: 1,3,5-trimethoxybenzene, 5
min, 84% yield; (b): ethyl 2-(4-hydroxyphenyl)acetate, K2CO3, and toluene, 55 °C, 2.5 h, 54% (0.5 mmol scale), 43% (3.6 mmol scale), and 48%
(10 mmol scale).
coupling with an appropriate phenol. Aryl(TMP)iodonium
salts (TMP = 2,4,6-trimethoxyphenyl) were a logical choice
given their established chemoselective aryl transfer,35 scalable
synthesis,36 and use in C−O coupling,37 and the low cost of
the auxiliary precursor, 1,3,5-trimethoxybenzene. Moreover,
process safety for the synthesis of aryl(TMP)iodonium salts
has been evaluated, including thermal stability of intermedi-
ates, and the synthesis was deemed safe within the operating
temperature window.32 Based on the cost and availability of
starting materials, aryl iodide 15 was selected as one of the
coupling partners (Scheme 4). Large-scale oxidation of 15
proceeded smoothly under slightly modified literature
conditions to deliver >200 g of unsymmetrical diaryliodonium
salt 16 in 84% yield. The reaction of 16 with ethyl 2-(4-
hydroxyphenyl)acetate in the presence of potassium carbonate
as a base provided access to 12 via metal-free C−O coupling in
moderate yield on various scales (43−54%, 48% average). The
overall yield of 12 using the iodonium salt route is lower than
the copper-catalyzed route (ca. 36−45% compared to 48−
71%), and the impurity profile required a column chromatog-
raphy step that was not required in the copper-catalyzed route.
Because of these disadvantages the iodonium route was not
pursued further. Additionally, other phenol coupling partners
that would provide direct access to 18 or a methyl ketone
analog of 12 were considered but led to complex mixtures of
products in coupling with 16.
proceeded only when freshly prepared LiHMDS was used to
deprotonate 12 at −20 °C. Preliminary attempts to acylate 12
suggested that C-acylation occurs initially followed by rapid O-
acylation of the newly introduced acetyl group, producing enol
acetate 17. If the reaction is quenched at −20 °C immediately
after addition of acetic anhydride, C-acylated 18, bis-acylated
17, and the starting material 12 can be detected by thin-layer
chromatography (TLC). This finding suggested that it was
necessary to use excess acetic anhydride with freshly prepared
LiHMDS in tetrahydrofuran (THF) to force the reaction to
consume all of the starting material 12, thereby forming only
the O-acylated β-keto ester 17. Under these conditions, 17 was
obtained in quantitative yield and in sufficient purity (>95%)
to be used in the next step without further purification.
Compound 17 exists as a mixture of equally reactive E- and Z-
isomers, which can be isolated by chromatography. The
identity of the Z-isomer was determined by 2D NOESY NMR,
and the percent of the Z-isomer was estimated to be 90−95%
1
using GC−MS and H-NMR.
Other acylating reagents were also explored. Ethyl acetate
and acetylimidazole have been reported to give 2-phenylaceto-
acetic esters in one step,38 and indeed, our model reactions
using ethyl 2-(4-bromophenyl)acetate provided direct access
to the corresponding β-keto ester. Unfortunately, reactions of
these reagents with diaryl ether 12 proved to be more complex.
In the case of ethyl acetate, we observed no reaction. In the
case of acetylimidazole, β-keto ester 18 was formed
predominantly along with some of the bis-acylated product
We next explored the most challenging part of this route, the
formation of the key intermediate, the substituted β-keto ester
18. The acylation reaction (Scheme 5) did not proceed using
strong bases such as NaH, n-butyllithium, lithium diisopropy-
lamide, and commercially prepared lithium hexamethyldisila-
zide (LiHMDS) solution. We found that the acylation
1
17 and some other uncharacterized products as shown by H-
NMR. Under these conditions, in order to obtain β-keto ester
18 in sufficient purity for the next reaction, chromatography
would be required. Thus, in our case, we found that acylation
with acetylimidazole is less desirable and may not be suitable
for large-scale synthesis, especially considering the relative ease
of work-up and low cost associated with acetic anhydride.
We found that it is possible to quantitatively convert the bis-
acylated 17 to the desired β-keto ester 18 using catalytic para-
toluenesulfonic acid (p-TsOH) in AcOH. The β-keto ester 18
exists in both keto and enol forms with a keto/enol ratio of
approximately 7:3 as determined by 1H-NMR. Since the β-keto
Scheme 5. Synthesis of Ethyl 3-oxo-2-(4-(4-
(trifluoromethoxy)phenoxy)phenyl) butanoate, β-keto ester
a
18
1
ester 18 could not be detected by GC−MS, TLC and H-
NMR were used to assess purity and characterize the product.
The isolated β-keto ester 18 was sufficiently pure for use in the
next reaction and already contained a 10% mole fraction of p-
TsOH, which is a suitable catalyst for the subsequent acid-
catalyzed condensation with anilines.
The target ELQ compounds were prepared from β-keto
ester 18 using a Conrad−Limpach reaction, which comprises a
Schiff base formation followed by high-temperature cyclization
(Scheme 6).15,16 Continuous removal of water using a Dean−
Stark trap and a water-carrying solvent afforded the desired
Schiff bases 20a−d via condensation with anilines 19a−d.
a
(a) LiHMDS, Ac2O, and THF, −20 °C to RT over 16 h, 99%; (b)
10% p-TsOH and AcOH, 100 °C, 60−90 min, 99%.
D
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a
Scheme 6. Synthesis of a Series of ELQ Compounds from β-Keto Ester Intermediate 18
a
(a) 10% p-TsOH, cyclohexane, and reflux, 24−72 h; (b) Dowtherm A, 230 or 250 °C, 0.5 h.
Traditionally, benzene has been used for such reactions.1,17,18
However, since benzene is not suitable for pharmaceutical
preparations, cyclohexane (which boils at nearly the same
temperature and also forms an azeotrope with water) was used
as an alternative. Anilines 19a−d were allowed to react with β-
keto ester 18 in the presence of catalytic p-TsOH in refluxing
cyclohexane with a Dean−Stark trap to give the imines 20a−d,
which were then used without further purification in the final
cyclization step.
