Discovery of 5-{2-[5-Chloro-2-(5-ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-4-methoxypyridine-2-carboxylic Acid, a Highly Selective in Vivo Useable Chemical Probe to Dissect MCT4 Biology

Article abstract of DOI:10.1021/acs.jmedchem.1c00448

Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.

Full text of DOI:10.1021/acs.jmedchem.1c00448

pubs.acs.org/jmc
Article
Discovery of 5‑{2-[5-Chloro-2-(5-ethoxyquinoline-8-
sulfonamido)phenyl]ethynyl}-4-methoxypyridine-2-carboxylic Acid,
a Highly Selective in Vivo Useable Chemical Probe to Dissect MCT4
Biology
Timo Heinrich,* Ada Sala-Hojman, Roberta Ferretti, Carl Petersson, Stefano Minguzzi,
Andrzej Gondela, Shivapriya Ramaswamy, Anna Bartosik, Frank Czauderna, Lindsey Crowley,
Pamela Wahra, Heike Schilke, Pia Böpple, Łukasz Dudek, Marcin Les,
Henryk Pawlik, Łukasz Włoszczak, Karol Zuchowicz, Jose Ramon Suarez Alvarez, Justyna Martyka,
Ewa Sitek, Maciej Mikulski, Joanna Szczęsniak, Sven Jäckel, Mireille Krier, Marcin Król, Ansgar Wegener,
Michał Gałęzowski, Mateusz Nowak, Frank Becker, and Christian Herhaus
́
Paulina Niedziejko, Kamila Olech,
́
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sı Supporting Information
ABSTRACT: Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport
to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate
transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors
(
hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n
with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation
spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular
viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and
antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its
effect on tumor biology.
5
INTRODUCTION
characterized. Overexpressions of MCT1 and MCT4 are
markers for poor prognosis of several cancer indications,
therefore they have been discussed as potential targets in
■
Enhanced glucose catabolism, a well-described feature of
growing and dividing cancer cells often referred to as the
Warburg effect, leads to the accumulation of metabolites in the
tumor microenvironment. Among these metabolites, lactic acid
is emerging as a key metabolic modulator responsible for
different cancer cell hallmarks including sustained angio-
genesis, evasion of immune surveillance, and reprogramming of
2,6
cancer therapy.
Several compounds have been described to non-specifically
inhibit MCTs (Figure 1). 4′-Diisothiocyanatostilben-2,2′-
disulfonate irreversibly binds to a lysine residue on MCT1
and MCT2 but not on MCT4, thus inactivating MCT1 and
MCT2. Organomercurial compounds, such as p-chloromer-
curibenzenesulfonate (p-CMBS), disrupt MCT interaction
1
energy metabolism. Due to the high consumption of glucose,
7
large quantities of lactic acid are transported out of tumor cells
and this process is mediated by monocarboxylate transporters
(
MCTs). In mammals, a family of MCT proteins containing at
2
Received: March 11, 2021
Published: August 12, 2021
least 14 members has been described so far. These
transporters also recognize other substrates, such as short-
chain fatty acids and pyruvate and ketone bodies, but lactate is
the most abundant one with up to 40 mM concentration in
tumors. Although many MCTs are still subject to deorpha-
nization, the physiological role for MCTs 1−4 is well
3
4
©
2021 American Chemical Society
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Figure 1. Selected structures of known MCT inhibitor tool compounds (upper panel) and more drug-like MCT1 inhibitors (lower panel).
Figure 2. Selected structures of known MCT4 inhibitors.
with chaperone CD147 interfering with MCT1, MCT3, and
MCT4 expression and activity, but not with MCT2. CHC (α-
with nanomolar viability activity in the highly MCT1
expressing Raji (Burkitt’s) lymphoma cell line. Recently,
8
18
cyano-4-hydroxy cinnamate) was described with 10-fold
selectivity for MCT1 compared to other MCTs. Special α-
the carboxylic acid BAY-8002 was reported as a novel selective
9
MCT1 inhibitor, with a 6-fold selectivity for MCT1 compared
19
cyano-4-amino-2-methoxy cinnamates have been published as
dual MCT1 and MCT4 inhibitors based on their activity in
to MCT2, no activity on MCT4, and no off-target effects. So
far, only 1 is reported in clinical trial (NCT01791595).
In 2015, a first selective MCT4 inhibitor (AZ93) was
10
RBE4 and MDA-MB-231 cell lines. Besides the mentioned
dual amino-methoxy-cinnamate-based compounds, the reser-
pine-derived antihypertensive drug syrosingopine was pub-
lished as the dual MCT1 and MCT4 inhibitor (with 60-fold
20
mentioned but not disclosed. Other carboxylic acids, derived
from CHC, were published in 2016 as selective MCT4
inhibitors, which inhibit lactate transport in cell lines that
express high levels of MCT4, such as MDA-MB-453, MDA-
MB-231, or NCI-H358, but not in cells that express high levels
11
higher potency on MCT4 compared to MCT1). In 2019, the
non-steroidal anti-inflammatory drug diclofenac was reported
12
21
to inhibit MCT1 and MCT4. These compounds have not yet
been investigated clinically for disrupting MCT functions in
of MCT1, such as BT20. In conference reports, the
carboxylic acid AZ1422 was described as a selective MCT4
13−15
22
cancer possibly due to polypharmacology issues.
inhibitor. Similar results were reported for compound
Currently, only a few MCT1 inhibitors (with some activity
against MCT2) have been described with candidate-stage
drug-like properties, such as AZD3965 (1) and AR-C155858
Example 62 with antitumoral activity in combination with
the VEGFR inhibitor Cediranib, αCTLA4, and αPD-1
antibodies in the MC38 MCT1 KO syngeneic cell line
1
6
23
(2). 1 was shown to inhibit lactic acid transport and cell
model. Phenex Pharmaceuticals compared their MCT4-
growth in vitro and in vivo in cancer cells that preferentially
express MCT1. Blocking of lactate transport led to a reduction
of the glycolytic rate and to intracellular acidification resulting
in the inhibition of cell proliferation or survival. 1 is six times
more selective for MCT1 than for MCT2 and does not inhibit
MCT4. Pteridine dione compounds (Scripps) were described
selective inhibitor PX-788 of unknown structure to an
undisclosed AZ MCT4 inhibitor and reported moderate
monotherapy effects on tumor growth by both compounds
in the NCI-H358 xenograft model. Drew University reported
another non-carboxylic compound CB-3 that exhibited in vivo
monotherapy effects in the MDA-MB-231 xenograft model,
17
24
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but the overall selectivity of this compound was not yet
derivatives with elongated saturated linker moieties followed
the strategy outlined in Scheme 1.
2
5
26
published. Pyrazole-(VB124) and quinolinone-(Niro-
gyone)-based carboxylic acids were described to inhibit
MCT4-dependent lactate transport and viability using SiHa
Iodo-carboxylate 3 was coupled under Sonogashira con-
ditions with alkynol 4 to intermediate 5, which was first
hydrogenated to the saturated analog 6a, and then converted
into the corresponding triphenyl phosphine 6c via the bromide
6b. After Wittig condensation with nitro-benzaldehyde 7,
comprehensive reduction of the nitro group and the double
bond in one step generated the amino building block 8, which
was used for amide couplings under standard conditions or
sulfonamide synthesis via the corresponding sulfonylchloride,
followed by ester saponification giving the target compounds
2
7
cells and claimed to interfere in lactate consumption.
Representative compounds are depicted in Figure 2.
This paper describes the identification and optimization of a
new class of selective MCT4 inhibitors leading to compound
28
18n with chemical probe characteristics.
RESULTS AND DISCUSSION
Design of MCT4-Selective Inhibitors. An analysis of a
■
9
a−m and 10. The preparation of compounds with shorter
panel of human cell lines for expression of MCTs yielded
MDA-MB 231, NCI-H358, and NCI-H441, among others, as
cells with high MCT4 and no/low MCT1 expression. The
MDA-MB-231 model does not express MCT1 at all, as result
of MCT1 gene promoter hypermethylation, and will hence-
linking moieties is exemplified in Scheme 2.
Scheme 2. Synthesis of Ethyl- and Ethenyl-Linked
a
Sulfonamides Described in Tables 2−4
29
forth be referred to as the MCT1 null cell line. In a cellular
screening approach on lactate efflux in MDA-MB-231 cells,
compound 9a was identified as one initial hit with
30
submicromolar activity. This compound has been published
earlier in the context of hyperglycemia without a description of
31
the mode of action. The submicromolar activity in the lactate
efflux assay in MDA-MB-231 prompted initial activity
optimization, followed by property adjustments. For opti-
mization attempts, different areas of the hit were considered
separately (Figure 3).
a
Reagents and conditions: (a) SOCl , MeOH, 0 °CRT, 82%; (b)
2
Pd(Ac) , P(o-tolyl) , TEA, 125 °C, 75%; (c) sulfonyl chloride, TEA,
2
3
DCM; (d) NaOH, MeOH, 65−85%; and (e) i. Pd/C, H2, MeOH,
9
5%.