The cyclization step of the Conrad−Limpach reaction is
classically performed at high temperatures (230 or 250 °C).39
We compared these two temperatures for a series of ELQ
derivatives and investigated the effect of a lower temperature of
200 °C for ELQ-300 (Table 1). All temperatures investigated
followed by cyclization at 250 °C. Under these conditions, the
reaction proceeded as expected and gave ELQ-300 in a yield of
57.1%, higher than at the 1−10 g (2−20 mmol) scale. The
1
crude product from this reaction was >99% pure by H-NMR
and HPLC. At all temperatures investigated, only a negligible
amount of the 6-chloro-5-methoxy regioisomer of ELQ-300
1
was formed, which was below the limit of detection by H-
NMR. To allow detection and quantification of the ELQ-300
regioisomer, the product was converted to its corresponding 4-
chloro derivative using phosphorus oxychloride (POCl3) and
analyzed by GC−MS. The results confirmed that a negligible
amount (<0.5%) of the regioisomer was formed. In this study,
when the reactions were performed at a 1−10 g (2−20 mmol)
scale, 230 °C appeared to be the optimal cyclization
temperature as it gave the highest yield. However, a higher
yield was obtained when the reaction was scaled up to 50.0 g
(131 mmol) of β-keto ester 18 at 250 °C. It is important to
emphasize that since starting materials 11 and 13 may also be
acetylated (Scheme 3, reaction b), diaryl ether 12 must be
obtained in a pure form without contamination from 11 or 13
in order to afford high purity of the final ELQ products. Other
solvents may work in this reaction.40 Tsoung et al. have shown
that 4(1H)-quinolones can be prepared by performing a
Conrad−Limpach reaction in a flow reactor, which indicates
that this approach should be amenable to implementation on
an industrial scale.41,42
As shown in Table 1, the β-keto ester 18 was found to be
equally useful as an intermediate for the synthesis of a series of
ELQ compounds in addition to ELQ-300, including ELQ-271,
ELQ-316, and ELQ-400.25,26,43 In all cases, the condensation
of β-keto ester 18 with the corresponding aniline 19a, 19c, and
19d, followed by thermal cyclization, afforded the desired
quinolones in relatively good yield. In the case of ELQ-316,
the reaction proceeded well using 115.2 g of β-keto ester 18 to
form the corresponding Schiff base 20c followed by cyclization
at 250 °C. Similar to the case of ELQ-300, in the cyclization of
20c to form ELQ-316, only trace formation of the undesired
regioisomer was detected by GC−MS of the corresponding 4-
chloro derivative. The copper content of this sample was below
the limit of detection (<17 ppm), as with 12 obtained in
Scheme 3 (<21 ppm), suggesting that removal of copper is not
a concern in this reaction sequence.
Table 1. Conrad−Limpach Reaction Conditions for the
Synthesis of a Series of ELQ Compounds
a
b
c
ELQ #
amount of 14 cyclization temperature yield
purity
ELQ-300
ELQ-300
ELQ-300
ELQ-300
ELQ-316
ELQ-316
ELQ-316
ELQ-400
ELQ-400
ELQ-271
131 mmol
21.5 mmol
250 °C
250 °C
230 °C
200 °C
250 °C
250 °C
230 °C
250 °C
230 °C
250 °C
57%
>99%
>99%
>99%
>99%
>99%
97%
d
43%
48%
30%
54%
53%
50%
32%
33%
63%
d
8.3 mmol
1.8 mmol
d
302 mmol
13.1 mmol
d
d
7.6 mmol
7.8 mmol
7.3 mmol
98%
d
>99%
>99%
>99%
d
d
8.1 mmol
a
Yields are calculated from the originally used 14, as the intermediate
Schiff bases 20a−d were used without purification or characterization
b
in the subsequent cyclization step. Purity determined by reversed-
c
d
phase HPLC. Yield and purity of recrystallized ELQ-300. Back-
calculated from the weight of Schiff bases 20a−d used.
led to acceptable yields of products with minimal impurity;
however, the reaction at 200 °C led to a somewhat lower yield
of ELQ-300. Although aniline 19b was used as the limiting
reagent, it was not completely consumed even after extending
the reaction time to 72 h. Excess aniline 19b, being poorly
soluble in cyclohexane, was partially recoverable by filtration.
To demonstrate the potential scalability of this synthetic route,
we performed the reaction using 50.0 g (131 mmol) of β-keto
ester 18 and 2.25 g of p-TsOH to form the Schiff base 20b,
E
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a
Scheme 7. New Efficient Synthesis of ELQ-300
a
Reaction (a) 1: CuCl, K2CO3, DMG, and DMF, 160 °C, 210 min, and 2: APDTC, 78%; (b) catalytic HCl and EtOH, 91%; (c) LiHMDS, Ac2O,
and THF, −20 °C to RT over 16 h, 100%; (d) 10%, p-TsOH and AcOH, 100 °C, 60−90 min, 100%; (e) 1: 10% p-TsOH and cyclohexane, reflux,
72 h, and 2: Dowtherm A, 230 or 250 °C, 30 min, 57%
At this early stage in development, it is difficult to obtain an
accurate estimate of the cost of goods for the industrial
production of ELQ-300 and ELQ-316. However, using ELQ-
300 as an example, it is possible to compare the relative cost of
the original synthesis (Scheme 2) to the new efficient synthesis
(Scheme 7). On one hand, the original synthesis comprises
seven reaction steps and has a 36% yield over its longest linear
sequence (five reaction steps), which does not take into
account the yield of the two non-linear steps. Additionally, it
requires the use of a relatively expensive palladium catalyst, at
least two chromatographic separations, and high vacuum
distillation. On the other hand, the new efficient synthesis
comprises five reaction steps and has a 41% overall yield. It
requires no expensive reagents, such as palladium, and no
chromatographic separations. Based on length, relative cost of
reagents, and simplicity of purification, we estimate the cost of
the new efficient synthesis of ELQ compounds to be 10−20%
of the cost of the original synthesis.
synthesis appears to be readily scalable and promises to
considerably reduce the cost of goods. This is especially
important in the case of ELQ-300 production given that it is
the active component of the preclinical candidate antimalarial
prodrug, ELQ-331, and the fact that many malaria-endemic
countries are among the world’s most impoverished.
In addition to the advantages described above, this efficient
synthetic route avoids the relatively harsh conditions (HBr in
AcOH) required to deprotect the 4-O-ethyl ether quinoline
intermediate in the previously described synthesis (Scheme
2).25,26 These conditions regularly resulted in the partial
demethylation of the 7-OMe ether of both ELQ-300 and
ELQ-316. Because the milder conditions of this new route do
not result in demethylation of the 7-OMe ether, we are
currently exploring using the new route to synthesize
quinolone analogs with sensitive functional groups that were
not accessible by the original route.
It is also noteworthy that ELQ-316 can be made efficiently
and with high purity and yield using this new late-stage
cyclization synthetic pathway. While ELQ-316 exhibits slightly
less antimalarial activity than ELQ-300 in vivo, it has superior
antiparasitic activity against a broader range of Apicomplexan
protozoan species including T. gondii and B. microti, which also
cause severe and potentially fatal disease in humans. Recent
studies show that both ELQ-300 and ELQ-316 are also active
against a range of Apicomplexan parasites of importance to
veterinary medicine.2,4,5
CONCLUSIONS
■
We have successfully developed an efficient late-stage
cyclization synthetic route to synthesize ELQ-300 and other
structurally related ELQ derivatives (Scheme 7), bypassing the
more expensive palladium-catalyzed Suzuki−Miyaura reaction
originally developed by our lab to explore the initial structure−
activity relationship of quinolone and side chain modifications
to the core scaffold (Scheme 2).25,26 Additionally, we have
found an efficient synthesis route to the key intermediate diaryl
ether 12 that does not require high vacuum distillation. The
overall yield in this two-step synthesis (71%, Scheme 3) is
better than the yield of the single-step synthesis of 12 involving
high vacuum distillation (48−60%, Scheme 3). The key to the
success of this efficient late-stage cyclization scheme is our
ability to synthesize the bis-acylated product 17 and then to
selectively convert it to the desired intermediate β-keto ester
18 using p-TsOH as a catalyst. This synthetic route is
inexpensive and contains five scalable steps, with purification
via a single recrystallization (as needed) and no chromatog-
raphy or distillation. ELQ-300 and other ELQ compounds are
obtained in a pure form with an overall yield of ca. 30−40%.
Further, the steps have not been fully optimized at an industrial
scale, and it is believed that improvements may be achieved
with the adjustment of reaction conditions (e.g., time and
temperature), solvents, and equipment. Compared to the
synthetic route to ELQ-300 and other ELQ compounds
originally described by us,25,26 this late-stage cyclization
METHODS
■
Unless otherwise stated, all chemicals and reagents were from
Sigma-Aldrich Chemical Company in St. Louis, MO (USA),
Combi-Blocks in San Diego (CA), or TCI America, Portland
(OR) and were used as received. Melting points were obtained
using an Optimelt Automated Melting point system from
Stanford Research Systems, Sunnyvale, CA (USA). Analytical
TLC utilized Merck 60F-254,250 micrometer precoated silica
gel plates, and spots were visualized under 254 nm UV light.
GC−MS was performed using an Agilent Technologies 7890B
gas chromatograph (30 m, DBS column set at either 100 or
200 °C for 2 min then at 30 °C/min to 300 °C with inlet
temperature set at 250 °C) using an Agilent Technologies
5977A mass-selective detector operating at 70 eV. Flash
chromatography on silica gel was performed using an Isolera
One flash chromatography system from Biotage, Uppsala,
Sweden. 1H-NMR spectra were obtained using a Bruker AMX-
400 NMR spectrometer operating at 400.14 MHz unless
F
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specified in the text. The NMR raw data were analyzed using
iNMR Spectrum Analyst software. 1H chemical shifts are
reported in parts per million (ppm) relative to an internal
(TMS) or residual solvent peak. Coupling constant values (J)
are reported in Hertz (Hz). Decoupled 19F operating at 376
MHz was also obtained for compounds containing fluorine
(data not shown). HPLC analyses were performed using an
Agilent 1260 Infinity instrument with detection at 254 nm and
a Phenomenex, Luna 5 μm C8(2) 100 Å reversed-phase LC
column (150 × 4.6 mm) at 40 °C, eluting with a gradient of A/
B at 25%/75% to A/B at 25% to 90% (A: 0.05% formic acid in
water, B: 0.05% formic acid in methanol). High-resolution
mass spectrometry (HRMS) was performed using a high-
resolution (30,000) Thermo LTQ-Orbitrap Discovery hybrid
mass spectrometry instrument (San Jose, CA) equipped with
an electrospray ionization source operating in the positive or
negative ion mode. The Orbitrap was externally calibrated
prior to data acquisition allowing accurate mass measurements
for [M + H]+ ions to be obtained within 4 ppm. Copper
content was determined by inductively coupled plasma−
atomic emission spectroscopy (ICP-AES) by Galbraith
Laboratories, Knoxville, TN.
19.9 g, 111.6 mmol, 1.2 equiv). The reaction mixture was
heated to 50 °C, degassed for 10 min, and purged with argon
for 5 min. The temperature was then raised and maintained at
160 °C for 3.5 h under argon, when 2-(4-bromophenyl)acetic
acid was consumed as shown by GC−MS analysis. The crude
mixture was cooled to room temperature, and 200 mL of water
and ammonium pyrrolidinedithiocarbamate (APDTC) (5.0 g,
30.6 mmol, 2.2 equiv with respect to CuCl used) were added
followed by stirring at 50 °C for 1 h. The resulting slurry was
passed through a Celite pad (50 g) and washed thoroughly
with water (250 mL). To the filtrate were added ice (150 g)
and concentrated HCl (12.1 N, 55 mL) until the pH was
around 2. The light yellow solid that precipitated out of the
solution was filtered, washed with hot water (100 mL), boiled
in 300 mL of water, cooled with 200 g of ice, filtered again, and
air dried. The above treatment with water was used to remove
water soluble residues and some of the remaining phenol 3.
The resulting 2-(4-(4-(trifluoromethoxy)phenoxy)phenyl)-
acetic acid 14 (22.7 g, 78% yield) was used in the subsequent
step without further purification. GC−MS showed one major
peak with M+ = 312 (53%), 267 (100%). 1H-NMR (400 MHz;
CDCl3): δ 7.29 (d, J = 8.4 Hz, 2H), 7.20 (dd, J = 9.1, 0.8 Hz,
2H), 7.04−6.99 (m, 4H), 3.67 (s, 2H). Copper <73 ppm.
HRMS calculated for C15H11F3O4 [M + H]+ = 313.0682,
observed for [M + H]+ = 313.0678, and calculated for
C15H11F3O4 [M + Na]+ = 335.0504, observed for [M + Na]+ =
335.0497. The product was at least 95% pure as shown by
Ethyl 2-(4-(4-(Trifluoromethoxy)phenoxy)phenyl)-
acetate (12) from 11 and 3. A round-bottom flask, stir
bar, and potassium carbonate were oven dried at 150 °C for at
least 24 h prior to use. Copper chloride (CuCl) (12.2 g, 123
mmol, 0.15 equiv), DMG (8.5 g, 82.3 mmol, 0.1 equiv), and
dimethylformamide (DMF, 200 mL) were placed into the hot
round-bottom flask and degassed for 20 min at 50 °C under
house vacuum, while stirring. To the intensely blue-colored
catalyst mixture were added K2CO3 (227 g, 1.65 mol, 2.0
equiv), ethyl 2-(4-bromophenyl)acetate 11 (200 g, 0.823 mol,
1.0 equiv) in 200 mL of degassed DMF, and 4-
(trifluoromethoxy)phenol 3 (161.7 g, 0.908 mol, 1.1 equiv).