Figure 3. Screening hit 9a and molecular sections: acid or isostere
bearing ring C, linking moiety, middle ring B, oxidized bridge, and
terminal moiety A.
Acid 10 was converted into the corresponding ester 11 and
coupled either under Heck or Herrmann−Beller’s phospha-
palladacycle conditions with bromo-aniline 12 to building
32
block 13. After sulfonamide formation (14), either the ester
was saponified (15) or the double bond was hydrogenated
concomitant with ester saponification, giving the free acid 16
Chemistry. The syntheses of the different scaffolds and
decorations is described in the following paragraphs. Access to
a
Scheme 1. Synthesis of Pentyl-Linked Carboxamides Described in Table 1 and Sulfonamide in Table 2
a
Reagents and conditions: (a) CuI, Pd(PPh ) Cl , diethylamine, RT, 90%; (b) Noblyst P1086, THF, RT, 73%; (c) PPh , NBS, THF, 0 °CRT,
3
2
2
3
6
2%; (d) PPh , AcCN, 82%, 65 °C; (e) 7, K CO , dioxane/water, reflux, 93%; (f) Noblyst P1086, H , THF, RT, 92%; and (g) i. acid chloride,
3
2
3
2
TEA, DCM, RT (48−92%); ii. NaOH, MeOH, RT (27−89%).
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a
Scheme 3. Synthesis of Ethynyl-Linked Sulfonamides Described in Tables 5−7
a
Reagents and conditions: (a) 2-ethynyl-R′-phenylamine, CuI, diisoproylamine, Pd(PPh ) , AcCN; (b) i: i. R″-sulfonyl chloride, NaHCO , AcCN,
3
4
3
RT or R″-sulfonyl chloride, pyridine, RT ii. NaOH or LiOH, MeOH, RT.
in one step. The synthesis of the alkynyl-linked compounds is
depicted in Scheme 3.
Table 2. Lactate Efflux Inhibition of Derivatives from
Geometry Investigations: Ring C, Bridge Y Alternatives, and
Linker X
Arylbromides D were coupled under Sonogashira conditions
with 2-ethynyl-R′-phenylamines to intermediates E. After
sulfonamide formation with the respective sulfonylchlorides,
the corresponding ester was saponified under basic conditions
to give the products shown in Tables 5−7. Derivative 18n
a
entry
COOH
R
Y
X
IC50 [μM]
Table 1. Lactate Efflux Inhibition of Analogs of Screening
Hit 9a
16a
3
3
4
4
2
4
1-naphthyl
1-naphthyl
1-naphthyl
2-naphthyl
1-naphthyl
1-naphthyl
CO
SO2
SO2
SO2
SO2
SO2
CH2
CH2
CH2
CH2
CH2
(CH2)4
>50
3
3.4
1.4
>50
>100
1
1
1
1
1
6b
6c
6d
6e
0
a
Efflux inhibition assay in MDA-MB-231 cells and geometric means
of three replicates.
Table 3. Lactate Efflux Inhibition of Ethyl Sulfonamides
a
Efflux inhibition assay in MDA-MB-231 cells and geometric means
of three replicates.
required a slight adaptation of the first step of this sequence as
the respective middle ring building block was not available.
Arylbromide D (methyl-5-Br-4-methoxypicolinate) was
coupled with trimethylsilylacetylene under Sonogashira con-
ditions, followed by desilylation with potassium carbonate in
methanol. The obtained ethynyl-intermediate was coupled
a
Efflux inhibition assay in MDA-MB-231 cells and geometric means
of three replicates.
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Table 4. Lactate Efflux Inhibition of Ethenyl Sulfonamides
Table 6. Screening of Pyridine, Pyrimidine, Pyrazine, and
Pyridazine Carboxylic Acids on Ethynyl Sulfonamides
a
entry
R
COOH
DB geometry
IC50 [μM]
15a
15b
15c
15d
15e
2,3-Me 4-OMe
3
3
3
3
4
trans
trans
trans
cis
2.9
1.2
0.9
2.7
1.8
2
1-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
trans
a
Efflux inhibition assay in MDA-MB-231 cells and geometric means
of three replicates.
Table 5. Lactate Efflux Inhibition of Ethynyl Sulfonamides
on Benzoic Acid
a
Efflux inhibition assay in MDA-MB-231 cells and geometric means
of three replicates.
again under Sonogashira conditions with 4-Cl-2-I-aniline to
intermediate of type E. The synthesis of derivatives from Table
8
(18p−u) followed the same strategy as outlined in Scheme 3.
Intermediate E was reacted with commercially available 4-
chloroquinoline-8-sulfonyl chloride, followed by substitution of
the chlorine with the respective alcohol function of the
different residues in the presence of potassium tert-butylate in
DMF at RT. Ester saponification occurred under these
conditions.
SAR Studies of Terminal Moiety A. From cellular MCT
33
expression profiles and literature evidence, the high MCT4/
no MCT1-expressing human breast cancer cell line MDA-MB-
2
31 was chosen to test compounds on their MCT4 transporter
inhibition potential. In addition, the effect of selected MCT4
inhibitors on MDA-MB-231 viability was tested to prove the
hypothesis that intracellular lactate accumulation might be
associated with reduced viability. The terminal moiety A was
systematically optimized first and a focused selection of
compounds is displayed in Table 1.
The submicromolar activity of the 2-naphthyl-screening hit
a could be confirmed with the 1-naphthyl isomer 9b. A
9
methyl extension para to the amide bridge like in 9c reduced
activity 4-fold. Being cognizant of the resulting compound
properties, the naphthyl group was replaced with an array of
substituted phenylamides. The unsubstituted phenyl 9d turned
out to be inactive while decoration with electron-donating
a
Efflux inhibition assay in MDA-MB-231 cells and geometric means
of three replicates.
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Table 7. Lactate Efflux Inhibition, Binding Constants, and PK Data of Ethynyl Sulfonamides on Picolinic Acid
IC
FCCS IC Ki
pred mouse CL microsomes/hepatocytes
[L/kg/h]
mouse CL
[L/kg/h]
mouse V
D
[L/kg]
5
0
50
a
b
entry
R2−R7
Y
X
[nM]
[nM]
1
1
1
1
1
1
1
1
8b
8c
8d
8e
8f
H
H
H
H
H
H
H
H
H
177
215
3
7
2-Me
3-Me
4-Me
5-Me
6-Me
7-Me
7-Me
H
H
H
H
H
H
H
245
153
209
283
234
34
579
9
9
139
2
9
154
3
2
1020
22
243
2
8g
8h
8i
5
1
79
0.2/0.4
1.9
1.9
1.8
1.9
1
7
3-Me
34
490
5
6
1
1
8j
8k
5-OMe
5-OMe
H
3-Me
H
H
91
29
≪0.03/0.1
0.4
0.6
0.6
0.6
99
1
1
1
1
1
1
8l
5-OEt
5-OEt
5-OEt
5-OEt
H
H
17
3
92
0.1/0.1
0.6
0.4
0.3
1.6
0.4
0.4
0.4
1.6
1
6
8m
8n
8o
4-OMe
4-OMe
3-NHMe
H
80
0.1/0.1
1
7
4-Cl
H
1
77
≪0.04/0.1
1
1
3
62
≪0.03/0.1
1
7
a
b
Efflux inhibition assay in MDA-MB-231 cells and geometric means of three replicates. Biophysical characterization via fluorescence cross-
correlation spectroscopy, mean of three replicates, and discussion of FCCS data vide infra.
(
9e−9g) or -withdrawing moieties (9h−9m) with different
steric demands only allowed a re-establishment of micromolar
activity. Screening hit 9a reduced MDA-MB-231 viability in
the micromolar range (GI : 5.7 μM; IC : 7.9 μM)
5
0
50
emphasizing the need to increase lactate transporter inhibition
potency. Microsomal turnover of compounds from Table 1
was in the high to very high range (data not shown), indicating
that optimization of metabolic stability was necessary as well.
SAR of Linker and Bridge. It was anticipated that a
reduced linker length would be beneficial for activity as
entropic penalties would be reduced. The relative positions of
the carboxylate to the amide and its residue were considered to
be important. An ortho-acid ring C together with a shorter
linker moiety would not match geometry requirements. In
silico analysis of preferred conformations (Figure 4A) and field
points (Figure 4B) confirmed the expectation that a
carboxylate in the meta position with an ethyl linker would
superimpose with the ortho-acid and pentyl linker quite well.
Specifically, field analysis showed that, despite a shorter linker
in ethyl compared to pentyl, the compounds’ negative field
points from carboxyl groups overlap very well, thus likely
retaining the same set of interactions.
Figure 4. Superimposition of preferred conformations (A) and field
points (B) of ortho-acid with pentyl linker 9a (magenta) and meta-
acid with an ethyl linker (olive). Square field points are derived from
pentyl linker compound, sphere field points are derived from the ethyl
linker compound. Cyan and red points indicate the maxima of
negative and positive electrostatic potentials, respectively; orange and
yellow points indicate the maxima of aromatic and hydrophobic
potentials, respectively.