The reaction mixture was heated to 50 °C, degassed for 10 min
under house vacuum, and purged with argon for 5 min. The
temperature was then raised and maintained at 160 °C for 90
min under argon, whereupon the GC−MS analysis showed
that 11 was consumed. Upon cooling, the solid residue was
filtered, boiled with 500 mL of ethyl acetate to extract the
products from the residue, cooled, and then filtered again. This
process was repeated once more. All of the filtrates were
combined and concentrated to give crude 12 as a black, oily
product (355 g) that was purified by distillation under high
vacuum (0.5−0.6 mTorr) at 140 °C to give 12 (134 g, 48%
yield) as a yellow oil. GC−MS shows one major peak with M+
1
GC−MS and H-NMR. The only impurity observed was the
phenol starting material 3.
Ethyl 2-(4-(4-(Trifluoromethoxy)phenoxy)phenyl)-
acetate (12) from 14. Into a round-bottom flask equipped
with a stir bar were added 14 (22.6 g, 72.4 mmol, 1.0 equiv),
absolute ethanol (167 g, 3.6 mol, 50 equiv), and 12.1 N HCl
(0.75 mL, 9.1 mmol, 0.13 equiv). The solution was heated to
reflux for 18 h, at the end of which time only ∼1% of the
starting material 14 was present. The solvent was removed in
vacuo, and the resulting thick slurry was passed through a silica
gel plug (70 g), rinsing with a mixture of hexanes/ethyl acetate
(3:1) until no more 12 came through as monitored by TLC.
After solvent removal, ethyl 2-(4-(4-(trifluoromethoxy)-
phenoxy)phenyl)acetate 12 was recovered (22.5 g, 91%
yield) as a light orange oil. GC−MS showed one major peak
1
with M+ = 340 (37%), 267 (100%). H-NMR (CDCl3): δ
7.32−7.28 (m, 2H), 7.22−7.18 (m, 2H), 7.04−6.98 (m, 4H),
4.20 (q, J = 7.1 Hz, 2H), 3.63 (s, 2H), 1.30 (t, J = 7.1 Hz, 3H).
Copper <12 ppm. HRMS calculated for C17H16F3O4 [M + H]+
= 341.0995, observed for [M + H]+ = 341.0993. The product
1
= 340 (37%), 267 (100%). H-NMR (CDCl3): δ 7.32−7.28
1
(m, 2H), 7.22−7.18 (m, 2H), 7.04−6.98 (m, 4H), 4.20 (q, J =
7.1 Hz, 2H), 3.63 (s, 2H), 1.30 (t, J = 7.1 Hz, 3H). Copper
<21 ppm. HRMS calculated for C17H16F3O4 [M + H]+ =
341.0995 observed for [M + H]+ = 341.0993.
was at least 95% pure as shown by GC−MS and H-NMR,
sufficient for use in the subsequent step without further
purification.
4 - ( T r i fl u o r o m e t h o x y ) p h e n y l ( 2 ′ , 4 ′ , 6 ′ -
trimethoxyphenyl)iodonium Tosylate (16) from 15. To a
1 L three-necked round-bottom flask at room temperature was
added toluenesulfonic acid monohydrate (73.04 g, 0.38 mol, 1
equiv), acetonitrile (384 mL), and 15 (62 mL, 0.39 mol, 1
equiv). An overhead stirrer was inserted into the middle neck
and stirring was commenced. meta-Chloroperbenzoic acid (m-
CPBA) (97.84 g, 0.43 mmol, 1.1 equiv) was then added slowly
to the flask. The flask was then lowered into a preheated oil
bath (65 °C) and a yellow slurry formed as the reaction
progressed. After 40 min, 1,3,5-trimethoxybenzene (64.58 g,
0.38 mol, 1.0 equiv) was added to the flask. The slurry
dissolved, and the reaction mixture became an orange liquid.
2-(4-(4-(Trifluoromethoxy)phenoxy)phenyl)acetic
Acid (14) from 13 and 3. A round-bottom flask, stir bar, and
K2CO3 were oven dried at 150 °C for at least 24 h, and DMF
was degassed under house vacuum for 1 h prior to use.
Copper(I) chloride (CuCl) (1.38 g, 13.9 mmol, 0.15 equiv),
DMG (0.96 g, 9.30 mmol, 0.10 equiv), and DMF (30 mL)
were placed into the hot round-bottom flask and degassed for
20 min at 50 °C, while stirring. To the blue-colored catalyst
mixture, DMF (100 mL) and K2CO3 (38.5 g, 279 mmol, 3.0
equiv) were added. Next, 2-(4-bromophenyl)acetic acid (13,
20 g, 93.0 mmol, 1.0 equiv) was slowly added to avoid
excessive foaming followed by 4-(trifluoromethoxy)phenol (3,
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The reaction was removed from the heat after 5 min and
cooled to room temperature. Trituration with diethyl ether
followed by filtration and drying afforded 16 (205.3 g, 0.33
mol, 84% yield) as a yellow solid. 1H-NMR (600 MHz;
DMSO-d6) δ 8.03 (d, J = 8.7 Hz, 2 H), 7.47 (m, 4 H), 7.10 (d,
J = 7.8 Hz, 2H), 6.48 (s, 2H), 3.95 (s, 6H), 3.87 (S, 3H), 2.28
(s, 3H) ppm. 13C NMR (151 MHz, DMSO-d6) δ 166.8, 159.8,
150.7, 146.1, 138.1, 137.2, 128.9, 125.9, 124.3, 120.3 (q, J =
171.2 Hz) 114.2, 92.6, 87.7, 57.8, 56.6, 21.2 ppm. FTIR (λ):
3054, 2984, 2945, 2845, 1581, 1263, 1228, 1161, 1121, 1031,
1008, 816, 732, 702 cm−1. HRMS (ESI) m/z: [M − OTs]+
Calculated: 454.9962; Observed: 454.9959. Melting point:
190−192 °C.
using a gradient of ethyl acetate/hexane (5:25) as the eluting
solvent mixture. Isomers of 17 were assigned using 2D-NOESY
1
NMR. H-NMR (400 MHz; CDCl3) of the Z-isomer of 17: δ
7.29−7.27 (m, 2H), 7.21−7.19 (m, 2H), 7.05−6.99 (m, 4H),
4.18 (q, J = 7.1 Hz, 2H), 2.23 (s, 3H), 1.94 (s, 3H), 1.23 (t, J =
7.1 Hz, 3H). 1H-NMR (400 MHz; CDCl3) of the E-isomer of
17: δ 7.21−7.17 (m, 4H), 7.05−6.96 (m, 4H), 4.23 (q, J = 7.1
Hz, 2H), 2.40 (s, 3H), 1.90 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H).
HRMS of the product 17 as an isomer mixture calculated for
C21H20F3O6 [M + H]+ = 425.1206, observed for [M + H]+ =
425.1199. HRMS of the 17 Z-isomer calculated for
C21H20F3O6 [M + H]+ = 425.1206, observed for [M + H]+
= 425.1200. HRMS of the 17 E-isomer calculated for
C21H20F3O6 [M + H]+ = 425.1206, observed for [M + H]+
= 425.1201.
Ethyl 3-Oxo-2-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)butanoate (18) from 17. A stirred solution of the
bis-acylated 17 (125.2 g, 295 mmol) in glacial AcOH (250
mL) and p-TsOH monohydrate (5.6 g, 29.5 mmol, 0.1 equiv)
was heated at 100 °C. After 2 h, starting material 17 was not
detected by TLC. The dark brown solution was cooled to
room temperature and concentrated under reduced pressure.
After most of the AcOH was eliminated, to remove the residual
AcOH, cyclohexane (2 × 50 mL) was added to the brown oil
and concentrated again to give 116.5 g of 18 as a dark brown
oil. Because this material still contained 5.6 g of p-TsOH, the
yield of 18 was 110.9 g (98% yield). The product can be used
without purification in the following Conrad−Limpach
reaction. If desired, pure 18 can be obtained by flash
chromatography using 2:8 ethyl acetate/hexanes as the eluting
solvent mixture. GC−MS cannot be used to characterize 18 as
it decomposes in the injection port. Both the keto and enol
forms can be detected by 1H-NMR. Integration of the aromatic
region does not provide an accurate count of the number of
protons in 18 because of the existence of the keto/enol form.
1H-NMR (400 MHz; CDCl3) δ 13.15 (s, 0.4), 7.37−7.34 (m,
2H), 7.23−7.20 (m, 3H), 7.16−7.14 (m, 1H), 7.08−7.00 (m,
7H), 4.71 (s, 1H), 4.27−4.20 (m, 4H), 2.24 (s, 3H), 1.90 (s,
1H), 1.31 (t, J = 7.1 Hz, 3H), 1.22 (t, J = 7.1 Hz, 2H). HRMS
calculated for C19H18F3O3 [M + H]+ = 383.1101, observed for
[M + H]+ = 383.1097.
General Procedure for the Preparation of the Schiff
Bases (20a−d, Scheme 6). A stirred mixture of substituted
anilines 19a−d and β-keto ester 18 containing 10% of p-TsOH
in cyclohexane was heated at reflux for 24−72 h using a Dean−
Stark trap to continuously remove the water formed during the
condensation. After cooling to room temperature, the mixture
was filtered and the solid thus removed (containing unreacted
anilines 19a−d and some unidentified materials) was washed
with cyclohexane. The filtrate combined with the cyclohexane
washes was concentrated in vacuo to give the products 20a−d
as yellow-brown, highly viscous oils, which were used without
purification in the next phase of the reaction.
General Procedure for the Conrad−Limpach Reac-
tion15,16 (ELQ, Scheme 6). The Conrad−Limpach reactions
were performed at different temperatures. The 250 °C
cyclization was conducted at the boiling point of Dowtherm
A. For other temperatures, the cyclization was conducted in
Dowtherm A maintained at the desired temperature,
determined by an internal thermometer. To facilitate addition
of the highly viscous Schiff base obtained above, it was diluted
with Dowtherm A with slight warming. This mixture was
added to the heated Dowtherm A over approximately 10 min
Ethyl-3-acetoxy-2-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)but-2-enoate (12) from 16. This reaction was
performed at three different scales using the following general
procedure. Compound 16 (1.0 equiv) was added to a round-
bottom flask containing toluene (5 mL per mmol, 7) and
potassium carbonate (3.0 equiv). Ethyl 2-(4-hydroxyphenyl)-
acetate (1.5 equiv) was added to the flask, and the reaction was
placed in a preheated (55 °C) oil bath and stirred for 2.5 h.
The reaction mixture was removed from the heat and
concentrated in vacuo. The resulting brown oil was subjected
to a short silica gel column using 5% ethyl ether in hexanes as
the eluting solvent mixture to yield the product in an impure
state. Product peaks corresponding to pure materials were used
1
to determine the yield of 12 based on H-NMR spectroscopy.
The yields of 12 obtained at the three scales used were as
follows: 54% (0.5 mmol scale), 43% (3.6 mmol scale), and
48% (10 mmol scale).
Ethyl-3-acetoxy-2-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)but-2-enoate (17) from 12. Temperatures given
were recorded by an internal thermometer. A stirred solution
of dry THF (350 mL) and hexamethyldisilazane (HMDS)
(109.1 g, 676 mmol, 2.3 equiv) under Ar was cooled to −20
°C in an 80% ethylene glycol, 20% ethanol, and dry ice bath.
While monitoring the temperature to ensure that it did not
exceed −20 °C, n-butyl-lithium (2.5 M) in hexane (n-BuLi)
(258.6 mL, 646 mmol, 2.2 equiv) followed by a solution of 12
(100 g, 293 mmol, 1.0 equiv) in THF (250 mL) were added
dropwise. After stirring for 35 min at −20 to −30 °C, acetic
anhydride (66.0 g, 74.2 mL, 646 mmol 2.2 equiv) was added
dropwise while monitoring the temperature to ensure that it
did not exceed −10 °C. The solution turned from intense
yellow to orange-yellow. At this point TLC showed no more
starting material and the presence of the products 17 and 18.