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Moving the acid-moiety into the meta position in ring C
together with 1-naphthyl amide ring A and ethyl linker resulted
in the inactive compound 16a (Table 2).
This inactivity was not anticipated from the analyses
depicted in Figure 4 but initiated optimization of the bridging
moiety. Changing the carboxamide bridge into sulfonamide
activity could also be achieved with 1- and 2-naphthyl
derivatives 17f, 17g, and 17h. Surprisingly, the meta-isomer
17i and para-regio isomer 17h showed equal potency, even
though the carboxylate is presented in different geometry. The
8-quinolinyl analog 17j and 1-naphthyl isomer 17f showed
comparable IC50 values of 0.7 and 0.8 μM, respectively.
Appropriate substitution of the 8-quinolinyl residue improved
potency even further. For the 7-methyl derivative 17k, an IC50
value of 50 nM was achieved for the first time. This analogue
showed high turnover in mouse liver microsomes (17k mouse
Clint: 416 μL/min/mg prot.), possibly caused by the elevated
lipophilicity and/or by the electron-rich, activated amino alkyl
substituent. The two derivatives 17j and 17l inhibited lactate
efflux with IC50 values of 200 and 100 nM, respectively, and
were stable in microsomes (17j and 17l mouse Clint: <10 μL/
min). Both were tested in the MDA-MB-231 viability assay and
were active with IC s of 7.2 μM and 1.7 μM and GI s of 4.4
(16b) reintroduced micromolar activity. Ring C geometry was
further investigated leading to the finding that the para-acid
isomers 16c and 16d resulted in equipotent micromolar
compounds. As expected, the ortho-benzoic acid 16e with an
ethyl linker and a sulfonamide bridge was inactive, in contrast
to the pentyl-linked carboxamide 9a, confirming the initial
considerations of the linker length and carboxylate position.
The pentyl-linked sulfonamide 10 was completely inactive as
well (Table 2). Accordingly, it was concluded that the
carboxamide bridge is required for the extended pentyl linker
activity, whereas the 2-atom-linker only yielded active
compounds when combined with the sulfonamide bridge.
Continued optimization of the ethyl-linked sulfonamide core is
shown in Table 3.
5
0
50
μM and 0.9 μM, respectively.
Nevertheless, combining suitable potency with high liver
microsomal stability was generally challenging for this series.
Under the assumption that heterocycles may be beneficial,
phenyl ring C replacements were studied (Table 6). A series of
the pyridine and diazines carboxylic acids bearing the
carboxylate in meta and para positions relative to the acetylene
linker were synthesized. The picolinic acid derivative 18a was
identified as the most active (1 μM) together with an
acceptable mouse microsomal turnover of 14 μL/min/mg prot.
and set the basis for further investigations (Tables 6 and 7).
The four derivatives, 19−22, contained a meta orientation of
the acid and the ethynyl linker. All derivatives were only active
in the upper micromolar range and confirmed that the
geometry is critical to maintain the activity. The remaining
four derivatives, 23−26, from Table 6 in addition to 18a with a
para orientation of the acid and the ethynyl linker
demonstrated that the position of the ring-nitrogen(s) was
also of importance. In all cases, where the nitrogen is
positioned meta to the acid, activity was reduced (23, 25,
and 26). Ortho orientation gave an IC value of 3.7 μM again
The micromolar activity of the 1-naphthyl derivative 16b
from Table 2 could be confirmed with the 2-naphthyl isomer
16f. Similar to the pentyl-carboxamide compound with an
unsubstituted phenyl ring A 9d (Table 1), also the ethyl
sulfonamide 16g (Table 3) was inactive. Notably, the ethyl-
linked 4-F-phenyl sulfonamide isomer 16h showed micromolar
activity, despite the analogues pentyl-linked carboxamide 9h
(Table 1) being inactive. Remarkably, the addition of a methyl
group ortho to the sulfonamide (16i) further increased
potency into the 100 nM range. Identifying the first analog
in this lead series with submicromolar activity underscored the
importance of having redesigned the central core of the
inhibitor series toward higher rigidity. Even though this
halogen alkyl substitution improved activity, the stability in
mouse liver microsome incubations was unfavorable (16i
mouse Clint: 167 μL/min/mg prot.). This was overcome by
the 2,3-Me2 4-OMe-phenyl residue (16j), which was
submicromolar active and showed acceptable metabolic
stability (16j mouse Clint: <10 μL/min/mg prot.). This
suggested that compound 16j would be an attractive starting
point for a further cycle of optimization (vide infra). The bis-
ortho chlorine substitution (16k) slightly increased the activity
but compromised the microsomal stability in mouse (89 μL/
min/mg prot.). More rigid ethenyl-linked sulfonamides were
investigated then (Table 4).
5
0
(24). Consequently, further optimization was based on these
ring A derivatives together with the optimized picolinic ring C
moiety (Table 7).
Compared to the unsubstituted analog 18b, permutation of
a methyl group on various positions on the quinoline (18c−
18h) only improved activity for the 7-yl isomer 18h.
Compound 18h was characterized further. It showed accept-
able solubility in PBS buffer (97 μg/mL) and turnover in
mouse microsomes (18 μL/min/mg prot.). The turnover was
much higher in human microsomes (82 μL/min/mg prot.),
further an unfavorable efflux ratio of 12 in the Caco-2 assay
was observed. Introduction of a methyl group on the picolinate
(18i) had no impact on the potency, but PBS solubility (384
μg/mL), as well as the mentioned liabilities of 18h, was
addressed: human Clint: 18 mg/min/mg prot.; efflux ratio: 3.
Ether substituents on position 5 of the quinoline were also
investigated. The methyl ether 18j was 3-fold more potent
than the aliphatic methyl analog 18f. Properties of 18j showed
a mixed profile with acceptable solubility and mouse
microsomal stability (PBS: 445 μg/mL; mouse Clint: <10
μL/min/mg prot.), but again turnover in human microsomes
was high (63 μL/min/mg prot.) as well as the efflux ratio (16).
Also, for the 5-yl methyl ether, the introduction of a methyl
group on the picolinate (18k) improved properties: PBS 876
μg/mL; human clint: 12 μL/min/mg prot.; ER 11. In this case,
In 16j (Table 3), the dimethyl-methoxy-phenyl residue
resulted in submicromolar activity, but the more rigid ethenyl
compound 15a was less active (Table 4). The naphthyl
isomers 16b (Table 2) and 16f (Table 3) had micromolar
activity, which could be confirmed with the ethenyl bridge in
15b and 15c (Table 4). The latter core was also investigated
with the acid in the para position to the double bond. In 15e,
activity dropped 2-fold. In addition, the 15d cis isomer of 15c
showed a 3-fold activity drop. Based on these observations, the
scaffold with an ethynyl bridge and 4-carboxylate was chosen
for further investigations (Table 5).
The fully unsubstituted starting point 17a showed only
micromolar activity. The meta methyl ether 17b did not much
alter activity but the 4-anisole analog 17c was 5−10-fold less
potent. The addition of two methyl groups in ortho and meta
positions to the 4-OMe moiety (17d) on the other hand
contributed to submicromolar activity, which was confirmed
with the bis-ortho chlorine substitution in 17e. Submicromolar
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Figure 5. Correlation (red line) of 4 h lactate efflux inhibition (X-axis) with a 6 d viability effect (Y-axis) of compounds from Table 7 in MDA-MB-
2
2
31 cells (Supporting Information Table S1). R : 0.87. 10-fold off-set to diagonal (green line).
the cellular activity also increased. Extension to the ethyl ether
8l improved activity even further. From benzoic acid pairs
metabolite. The acid function in ring C, in combination with
the sulfonamide bridge, resulted in low to moderate
1
−
6
such as 17m/17n (Table 5), it was learned that methoxy
substitution of ring C could have a negative impact on potency.
The methylamine, on the other hand, suggested a favorable
impact (17k). Because of this inconsistency, similar sub-
stituents were investigated on the picolinic acid ring C. For
both the methyl ether 18m as wells as the methylamine 18o,
permeability (frequently below 1 × 10 × 10 cm/s in Caco-
2 cells). This caused transporter-mediated elimination,
manifested by underestimation of in vivo clearance ranging
from 4- to 16-fold from in vitro systems. This was further
supported by elevated fecal levels (20−40% of the dose) after
an IV dose indicating biliary elimination. 18h and 18o also
showed a higher volume of distribution than expected for the
potency could be increased to an IC value of 3 nM. Halogen
50
substitution on ring B improved activity for the ring C
benzoates (not discussed above) and was analyzed for the
picolinates, too. The 18n chlorine-substituted analog of 18m
was tested with an IC of 1 nM in the lactate efflux assay and
ion-class, presumably due to enterohepatic recirculation (Table
34
7
).
To optimize the still limited permeability of derivatives
50
based on the acetylene scaffold, both moieties, the acid and the
sulfonamide, were probed with isosteres and bridge alternatives
to reduce the H-bond donor count. This work did not provide
compounds with improved properties and is described in the
Supporting Information.
proved to have a favorable property profile (vide infra).
Explanations for the high activity could be a specific van der
Waals interaction or the appropriate positioning of transporter-
interacting moieties on a rigid scaffold, which avoids negative
entropic penalties (preferred conformation of sulfonamide
discussion in Supporting Information, Figure S1).
Biophysical Target-Binding Confirmation. Solute car-
riers such as MCT4 are challenging treatment targets not only
due to subtype plurality and linked selectivity issues, but also
because, as membrane-bound targets, no enzymatic assay could
be established, and only cellular testing was possible. In
addition, confirmation of target engagement was difficult given
that biophysical measurements for targets that require specific
cellular environments often also require target-specific
experimental conditions. Thus, fluorescence cross-correlation
spectroscopy (FCCS) was selected to investigate target
The original screening hit 9a (Table 1) had demonstrated
that lactate accumulation (IC : 0.7 μM) could lead to a
50
viability effect (GI : 5.9 μM). Accordingly, optimized
5
0
derivatives from Table 7 were investigated with respect to
their effect on MDA-MB-231 cell viability. Figure 5 depicts
that in MDA-MB-231, inhibition of lactate efflux (IC ; x-axis)
5
0
correlates well with cell viability (GI after 144 h; y-axis) with
50
an off-set of around 10 (Supporting Information, Table S1).
Virtually all derivatives from Table 7 had intrinsic clearance
values below 10 μL/min/mg prot. in mouse liver microsomes.
Only derivatives 18h and 18n were in the range of 20 μL/min/
mg prot. and the 5-methyl derivative 18f showed high intrinsic
clearance. Most analogues showed a higher clearance in
hepatocyte incubation than with microsomes, indicating phase
II metabolism, possibly via formation of an acyl glucuronide
35
interaction. Although GFP-tagged expression of the MCT4
transporter and solubilization of an active conformation were
two major hurdles, the SAR work around compound 18b
suggested that the quinoline moiety could provide an exit
vector for fluorescent label attachment. To test this exit vector
hypothesis, some probe molecules 18r−18u with different
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linkers and terminal moieties were prepared and proven to be
still active in the cellular assay (Table 8).
lactate efflux and accumulation were measured in the MDA-
MB-231 cellular assay with the disclosed compounds. The
measured binding data for compounds from Table 7 correlated
very well with the inhibition of lactate efflux in MDA-MB-231
cells as shown in Figure 6. Considering the significant
difference in the complexity of the correlated experiments,
functional transporter inhibition in a cellular assay context
versus binding to the solubilized protein in a biophysical
experimental setup, the on−target activity in the cellular assay
could be considered a strong possibility.
Specific Binding of Compounds to the MCT4 Lactate
Transporter. HEK293 cells expressing human-MCT4-eGFP
were tested by imaging to determine if compounds bind to the
cell surface or if they can passively penetrate the membrane.
The addition of 10 μM labeled 18u (Supporting Information,
Figure S2) to the cells did result in binding to MCT4-GFP, but
only in the presence of 0.05% Triton, which allowed
permeabilization of the cells (Figure 7). This binding was
shown to be specific because it was displaced by the addition of
Table 8. Probe Molecules and the FCCS Tool 18u
a
entry
R
IC50 [μM]
1
1
1
1
1
1
8p
8q
8r
8s
8t
(C H O) -Phe
0.04
0.24
0.57
0.96
>25
8.8
2
4
2
(C H O) C H O-Bzl
2
4
3
6
12
C H O -(4-NO -Phe)
2
4
2
2
(C H O) C H NH-BOC
2
4
3
2
4
(C H O) C H -NH
2
2
4
3
2
4
8u
(C H O) C H -NH-Dye
2 4 3 2 4
a
Efflux inhibition assay in MDA-MB-231 cells and geometric means
of three replicates.
1
0 μM of unlabeled parental compound 18t (Figure 7C). This
The PEG-linked phenyl ether 18p showed an IC50 value of
0 nM. Different linking moieties and terminal functionalities
result suggested that binding of the compounds to MCT4
occurred at the cytosolic domain of MCT4.
4
also provided cellular activity in the micromolar range or even
lower (18q−18s). Based on these results, a fluorescent tracer
molecule (18u; chemical structure depicted in Supporting
Information; Figure S2) was prepared, built on amine 18t.
For the FCCS experiment, membranes of a stable cell line
expressing a C-terminal GFP-fusion of MCT4 were prepared
and solubilized using Lauryl Maltose Neopentyl Glycol
Binding of the compound to the mouse target was also
tested in live cells expressing a GFP-mouse-MCT4 clone by
confocal microscopy (Supporting Information, Figure S3) and
FCCS data confirmed a binding affinity of 301 nM
(
Supporting Information, Figure S4).
Mechanistic Confirmation by Measurement of Intra-
cellular Lactate Accumulation. Whether the observed
modulation of extracellular lactate levels was a result of
blocking lactate efflux from the cells was verified by
determining intracellular lactate accumulation by a HPLC-
based analytical method. As expected, increased intracellular
lactate accumulation was observed for compounds that
reduced extracellular lactate levels in a dose-dependent manner
(data not shown). This method was also used to exclude
(
LMNG). The addition of increasing amounts of 18u in a
titration experiment did confirm a specific or dose-dependent
interaction of the tracer with MCT4-GFP. The equilibrium
dissociation constant of the interaction was determined at 9.6
nM (data not shown). This interaction was used to determine
the IC s and K s of the test compounds in competition
5
0
i
experiments. The biophysical orthogonal target-binding results
support the assumption that MCT4 was inhibited when altered
Figure 6. Correlation of cellular lactate efflux inhibition in MDA-MB-231 cells (y-axis) with hMCT4 FCCS binding (x-axis) of compounds from
2
Table 7. Diagonal in green. Regression line in red, R : 0.8.
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Figure 7. Imaging of HEK293 cells expressing hMCT4-GFP (green) after the addition of 10 μM 18u (red). (A): no detergent was added, while in
B,C), 0.05% Triton X 100 was added to permeabilize the cells. (C): 10 μM of unlabeled parental 18t was added. Bars correspond to 50 μm.
(
compounds showing off-target related extracellular lactate
modulation.
addition, MiaPaCa2 CRISPR Knockout clones for MCT4 and
MCT1 were tested. The initially discovered hit compound 9a
inhibited lactate secretion exclusively in cell lines expressing
predominantly MCT4 transporter. The trend was maintained
during optimization of ethyl-, ethenyl-, and ethynyl linkers
(Supporting Information, Table S6).
In addition, modulation of lactate flux was confirmed by
monitoring compound-dependent intracellular concentration
1
4
changes of C-labeled lactic acid (radioactive lactate uptake
assay, Evotec) in MDA-MB-231 and in the high MCT1/low
MCT4 expressing SNU-398 cells (Table 9). The data
correlated well with the results of the extracellular lactate
efflux assay, in line with an MCT4-selective profile for the
newly synthesized compounds (Table 9).
In Vivo Testing. Compounds 18i and 18k were the first
seen to be applicable for in vivo studies because of their IC50
values for lactate efflux inhibition in MDA-MB-231 of 34 and
29 nM, respectively (Table 7). Derivative 18k also showed
favorable activity in alternative MCT4-high/MCT1-low cell
lines (NCI-H358, IC : 137 nM; NCI-H441, IC : 239 nM).
Table 9. Confirmation of Compound Selectivity by
5
0
50
Transporter Assay
In addition, the PK profiles of these derivatives were
considered to be acceptable. Oral administration of 18i as
homogeneous suspension in 0.5% methocel/0.25% tween20 in
radioactive lactate transporter
assay IC [nM]
lactate efflux assay IC50 [nM]
5
0
50 mM acetate buffer (pH 5.5) resulted in 45% bioavailability.
entry
MDA-MB-231
SNU-398
MDA-MB-231
SNU-398
Dosed at 30 mg/kg, 18i showed a free exposure exceeding the
cellular IC50 for approximately 3 h (Supporting Information,
Figure S5A). 18k administered orally as a homogeneous
suspension in 0.5% methocel/0.25% tween20 in 50 mM
phosphate buffer (pH 7.4) resulted in 35% bioavailability, and
administration at 30 mg/kg yielded a free exposure exceeding
the MDA-MB-231 IC50 for approximately 4 h (Supporting
Information, Figure S5B).
1
2
>30,000
>30,000
18
21
13
7.1
2.5
13,000
2700
>5000
>50,000
100
150
40
22
6.2
>50,000
>50,000
10,000
a
1
1
1
8j
b
7l
8h
a
8200
a
b
Structures shown in Table 7. Structures shown in Table 5.
MCT Isoform Selectivity. The selectivity of described
Compound tolerability was tested in SCID beige mice upon
administration of 18i and 18k up to 200 mg/kg once-per-day
(QD) for 10 days. Administration of either compound was well
tolerated and did not result in body weight loss or any adverse
clinical sign (data not shown).