GC−MS showed the presence of the desired product 17. The
solution was then allowed to warm gradually to room
temperature, and after 16 h, the solution took on the
appearance of a yellow gel. The reaction mixture was then
added to 700 mL of 10% HCl and 200 g of ice. The organic
layer was separated and the aqueous layer was extracted with
ethyl acetate (2 × 250 mL). The organic layers were
combined, washed with water (200 mL) and brine (200
mL), dried over Na2SO4 (100 g), filtered, and concentrated to
give crude 17 (129.2 g, 104% yield) as a yellow oil. GC−MS
showed one major peak at 7.18 min with M+ = 424 (22%), 336
(100%) corresponding to the Z-isomer of 17 and one minor
peak at 6.97 min with M+ = 424 (30%), 336 (100%)
corresponding to the E-isomer. The crude product did not
contain the starting material 12 and was approximately 95%
pure by GC−MS, sufficient for further reaction. For analysis,
pure Z and E isomers were obtained by flash chromatography
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with vigorous stirring, so that the desired temperature was
always maintained. After stirring for a further period of time
(specified for individual reactions below), the reaction mixture
was cooled to room temperature and diluted with hexanes,
resulting in the formation of a white precipitate. The
precipitate was recovered by filtration, washed with hexanes,
and air dried to give the crude ELQ compound. Yields were
determined over two steps based on the β-keto ester 18 used
to form the initial Schiff base.
2-Methyl-3-(4-(4-(trifluoromethoxy)phenoxy)-
phenyl)quinolin-4(1H)-one (ELQ-271). 1-Ethoxy-3-(phe-
nylimino)-2-(4-(4-(trifluoromethoxy)phenoxy)phenyl)but-1-
en-1-ol (20a, Scheme 6). Following the general procedure for
the preparation of the Schiff base, a mixture of aniline 19a
(1.85 g, 19.9 mmol, 1.0 equiv) and 8.07 g of β-keto ester 18
containing 10% of p-TsOH (7.69 g, 20.1 mmol, 1.0 equiv of 18
and 0.38 g, 2.6 mmol, 0.1 equiv of p-TsOH) in cyclohexane
(200 mL) was heated at reflux for 24 h. Cyclohexane (30 mL)
was used to wash the precipitate. The filtrate was concentrated
in vacuo to give the crude Schiff base 20a (9.60 g) as a yellow,
highly viscous oil.
Conrad−Limpach Cyclization of 20a. A portion of the
Schiff base 20a (3.90 g) was diluted with Dowtherm A (5 mL)
with slight warming. This Schiff base solution was added over 5
min to boiling Dowtherm A (100 mL, 250 °C) with vigorous
stirring so that boiling was always maintained. After stirring for
another 20 min at 250 °C, the stirred reaction mixture was
cooled to room temperature resulting in the formation of a
white precipitate. Hexane (150 mL) was then added to the
mixture. The resulting solid was filtered, washed with hexanes
(30 mL) and acetone (100 mL), and air-dried to give crude
ELQ-271 (2.11 g, 63%) as a white solid. 1H-NMR (400 MHz;
DMSO-d6): δ 11.66 (s, 1H), 8.10−8.08 (m, 1H), 7.64 (ddd, J
= 8.4, 6.9, 1.5 Hz, 1H), 7.54 (ddd, J = 8.3, 1.1, 0.6 Hz, 1H),
7.44−7.41 (m, 2H), 7.32−7.28 (m, 3H), 7.19−7.15 (m, 2H),
7.10−7.07 (m, 2H), 2.27 (s, 3H). HPLC analysis indicated
that the obtained ELQ-271 was >99% pure. GC−MS analysis
of the 4-chloro derivative of ELQ-271 obtained by
chlorination with POCl3 showed only a few very small
uncharacterized impurities besides the major component
with M+ = 429.5.
stirred reaction mixture was cooled to room temperature
resulting in the formation of a thick white precipitate. The
mixture was then allowed to stand without stirring at room
temperature overnight. Hexane (1000 mL) was added and the
mixture was stirred for 15 min and filtered. The resulting white
precipitate was washed with ethyl acetate (500 mL) and
acetone (250 mL) and air dried to give ELQ-300 (35.5 g, 57%
yield). 1H-NMR (400 MHz; DMSO-d6): δ 11.66 (s, 1H), 8.00
(s, 1H), 7.43−7.41 (m, 2H), 7.30−7.26 (m, 2H), 7.18−7.14
(m, 2H), 7.09−7.05 (m, 3H), 3.97 (s, 3H), 2.24 (s, 3H).
HPLC analysis indicated that ELQ-300 was >99% pure. No
1
ELQ-300 regioisomer was observed by H-NMR, and GC−
MS analysis of the 4-chloro derivative of ELQ-300 obtained by
chlorination with POCl3 showed only a single compound to be
present, M+ = 493.1.
To assess the performance of the Conrad−Limpach reaction
at temperatures below 250 °C using the Schiff base 20b, 10.0 g
of β-keto ester 18 containing 10 mol % of p-TsOH (9.5 g, 25
mmol, 1.0 equiv of 14 and 0.5 g, 2.9 mmol, 0.1 equiv of p-
TsOH) and 1.0 equiv of aniline 19b (4.0 g, 25 mmol) were
condensed as described above, and the cold cyclohexane
solution was filtered and diluted to a total volume of 300 mL.
From this stock solution (Solution A), aliquots of specific
volume were removed, stripped of the solvent, diluted with
Dowtherm A, and cyclized at the specified temperatures.
The cyclization at 230 °C was performed similarly to that at
250 °C above. To 100 mL of rapidly stirred Dowtherm A held
at 230 °C, the residue of a 100 mL aliquot of Solution A
(corresponding to 8.3 mmols of 18 condensed with 19b) was
dissolved in Dowtherm A (10 mL) and added dropwise over
the course of 10 min. The temperature was carefully monitored
and the heat reservoir was sufficiently large to keep
temperature changes within 1 °C. After stirring for another
20 min at 230 °C, the stirred reaction mixture was cooled to
room temperature resulting in the formation of a white
precipitate. The mixture was filtered, washed with hexanes and
acetone, and air dried to give crude ELQ-300 (1.88 g, 48%
yield) as a white solid. As in the case of the cyclization at 250
1
°C, no ELQ-300 regioisomer was observed by H-NMR and
GC−MS analysis of the 4-chloro derivative of ELQ-300
1
obtained by chlorination with POCl3. The H-NMR spectrum
6 - C h l o r o - 7 - m e t h o x y - 2 - m e t h y l - 3 - ( 4 - ( 4 -
(trifluoromethoxy)phenoxy)phenyl)quinolin-4(1H)-one
(ELQ-300). 3-(4-Chloro-3-methoxyphenyl)imino)-1-ethoxy-
2-(4-(4-(trifluoromethoxy)phenoxy)-phenyl)but-1-en-1-ol
(20b). Following the general procedure for the preparation of
the Schiff base, a mixture of 4-chloro-3-methoxyaniline 19b
(20.63 g, 131 mmol, 1.0 equiv) and 52.25 g of keto ester 18
containing 10 mol % of p-TsOH (50.0 g, 131.0 mmol, 1.0
equiv of 18 and 2.25 g, 1.31 mmol, 0.1 equiv of p-TsOH) in
cyclohexane (300 mL) was heated at reflux for 44 h.