MCT4 inhibitors toward the other MCT isoforms, especially
MCT1 and MCT2, was verified by testing extracellular lactate
secretion inhibition in selected cell lines with proven
predominant expression of given transporters: MDA-MB-231
and NCI-H358 for MCT4, SNU-398 for MCT1, MiaPaCa2
for MCT1 + MCT4, and RT4 for MCT2 + MCT3. In
To examine the capability of ethynylsulfonamide com-
pounds to modulate lactate levels and tumor growth in vivo,
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compounds 18i and 18k were tested in MCT4-high/MCT1-
low/no expressing xenograft models. Treatment of NCI-H358
or NCI-H441 MCT4-high/MCT1-low tumors did not result
in the tumoral lactate modulation or antitumor effect (data not
shown), suggesting that MCT4 inhibition alone was not
sufficient to elicit an effect in MCT1 expressing models.
Notably, while a slight to moderate increase of total tumoral
lactate could be observed as a consequence of intracellular
lactate accumulation in the MCT4-high/MCT1-no MDA-MB-
2
31 model for both compounds 18i and 18k (Supporting
Information Figure S7A and B), tumor growth inhibition could
not be achieved (Supporting Information Figure S7C and D).
It was hypothesized that the degree and/or duration of target
inhibition was insufficient to obtain a therapeutic effect.
Therefore, the search for compounds with improved properties
was continued.
Further optimization of acid ring C, linker, middle ring B,
bridge, and terminal moiety A with appropriate decoration
culminated in compound 18n. Lead 18n inhibited lactate efflux
in the MDA-MB-231 cell line with an IC50 of 1 nM. The on-
Figure 8. Free plasma concentration in female CD1 mice after a 30
mg/kg oral administration of 18n formulated in 20% kleptose in 50
mM PBS (pH 7.4). Blue and green lines correspond to the free
concentration, related to lactate efflux inhibition IC50 in MDA-MB-
231 and 10-fold of this value, respectively.
target activity was confirmed with a K of 11 nM, determined
i
via FCCS measurements (Table 7). MCT4 selectivity was
validated, as the compound did not inhibit lactate efflux to a
similar extent in SNU-398 and MiaPaca2, and only 600-fold
less in RT-4 cell lines (Supporting Information; Table S7). No
PGE2 affinity could be determined as a main off-target effect
for benzoic series (Supporting Information, Table S6). The
solubility in PBS (pH: 7.4) was acceptable (58 μg/mL) but
dropped significantly at lower pH (SGF; pH: 1; 12 μg/mL).
Passive permeability in the Caco-2 assay was low (2.2 10 ×
Table 10. Technical Profile of Selective MCT4 Inhibitor
18n
parameter
value
Phys Chem
MW
log P/D
tPSA
538
3.5/1.5
136
−
6
1
0
cm/s) and an efflux ratio of 10 was observed in the same
pKa
4.6
system. In vitro metabolic stability was good in the mouse
systems: the compound was stable in mouse microsomes and
PBS pH 7.4
24 h [μg/mL]
58
SGF pH 1.2
12
showed acceptable metabolic stability in mouse hepatocytes
6
FaSSIF pH 6.5
FeSSIF pH 5.0
268
232
(
18 μL/min/1 × 10 cells). In line with these results and the
modest free fraction in mouse plasma (0.6%), a low clearance
was observed in mouse (0.33 L/h/kg), corresponding to
approximately 5% of the liver blood flow.
properties
Mic (m/h) [μL/min/mg prot.]
Hep (m/h) [μL/min/1 × 10 cells]
23/250
18/29
0.6/0.5
2.2; 10
−11
6
Fu (m/h) [%]
Attempts to administer 18n via the oral route in mouse
using suspensions and unbuffered solutions resulted in modest
bioavailability (20%). First pass effects could be excluded as
the clearance was low. It was hypothesized that the low
solubility at low pH caused the compound to precipitate in the
stomach with a dissolution further down in the gastrointestinal
tract, inadequate for complete absorption. Therefore, it was
transformed into a potassium salt and administered as a
solution in 20% kleptose in 50 mM PBS (pH 7.4), which
resulted in an increased bioavailability (65%). Dosed at 30 mg/
kg, 18n showed a free exposure exceeding the MDA-MB-231
IC50 for 24 h (Figure 8). It is possible that free concentration
in tumor was lower as 18n was a substrate for efflux
transporters (Table 10). Assessing free levels in tumors is
currently not possible due to heterogeneity (vascularization
etc.), but pharmacodynamic effects (see below) were observed
in line with observed free exposure. Based on this extensive
characterization, we conclude that 18n is an excellent selective
MCT4 inhibitor tool to investigate the biological role of the
MCT4 transporter inhibition (Table 10).
Caco-2 PP [10 × 10− cm/s]; ER
6
HERG [% effect @ 10 μM]
cellular
MDA-MB-231
NCI-H358
SNU-398
RT4
MiaPaCa
IC /GI
50
1/11
3/−
>25,000/−
638/−
>25,000/4720
0.33
5
0
profile
[nM]
PK
CL [L/h/kg]
AUC [ng/mL × h]
iv
(
0.2 mg/kg)
601
C
[ng/mL]
489
1
max
/2
t1 [h]
Vss [L/kg]
0.4
methocel/tween20 in
water
F [ %] po
(10 mg/kg)
19
methocel/tween20 in
50 mM Na-citrate pH 3
20
20% glycofurol in water
20% kleptose in 50 mM
phosphate pH 7.4
27
65
Based on the results from 18i and 18k in high MCT4
expressing xenograft models, an alternative approach was
followed for the optimized compound 18n to also address the
well-described immunosuppressive role of lactate in the tumor
model was selected as the syngeneic model for its high
MCT4/low MCT1 expression. In addition, we found that
MC38 tumors accumulate higher levels of lactic acid in
comparison to other syngeneic models tested (data not
2
2
microenvironment. The MC38 murine colorectal cancer
shown). 18n activity was first characterized in vitro in
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Figure 9. Effect of 18n treatment on tumoral lactate and tumor growth. (A) Tumoral intracellular lactate levels were measured in MC38 tumor
samples collected 2 h after single administration of the indicated compounds or combination. Bars indicate means ± SD. Statistical significance was
assessed with One-way ANOVA, followed by Tukey’s multiple comparison test. (B) Mice bearing MC38 tumors (n = 10) were treated with vehicle,
18n, or 1 as a single agent and in combination at the indicated concentrations. Statistical significance was tested with Two-way RM-ANCOVA,
followed by post-hoc Tukey multiple pairwise comparisons. po, oral administration; QD, once daily; SD, standard deviation; and SEM, standard
error of the mean.
MC38 cells, alone or in combination with MCT1/2 inhibitor
, demonstrating that, in this model, modulation of lactate
capable sulfonamide 18n. This highly potent derivative
demonstrated lactate efflux inhibition and intracellular lactate
accumulation in high MCT4-expressing cell lines in vitro and
was found to be inactive in cells, which do not highly express
this transporter subtype in comparison to others. Target
binding of the compounds to the cytosolic domain of MCT4
was confirmed by FCCS, while off-target activity was not
detected. Salt formation and application as solution-formula-
tion with an appropriate vehicle in PK studies demonstrated a
free exposure exceeding cellular IC50 for 24 h. Thus, 18n is
considered an attractive compound to investigate pharmaco-
logical effects of selective MCT4 inhibition in vitro and in vivo.
This compound is available upon request.
1
transport and decrease of cellular viability can be achieved
exclusively when both MCT1 and MCT4 are inhibited. This
effect was confirmed in MC38 MCT1 and MCT4 CRISPR
Knockout clones (Supporting Information; Figure S6). In vivo
tolerability of 18n was tested in non-tumor-bearing C57Bl/6
mice; oral administration of 18n up to 30 mg/kg QD for 10
days was well tolerated without body weight loss or side effects
(
data not shown). 18n was then tested in vivo for its ability to
modulate tumoral intracellular lactate accumulation. MC38
tumor-bearing mice were treated with a single administration
of 18n as potassium salt, MCT1/2 inhibitor 1, or a
combination of the two at 30 and 150 mg/kg, respectively;
animals in the control group were administered with both
vehicles. Tumor samples were collected 2 h after single
administration and lactate was measured. Only the 18n + 1
combination group exhibited a significant tumoral intracellular
lactate accumulation (Figure 9A), as previously observed in
vitro (Supporting Information; Figure S6). Administered
compounds were quantified in plasma and tumor from the
dosed animals, to confirm exposure (data not shown). To test
the antitumor effect of MCT4 inhibition alone or in
combination with MCT1/2 inhibition, the efficacy of 18n as
a single agent or in combination with 1 was evaluated using the
MC38 model. 18n showed no significant antitumor activity,
neither when administered as monotherapy nor in combina-
tion with 1 (Figure 9B).