Cyclohexane (100 mL) was used to wash the precipitate.
The condensation was practically complete after 20 h, as no
substantial increase in the volume of water occurred after this
time. The combined filtrates were concentrated in vacuo to
give the crude Schiff base 20b (67.5 g) as a yellow, highly
viscous oil.
of ELQ-300 obtained from the reaction at 230 °C was
identical to that of ELQ-300 obtained at 250 °C. The HPLC
analysis indicated that the obtained ELQ-300 was >99% pure.
The cyclization at 200 °C was performed similarly to the
above. To 100 mL of rapidly stirred Dowtherm A held at 200
°C, the residue of a 22 mL aliquot of Solution A
(corresponding to 1.8 mmol 18 condensed with 19b) in
Dowtherm A (5 mL) was added dropwise over the course of
10 min. The temperature was carefully monitored and the heat
reservoir was sufficiently large to keep temperature changes
within 1 °C. After stirring for another 60 min at 200 °C, the
stirred reaction mixture was allowed to cool to room
temperature and let stand overnight, resulting in the formation
of a white precipitate. Hexane (100 mL) was added and the
mixture was filtered, washed with hexanes, and air-dried to give
ELQ-300 (0.26 g, 30% yield) as a white solid. As in the cases
of the cyclizations at 250 and at 230 °C, no ELQ-300
Conrad−Limpach cyclization of 20b. The Schiff base 20b
(67.5 g) was diluted with Dowtherm A (30 mL) with slight
warming. This Schiff base 20b solution was added in small
portions over 25 min to boiling Dowtherm A (550 mL, 250
°C) with vigorous stirring so that boiling was always
maintained. After stirring for another 10 min at 250 °C, the
1
regioisomer was observed by H-NMR and GC−MS analyses
of the 4-chloro derivative of ELQ-300 obtained by
1
chlorination with POCl3. The H-NMR spectrum of ELQ-
300 obtained from the reaction at 200 °C was identical to that
I
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of ELQ-300 obtained at 250 °C. The HPLC analysis indicated
that the obtained ELQ-300 was >99% pure.
the clear solution was separated from the residue stuck to the
walls of the reaction flask and was concentrated in vacuo to
give the crude Schiff base 20d (13.0 g) as a yellow, highly
viscous oil.
6 - F l u o r o - 7 - m e t h o x y - 2 - m e t h y l - 3 - ( 4 - ( 4 -
(trifluoromethoxy)phenoxy)phenyl)quinolin-4(1H)-one
(ELQ-316). 1-Ethoxy-3-((4-fluoro-3-methoxyphenyl)imino)-
2-(4-(4-(trifluoromethoxy)-phenoxy)phenyl)but-1-en-1-ol
(20c). Following the general procedure for the preparation of
the Schiff base, a mixture of 4-fluoro-3-methoxyaniline 19c
(42.5 g, 0.301 mol, 1 equiv) and 120.7 g of β-keto ester 18
containing 10% of p-TsOH (115.2 g, 0.302 mol, 1.0 equiv of
18 and 5.5 g, 0.1 equiv of p-TsOH) in cyclohexane (600 mL)
was heated at reflux for 72 h. Cyclohexane (50 mL) was used
to wash the precipitate. The combined filtrates were
concentrated in vacuo to give the crude Schiff base 20c as a
yellow, highly viscous oil.
Conrad−Limpach cyclization of 20d. A portion of the
Schiff base 20d (4.00 g) was diluted with Dowtherm A (2 mL)
with slight warming. The Schiff base solution was added over 5
min to boiling Dowtherm A (90 mL, 250 °C) with vigorous
stirring so that boiling was always maintained. After stirring for
another 15 min at 250 °C, the stirred reaction mixture was
cooled to room temperature, resulting in the formation of a
white precipitate. When cold, hexane (100 mL) was added to
the mixture. After brief stirring, it was filtered, washed with
hexanes (30 mL), and air-dried to give crude ELQ-400 (1.90
1
g, 32%) as a white solid. H-NMR (400 MHz; DMSO-d6): δ
11.74 (s, 1H), 7.43−7.40 (m, 2H), 7.28−7.25 (m, 2H), 7.18−
7.14 (m, 2H), 7.10−7.00 (m, 4H), 2.21 (s, 3H). The HPLC
analysis indicated that the obtained ELQ-400 was >99% pure.
The cyclization at 230 °C was performed similarly to the
above. To 100 mL of rapidly stirred Dowtherm A held at 230
°C, 3.80 g of the condensation product 20d in Dowtherm A (5
mL) was added dropwise over the course of 10 min. The
temperature was carefully monitored and the heat reservoir
was sufficiently large to keep temperature changes within 1 °C.
After stirring for another 30 min at 230 °C, the stirred reaction
mixture was cooled to room temperature resulting in the
formation of a white precipitate. Hexane (150 mL) was then
added, and the precipitate was collected by filtration, washed
with hexanes (50 mL) and acetone (10 mL), and air-dried to
give crude ELQ-400 (1.14 g, 33%) as a slightly yellow solid.
The 1H-NMR spectrum of ELQ-400 obtained from the
reaction at 230 °C was identical to that of ELQ-400 obtained
at 250 °C. HPLC analysis indicated that the obtained ELQ-
400 was >99% pure.
Conrad−Limpach Cyclization of 20c. The Schiff base 20c
was diluted with Dowtherm A (50 mL) with slight warming.
This Schiff base solution was added over 30 min to boiling
Dowtherm A (700 mL, 250 °C) with vigorous stirring so that
boiling was always maintained. After stirring for another 15
min at 250 °C, the reaction mixture was cooled to room
temperature, resulting in the formation of a firm, white cake.
Ethyl acetate (2 L) was added, and the cake was broken up
with a glass rod and stirred until no large pieces remained. The
mixture was filtered, washed with methanol (500 mL) and then
with acetone (250 mL), and air-dried to give crude ELQ-316
(73 g, 54%) as a white solid. 1H-NMR (400 MHz; DMSO-d6):
δ 11.63 (s, 1H), 7.70 (d, J = 11.8 Hz, 1H), 7.43−7.40 (m, 2H),
7.30−7.26 (m, 2H), 7.18−7.14 (m, 2H), 7.11−7.05 (m, 3H),
3.96 (s, 3H), 2.24 (s, 3H). The HPLC analysis indicated that
the obtained ELQ-316 was >99% pure. Copper <17 ppm. No
1
ELQ-316 regioisomer was observed by H-NMR, and GC−
MS analysis of the 4-chloro derivative of ELQ-316 obtained by
chlorination with POCl3 showed only a single compound to be
present, M+ = 477.5.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.1c00099.