Initial in vivo studies assessing PD biomarker modulation by
1
8n in murine MC38 tumors (MCT4-high/MCT1-low)
revealed that an increase in lactate accumulation in tumor
cells could only be achieved by concomitant inhibition of
MCT4 and MCT1/2. However, even though the combined
inhibition of MCT1/2 and MCT4 led to decreased viability in
MC38 cell viability in vitro, combination of 18n with MCT1/2
inhibitor 1 did not translate into a significant tumor growth
inhibition in this tumor model. Similar observations were made
in the MCT4-high/MCT1-no xenograft model MDA-MB-231,
where in vitro findings translated into in vivo tumoral lactate
modulation but not into the antitumor effect. Taken together,
these data suggest that MCT1 plus MCT4 lactate transport
inhibition, while resulting in tumoral lactate modulation, may
not be sufficient to achieve antitumor activity in these models.
In line with these results, in vivo tumor growth inhibition
was reported for the MCT4 inhibitor AZD0095, not as a single
agent but only if combined with VEGFR TKI, αCTLA4, or
Similarly, modulation of tumoral lactate with concomitant
lack of antitumor response was observed for 18i and 18k in the
MDA-MB-231 model as well (Supporting Information, Figure
S7).
23
αPD1 antibodies. More studies are required to establish if
this effect is a general limitation of MCT4 inhibitors, with or
without MCT1 inhibition, or if a combination approach is
required to achieve reduction of tumor growth. In fact,
extensive literature has described rationales for combining
CONCLUSIONS
■
9
In summary, the flexible micromolar active amide-screening hit
a was optimized to a rigid nanomolar active and in vivo
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specific MCT inhibitors with investigational or approved
treatments with different mechanisms of action, such as
7: Agilent Series; Kinetex EVO C18 5.0 μm; 2.0 mL/min; solvent A:
H O + 0.05% HCOOH; solvent B: acetonitrile + 0.04% HCOOH;
2
9
,12
19,20
220 nm; 0 to 6.5 min:1% B to 99% B; 6.5 to 7.0 min: 100% B. LCMS
8: column: CORTECS C18 100A, 2.1 × 50 mm, 2.7 μm; mobile
phase A: water/0.1% FA, mobile phase B: acetonitrile/0.1% FA; flow
rate: 1.0 mL/min; gradient:10% B to 100% B in 2.0 min, hold 0.6
min; 254 nm; and LCMS 9: column: Ascentis Express C18, 3.0 × 50
checkpoint inhibitors,
radiation. Moreover, combination of MCT inhibitors with
molecules able to interfere with cellular metabolism, such as
tyrosine kinase inhibitors,
or
3
6
8
the antidiabetic drug metformin or glutaminase inhibitor CB-
2
2
8
39, results in reduction or loss of cellular viability.
mm, 2.7 μm; mobile phase A: H O/0.05% TFA, mobile phase B:
2
Consequently, a sequence of experiments was initiated to
identify compounds synergistic with MCT4 inhibitors.
Furthermore, data for 18n together with published data
indicate that the MCT1/4 expression profile of targeted tumor
cells needs to be considered. Overall, 18n is considered an
attractive compound to investigate the pharmacological effects
of selective MCT4 inhibition on lactate modulation, but it may
require additional investigation into target coverage, tumor
model selection, and/or combination partners in order to
achieve substantial tumor growth inhibition in vivo.
ACN/0.05% TFA; flow rate: 1.5 mL/min; gradient: 5% B to 100% B
in 1.2 min, hold 0.5 min; 254 nm.
2-(5-{2-[(Naphthalene-2-carbonyl)-amino]-phenyl}-pentyl)-ben-
zoic Acid (9a). Methyl 2-iodobenzoate (1.7 mL; 11.22 mmol) was
added into a microwave vial and dissolved in diethylamine (20 mL).
Under nitrogen, 3-butin-1-ol 4 (2.6 mL; 33.66 mmol), copper(I)
iodide (1.3 g; 6.73 mmol), and bis(triphenylphosphine)palladium(II)
chloride (15,2% Pd, 2.4 g; 3.37 mmol) were added and the reaction
was stirred for 15 h at RT. The reaction mixture was filtered, the
solvent was removed under reduced pressure, and the residue was
purified by flash chromatography giving the product 5 as a brown oil
23
(
2.2 g; 90% yield).
Methyl 2-(4-hydroxybut-1-yn-1-yl)benzoate 5 (1.8 g; 7 mmol) was
EXPERIMENTAL SECTION
■
dissolved in THF (20 mL). Noblyst P1086 (Pd/C-5%; E101 R;
50% H O; 14 g; 45 mmol) was added, and the mixture was stirred at
Chemistry. General Information. All reactions were carried out
under a nitrogen atmosphere or in sealed vials unless noted otherwise.
Dry solvents and reagents were of commercial quality and were used
as purchased. Reactions were magnetically stirred and monitored by
thin-layer chromatography (TLC) using Merck silica gel 60 F254 by
fluorescence quenching under UV light or by high-pressure liquid
chromatography with subsequent mass detection, except if indicated
otherwise. Oxidation reactions carried out with peroxides such as tert-
butyl hydroperoxide, magnesium monoperoxyphthalate hydrates, or
others were analyzed before workup with Merck test MQuant. In the
case of detected traces of peroxide, the reaction mixture was treated
with sodium thiosulfate accordingly. The purity of the compounds
reported in the manuscript was analyzed through the HPLC-MS
methodology. In addition, TLC plates were stained using
phosphomolybdic acid or potassium permanganate stain. Chromato-
graphic purification of products (flash chromatography) was
performed on Isco Combiflash systems using Redisep columns and
ethyl acetate (EtOAc)/heptane gradients. Concentration under
reduced pressure was performed by rotary evaporation at 40 °C at
∼
2
RT under hydrogen (500 mL, 22 mmol). The catalyst was filtered and
the solution evaporated to dryness under reduced pressure giving
methyl 2-(4-hydroxybutyl)benzoate 6a as a colorless oil (1.6 g; 73%
yield).
2-(4-Hydroxy-butyl)-benzoic acid methyl ester (1.6 g; 6.12 mmol)
was dissolved in THF (50 mL). The solution was cooled to 0 °C and
triphenylphosphine (2.4 mL; 10.71 mmol) and NBS (1.6 g; 9.18
mmol) were added. The reaction was stirred for 15 h at RT. The
solvent was removed under reduced pressure and the residue was
purified by flash chromatography giving the product 6b as a colorless
oil (1.9 g; 62% yield).
2-(4-Bromo-butyl)-benzoic acid methyl ester (1.9 g; 3.80 mmol)
was dissolved in acetonitrile (20 mL). To the solution, triphenyl-
phosphine (0.9 mL; 4.18 mmol) was added, and the reaction was
stirred for 4 days at 65 °C. The solvent was removed under reduced
pressure and the residue was washed with toluene and dried giving the
crude product 6c as a colorless solid (3 g; content 82%).
1
4
-(2-Methoxycarbonyl-phenyl)-butyl]-triphenyl-phosphonium bro-
mide 6c (2, 50 g; 3.86 mmol) was dissolved in 1,4-dioxane (50 mL).
-Nitrobenzaldehyde 7 (5.6 g; 3.86 mmol), potassium carbonate (0.7
an appropriate pressure unless otherwise stated. H NMR (in DMSO-
d6) and mass spectra are in agreement with the structures and were
recorded on a Bruker NMR 400 MHz spectrometer if not stated
differently (TMS as an internal standard) and Vacuum Generators
VG 70−70 or 70−250 at 70 eV, respectively. High-resolution mass
spectroscopy (HRMS) analyses (obtained with a Bruker maXis
analyzer) for the final products were within 0.4% of calculated values
if not stated otherwise. All compounds reported in the manuscript
have a purity ≥95% unless noted otherwise.
2
g; 5.56 mmol), and water (0.17 mL) were added to the solution and
the reaction was stirred under nitrogen overnight at reflux. The
reaction mixture was diluted with water and the layers were separated.
The water layer was extracted with ethyl acetate two times, the
combined organic layers were washed with water and brine, dried over
sodium sulfate, filtered, and evaporated to dryness. The residue was
purified by flash-chromatography giving the product 8a as a yellow oil
as a cis/trans mixture (1.2 g; 93% content).
General Conditions. LCMS method 1: HPLC; MERCK-
HITACHI LaChrom; chromolith performance RP-18e 100−4.6; 3.3
mL/min; solvent A: H O + 0.05% HCOOH; solvent B: acetonitrile +
Methyl 2-[(4E)-5-(2-nitrophenyl)pent-4-en-1-yl]benzoate 8a (1.1
g; 3.2 mmol) and Noblyst P1086 (1.1 g; Pd/C-5%; E101 R; ∼50%
H O) were suspended in THF (10 mL) and hydrogen (350 mL) was
2
0
.04% HCOOH; 220 nm; 0 to 2.0 min: 0% B to 100% B; 2.0 to 2.5
min: 100% B; LCMS method 2: Agilent 1200 Series; Chromolith RP-
2
1
8e 50−4.6 mm; 3.3 mL/min; solvent A: H O + 0.05% HCOOH;
added at RT overnight. After filtration of the catalyst, the remainder
was evaporated to dryness under reduced pressure giving the product
8b as a yellow oil (921 mg; 92% content).
2
solvent B: acetonitrile + 0.04% HCOOH; 220 nm; 0 to 2.0 min: 0% B
to 100% B; 2.0 to 2.5 min: 100% B; LCMS Method 3: Column:
Chromolith RP-18e 50−4.6 mm; A: H O + 0.05% HCOOH |B:
2-[5-(2-Amino-phenyl)-pentyl]-benzoic acid methyl ester 8b (50
mg; 0.14 mmol) was dissolved in DCM (3 mL), triethylamine (54
μL; 0.42 mmol) was added, and the mixture was stirred at RT for 20
min. Then, 2-naphthoyl chloride (28 mg; 0.15 mmol) was added in
one portion. The reaction mixture was stirred overnight at RT and
then diluted with ethyl acetate and water. The layers were separated
and the water layer was extracted two times with ethyl acetate. The
combined organic layers were washed with water and brine, dried over
Na SO , filtered, and evaporated to dryness giving the product as a
2
MeCN + 0.04% HCOOH/4% → 100% B: 0 → 2,8 min|100% B: 2.8
→
3,3 min; LCMS methods 4: column: Waters Acquity UPLC HSS
C18 (2.1 × 50 mm, 1.8 μm); Solvent A: H O + 0.1% formic acid;
2
Solvent B: ACN + 0.1% formic acid; Flow: 0.5 mL/min; gradient: 0
min: 95% B, 4 min: 5% B; 5 min: 5% B; 5.2 min: 95% B; 6 min: 95%
B; LCMS method 5: column: Kinetex XB C18 (4.6 × 50 mm, 2.6
μm); solvent A: H O + 0.1% formic acid; solvent B: ACN + 0.1%
2
formic acid; flow: 0.5 mL/min; gradient: 0 min: 20% B, 6.7 min: 80%
2
4
B; 7.5 min: 80% B; 7.8 min: 95% B; 9.5 min: 95% B; LCMS method
white sticky oil (65 mg; 89% yield).
6
: column: Waters Symmetry C18 (3.9 × 150 mm × 5 μm); solvent
2-(5-{2-[(Naphthalene-2-carbonyl)-amino]-phenyl}-pentyl)-ben-
zoic acid methyl ester (65 mg; 0.12 mmol) was dissolved in methanol
(3 mL), and sodium hydroxide solution (c(NaOH) = 1 mol/l (1 N)
A: H O + 0.1% formic acid; solvent B: ACN + 0.1% formic acid; flow:
2
1.2 mL/min; gradient: 0 min: 20% B; 20 min: 80% B; 22 min; LCMS
1
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Article
0
.50 mL) was added. The reaction was stirred at RT for 2 days, and
2-{5-[2-(4-methylbenzamido)phenyl]pentyl}benzoate as a light
brown oil (60 mg; 79%). Saponification of this intermediate with
sodium hydroxide yielded the product as a colorless solid (20 mg;
then HCl was added. After dilution with water and ethyl acetate, the
organic layer was washed with water and brine, dried over Na SO ,
filtered, and evaporated to dryness. The residue was purified by
2
4
1
27%). LCMS method 2: 1.826 min; 402.1 (M + H). H NMR (700
chromatography giving the product as a white solid (17 mg 31%).
LCMS method 1: 1.93 min; 438.00 (M + H). H NMR (400 MHz,
MHz, DMSO-d ): δ 12.76 (s, 1H), 9.77 (s, 1H), 7.87−7.83 (m, 2H),
6
1
7.74 (dd, J = 7.7, 1.4 Hz, 1H), 7.37 (td, J = 7.5, 1.5 Hz, 1H), 7.31−
7.15 (m, 8H), 2.86−2.81 (m, 2H), 2.61−2.56 (m, 2H), 2.38 (s, 3H),
1.57−1.46 (m, 4H), 1.29 (p, J = 7.5 Hz, 2H). HRMS calcd for
C H NO , 402.2064; found, 402.2059.
DMSO-d ): δ 12.72 (s, 1H), 10.03 (s, 1H), 8.57 (s, 1H), 8.05−7.98
6
(
m, 4H), 7.71 (dd, J = 7.7, 1.5 Hz, 1H), 7.67−7.58 (m, 2H), 7.37−
7
.33 (m, 1H), 7.32−7.18 (m, 5H), 7.15−7.10 (m, 1H), 2.87−2.80
m, 2H), 2.68−2.61 (m, 2H), 1.63−1.47 (m, 4H), 1.37−1.27 (m,
H). HRMS calcd for C H NO , 438.2064; found, 438.2057.
2
6
27
3
(
2
2-{5-[2-(4-Ethyl-benzoylamino)-phenyl]-pentyl}-benzoic Acid
(9g). The synthesis was carried out as described for 9a. 2-[5-(2-
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 4-ethyl-benzoic acid (27 mg; 0.18 mmol) gave methyl
2-{5-[2-(4-ethylbenzamido)phenyl]pentyl}benzoate as a brown oil
(57 mg; 74%). Saponification of this intermediate with sodium
hydroxide yielded the product as a colorless solid (35 mg; 47%).
2
9
27
3
2
-(5-{2-[(Naphthalene-1-carbonyl)-amino]-phenyl}-pentyl)-ben-
zoic Acid (9b). The synthesis was carried out as described for 9a. 2-
5-(2-Amino-phenyl)-pentyl]-benzoic acid methyl ester (63 mg; 0.17
mmol) and naphthalene-1-carbonyl chloride (28 μL; 0.18 mmol) gave
-(5-{2-[(Naphthalene-1-carbonyl)-amino]-phenyl}-pentyl)-benzoic
[
2
1
acid methyl ester (65 mg; 83% yield). Saponification of this
LCMS Method 2: 1.890 min; 416.2 (M + H). H NMR (700 MHz,
intermediate (72 mg; 0.13 mmol) with sodium hydroxide yielded
DMSO-d ): δ 9.79 (s, 1H), 7.89−7.85 (m, 2H), 7.74 (dd, J = 7.8, 1.4
6
the product as a white solid (20 mg; 36% yield). LCMS method 2;
Hz, 1H), 7.37 (m, 1H), 7.34−7.16 (m, 9H), 2.86−2.82 (m, 2H), 2.68
(q, J = 7.6 Hz, 2H), 2.62−2.57 (m, 2H), 1.57−1.47 (m, 4H), 1.29 (p,
J = 7.5 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H). HRMS calcd for
C H NO , 416.2220; found, 416.2215.
1
1
1
4
(
2
.92 min; 438.00 (M + H). H NMR (400 MHz, DMSO-d ): δ
6
2.98−12.43 (m, 1H), 10.02 (s, 1H), 8.56 (s, 1H), 8.05−7.99 (m,
H), 7.71 (dd, J = 7.7, 1.5 Hz, 1H), 7.67−7.58 (m, 2H), 7.37−7.33
2
7
29
3
m, 1H), 7.32−7.17 (m, 5H), 7.14−7.10 (m, 1H), 2.87−2.81 (m,
2-{5-[2-(4-Fluoro-benzoylamino)-phenyl]-pentyl}-benzoic Acid
H), 2.68−2.61 (m, 2H), 1.63−1.47 (m, 4H), 1.37−1.27 (m, 2H).
(9h). The synthesis was carried out as described for 9a. 2-[5-(2-
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 4-fluoro-benzoic acid (25 mg; 0.18 mmol) gave methyl
2-{5-[2-(4-fluorobenzamido)phenyl]pentyl}benzoate as a brown oil
(69 mg; 92%). Saponification of this intermediate with sodium
HRMS calcd for C H NO , 438.2064; found, 438.2059.
29
27
3
2
-(5-{2-[(4-Methyl-naphthalene-1-carbonyl)-amino]-phenyl}-
pentyl)-benzoic Acid (9c). The synthesis was carried out as described
for 9a. 2-[5-(2-Amino-phenyl)-pentyl]-benzoic acid methyl ester (57
mg; 0.18 mmol) and 4-methyl-1-naphthoic acid (33 mg; 0.18 mmol)
gave 2-(5-{2-[(4-methyl-naphthalene-1-carbonyl)-amino]-phenyl}-
pentyl)-benzoic acid methyl ester as a clear sticky oil (50 mg;
hydroxide yielded the product as a white solid (51 mg; 70%). LCMS
1
method 2: 1.794; 406.1 (M + H). H NMR (700 MHz, DMSO-d ): δ
6
12.76 (s, 1H), 9.90 (s, 1H), 8.05−7.99 (m, 2H), 7.74 (dd, J = 7.7, 1.4
Hz, 1H), 7.38 (td, J = 7.5, 1.5 Hz, 1H), 7.35−7.30 (m, 2H), 7.30−
7.16 (m, 6H), 2.86−2.81 (m, 2H), 2.61−2.56 (m, 2H), 1.57−1.46
(m, 4H), 1.29 (p, J = 7.5 Hz, 2H). HRMS calcd for C H FNO ,
48%). Saponification of this intermediate with sodium hydroxide
yielded the product as white solid (9.40 mg; 24%). LCMS method 3:
1
2
.63 min; 452.00 (M + H). H NMR (500 MHz, DMSO-d ): δ 12.78
6
25 24
3
(
7
(
2
s, 1H), 9.96 (s, 1H), 8.31−8.25 (m, 1H), 8.10 (d, J = 8.4 Hz, 1H),
.75 (dd, J = 7.8, 1.5 Hz, 1H), 7.65−7.58 (m, 2H), 7.55 (m, 1H), 7.45
d, J = 7.6 Hz, 1H), 7.43−7.36 (m, 2H), 7.32−7.18 (m, 5H), 2.92−
.86 (m, 2H), 2.71 (s, 3H), 2.69−2.63 (m, 2H), 1.59 (m, 4H), 1.38
m, 2H), 1.24 (s, 1H). HRMS calcd for C H NO , 452.2220; found,
406.1813; found, 406.1811.