The cyclization at 230 °C was performed similarly to the
above. To 100 mL of rapidly stirred Dowtherm A held at 230
°C, 3.84 g of the condensation product 20c in Dowtherm A (2
mL) was added dropwise over the course of 10 min. The
temperature was carefully monitored and the heat reservoir
was sufficiently large to keep temperature changes within 1 °C.
After stirring for another 30 min at 230 °C, the stirred reaction
mixture was cooled to room temperature resulting in the
formation of a white precipitate. Hexane (100 mL) was then
added to the mixture. It was then filtered, washed with hexanes
(30 mL) and ethyl acetate (2 × 10 mL), and air-dried to give
ELQ-316 (1.75 g, 50%) as a white solid. No ELQ-316
■
sı
Synthetic details for the optimized original synthesis of
ELQ-300 (Scheme 2) (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Sovitj Pou − VA Portland Healthcare System, Portland,
Oregon 97239, United States; Email: sovitjpou@gmail.com
Rolf W. Winter − VA Portland Healthcare System, Portland,
Oregon 97239, United States; Email: rolf.w.winter@
gmail.com
1
regioisomer was observed by H-NMR and GC−MS analyses
of the 4-chloro derivative of ELQ-316 obtained by
1
chlorination with POCl3. The H-NMR spectrum of ELQ-
316 obtained from the reaction at 230 °C was identical to that
of ELQ-316 obtained at 250 °C. HPLC analysis indicated that
the obtained ELQ-316 was 98% pure.
Authors
Rozalia A. Dodean − VA Portland Healthcare System,
Portland, Oregon 97239, United States; orcid.org/0000-
0001-5002-4607
Lisa Frueh − VA Portland Healthcare System, Portland,
Oregon 97239, United States
Katherine M. Liebman − VA Portland Healthcare System,
Portland, Oregon 97239, United States
Rory T. Gallagher − Department of Chemistry, Portland State
University, Portland, Oregon 97201, United States
Haihong Jin − Medicinal Chemistry Core, Oregon Health &
Science University, Portland, Oregon 97239, United States
5. 7-Difluoro-7-methoxy-2-methyl-3-(4-(4-
(trifluoromethoxy)phenoxy)phenyl)quinolin-4(1H)-one
(ELQ-400). 1-Ethoxy-3-((3,5-difluorophenyl)imino)-2-(4-(4-
(trifluoromethoxy)phenoxy)phenyl)but-1-en-1-ol (20d). Fol-
lowing the general procedure for the preparation of the Schiff
base, a mixture of 3,5-difluoro aniline 19d (3.26 g, 25.3 mmol,
1.0 equiv) and 10.14 g of β-keto ester 18 containing 10% of p-
TsOH (9.66 g, 25.3 mmol, 1.0 equiv of 18 and 0.48 g, 2.8
mmol, 0.1 equiv of p-TsOH) in cyclohexane (200 mL) was
heated at reflux for 24 h. After cooling to room temperature,
J
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Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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Article
Robert T. Jacobs − Medicines for Malaria Venture, 1215
Geneva, Switzerland
thyldisilazide; THF, tetrahydrofuran; NOESY NMR, nuclear
Overhauser effect spectroscopy nuclear magnetic resonance;
1H-NMR, proton nuclear magnetic resonance; GC−MS, gas
chromatography−mass spectrometry; p-TsOH, para-toluene-
sulfonic acid; AcOH, acetic acid; OMe, o-methyl; HPLC, high-
performance liquid chromatography; POCl3, phosphorus
oxychloride; TMS, tetramethylsilane; HRMS, high-resolution
mass spectrometry
Aaron Nilsen − VA Portland Healthcare System, Portland,
Oregon 97239, United States; Medicinal Chemistry Core,
Oregon Health & Science University, Portland, Oregon
97239, United States; orcid.org/0000-0002-3827-8222
David R. Stuart − Department of Chemistry, Portland State
University, Portland, Oregon 97201, United States;
orcid.org/0000-0003-3519-9067
J. Stone Doggett − VA Portland Healthcare System, Portland,
Oregon 97239, United States; School of Medicine Division of
Infectious Diseases, Oregon Health & Science University,
Portland, Oregon 97239, United States; orcid.org/0000-
0002-6098-1520
Michael K. Riscoe − VA Portland Healthcare System,
Portland, Oregon 97239, United States; Department of
Microbiology and Molecular Immunology, Oregon Health &
Science University, Portland, Oregon 97239, United States;
orcid.org/0000-0002-1343-5279
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The authors declare the following competing financial
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M.R., and R.W. have a financial interest in a company that may
have a commercial interest in the results of this research and
technology. A patent application on the intellectual property
described herein has been filed by OHSU.
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ACKNOWLEDGMENTS
■
This project was supported with funds from the United States
Department of Veterans Affairs, Veterans Health Adminis-
tration, Office of Research and Development Program, with
additional supplemental funding from VA Technology Trans-
fer, Merit Review Grant award number i01 BX003312
(M.K.R.). M.K.R. is a recipient of a VA Research Career
Scientist Award (14S-RCS001). Research reported in this
publication was also supported by the US National Institutes of
Health under award numbers R01AI100569 and R01AI141412
(M.K.R.) and by the US Department of Defense Peer
Reviewed Medical Research Program (Log # PR181134)
(M.K.R.). This work was also funded by the Career
Development Award BX002440 and VA Merit Review
Award BX004522 to J.S.D. from the US Department of
Veterans Affairs Biomedical Laboratory Research and Develop-
ment. The National Science Foundation provided instrument
funding for the BioAnalytical Mass Spectrometry Facility at
Portland State University (NSF, MRI 1828573). The
Medicines for Malaria Venture supported the discovery of
ELQ-300, and the authors would like to acknowledge their
continued involvement in this project. The authors would also
like to acknowledge the contributions of the OHSU Medicinal
Chemistry Core.
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ABBREVIATIONS
■
ELQ, endochin-like quinolone; HBr, hydrobromic acid; DMG,
N,N-dimethylglycine; K2CO3, potassium carbonate; DMF,
N,N-dimethylformamide; TMP, 2,4,6-trimethoxyphenyl;
TLC, thin-layer chromatography; LiHMDS, lithium hexame-
K
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