2-{5-[2-(2-Fluoro-benzoylamino)-phenyl]-pentyl}-benzoic Acid
(9i). The synthesis was carried out as described for 9a. 2-[5-(2-
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 2-fluoro-benzoic acid (25 mg; 0.18 mmol) gave methyl
2-{5-[2-(2-fluorobenzamido)phenyl]pentyl}benzoate as a brown oil
(58 mg; 77%). Saponification of this intermediate with sodium
hydroxide yielded the product as a white solid (49 mg; 68%). LCMS
(
3
0
27
3
4
52.2218.
2
-[5-(2-Benzoylamino-phenyl)-pentyl]-benzoic Acid (9d). The
synthesis was carried out as described for 9a. 2-[5-(2-Amino-
phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18 mmol) and
benzoic acid (22 mg; 0.18 mmol) gave methyl 2-[5-(2-
benzamidophenyl)pentyl]benzoate as a brown oil (61 mg; 84%).
Saponification of this intermediate with sodium hydroxide yielded the
1
method 2: 1.872 min; 388.1 (M + H). H NMR (700 MHz, DMSO-
d ): δ 12.78 (s, 1H), 9.80 (s, 1H), 7.75 (dd, J = 7.7, 1.4 Hz, 1H), 7.67
6
(m, 1H), 7.57 (tdd, J = 7.4, 5.1, 1.8 Hz, 1H), 7.44−7.38 (m, 2H),
7.36−7.29 (m, 2H), 7.26 (m, 3H), 7.21 (m, 2H), 2.92−2.87 (m, 2H),
2.65−2.60 (m, 2H), 1.60−1.52 (m, 4H), 1.35 (p, J = 7.5 Hz, 2H).
HRMS calcd for C H FNO , 406.1813; found, 406.1809.
product as a white solid (25 mg; 36%). LCMS method 2: 1.772 min;
1
3
9
1
7
1
3
88.1 (M + H). H NMR (700 MHz, DMSO-d ): δ 12.76 (s, 1H),
6
25 24
3
.87 (s, 1H), 7.95 (dd, J = 7.9, 1.5 Hz, 2H), 7.73 (dd, J = 7.9, 1.4 Hz,
H), 7.60−7.55 (m, 1H), 7.50 (t, J = 7.7 Hz, 2H), 7.37 (m, 1H),
.31−7.16 (m, 6H), 2.87−2.82 (m, 2H), 2.62−2.57 (m, 2H), 1.58−
.47 (m, 4H), 1.30 (p, J = 7.6 Hz, 2H). HRMS calcd for C H NO ,
88.1907; found, 388.1899.
-{5-[2-(2-Methyl-benzoylamino)-phenyl]-pentyl}-benzoic Acid
9e). The synthesis was carried out as described for 9a. 2-[5-(2-
2-{5-[2-(2-Chloro-benzoylamino)-phenyl]-pentyl}-benzoic Acid
(9j). The synthesis was carried out as described for 9a. 2-[5-(2-
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 2-chloro-benzoic acid (28 mg; 0.18 mmol) gave methyl
2-{5-[2-(2-chlorobenzamido)phenyl]pentyl}benzoate as a brown oil
(70 mg; 90%). Saponification of this intermediate with sodium
hydroxide yielded the product as a white solid (68 mg; 89%). LCMS
2
5
25
3
2
(
1
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 2-methyl-benzoic acid (24 mg; 0.18 mmol) gave methyl
method 2: 1.818 min; 404.1 (M + H). H NMR (700 MHz, DMSO-
d6): δ 12.79 (s, 1H), 9.95 (s, 1H), 7.76 (dd, J = 8.0, 1.5 Hz, 1H), 7.54
(m, 2H), 7.49 (m, 1H), 7.46−7.40 (m, 2H), 7.38 (dd, J = 7.7, 1.5 Hz,
1H), 7.29−7.24 (m, 3H), 7.21 (m, 2H), 2.93−2.88 (m, 2H), 2.66−
2.61 (m, 2H), 1.57 (h, J = 7.5 Hz, 4H), 1.36 (p, J = 7.5 Hz, 2H).
HRMS calcd for C H ClNO , 422.1517; found, 422.1508.
2
-{5-[2-(2-methylbenzamido)phenyl]pentyl}benzoate as a light
brown oil (59 mg; 79%). Saponification of this intermediate with
sodium yielded the product as a colorless solid (23 mg; 32%). LCMS
1
method 2: 1.821 min; 402.1 (M + H). H NMR (700 MHz, DMSO-
2
5
24
3
d6): δ 12.79 (s, 1H), 9.75 (s, 3H), 7.76 (dd, J = 7.8, 1.5 Hz, 3H),
2-{5-[2-(3-Chloro-benzoylamino)-phenyl]-pentyl}-benzoic Acid
(9k). The synthesis was carried out as described for 9a. 2-[5-(2-
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 3-chloro-benzoic acid (28 mg; 0.18 mmol) gave methyl
2-{5-[2-(3-chlorobenzamido)phenyl]pentyl}benzoate as a yellow oil
(69 mg; 90%). Saponification of this intermediate with sodium
hydroxide yielded the product as a white solid (67 mg; 89%). LCMS
7
1
2
.45−7.39 (m, 6H), 7.39−7.33 (m, 6H), 7.29 (s, 2H), 7.29−7.24 (m,
3H), 7.24−7.16 (m, 6H), 2.92−2.88 (m, 6H), 2.64−2.59 (m, 6H),
.40 (s, 9H), 1.56 (h, J = 7.7 Hz, 12H), 1.36 (p, J = 7.5 Hz, 6H).
HRMS calcd for C H NO , 402.2064; found, 402.2058.
26
27
3
2
-{5-[2-(4-Methyl-benzoylamino)-phenyl]-pentyl}-benzoic Acid
(
9f). The synthesis was carried out as described for 9a. 2-[5-(2-
1
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 4-methyl-benzoic acid (24 mg; 0.18 mmol) gave methyl
method 2: 1.866 min; 404.0 (M + H). H NMR (700 MHz, DMSO-
d6): δ 12.76 (s, 1H), 10.01 (s, 2H), 7.98 (t, J = 1.9 Hz, 2H), 7.90 (d, J
1
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Article
=
=
7.7 Hz, 2H), 7.74 (dd, J = 7.8, 1.4 Hz, 2H), 7.65 (m, 2H), 7.54 (t, J
7.9 Hz, 2H), 7.37 (m, 2H), 7.30−7.19 (m, 10H), 7.18 (dd, J = 7.6,
the precipitate was filtered and washed using a funnel with a small
volume of diethyl ether to afford 3-((E)-2-{2-[(naphthalene-1-
carbonyl)-amino]-phenyl}-vinyl)-benzoic acid methyl ester (84 mg;
85% yield) as a white solid. LC−MS 4: 4.02 min; product not ionized.
To a solution of 3-((E)-2-{2-[(naphthalene-1-carbonyl)-amino]-
phenyl}-vinyl)-benzoic acid methyl ester (50 mg; 0.12 mmol) in
methanol (10 mL), palladium on carbon (10% dry, 10 mg; 0.01
mmol) was added. The resulting mixture was subjected to
hydrogenation using a Parr apparatus for 3 h. Then, the reaction
mixture was filtered through a pad of Celite and silica gel, which was
then washed several times with ethyl acetate. The filtrate was
evaporated to dryness under reduced pressure, redissolved in a small
volume of DCM, and once again evaporated to dryness under reduced
pressure to afford 3-(2-{2-[(naphthalene-1-carbonyl)-amino]-phe-
nyl}-ethyl)-benzoic acid methyl ester (49 mg; 95% yield) as a white
1
8
4
.3 Hz, 2H), 2.87−2.82 (m, 4H), 2.61−2.56 (m, 4H), 1.58−1.47 (m,
H), 1.30 (p, J = 7.5 Hz, 4H). HRMS calcd for C H ClNO ,
2
5
24
3
22.1517; found, 422.1515.
-{5-[2-(3,4-Dichloro-benzoylamino)-phenyl]-pentyl}-benzoic
2
Acid (9l). The synthesis was carried out as described for 9a. 2-[5-(2-
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 3,4-dichloro-benzoic acid (34 mg; 0.18 mmol) gave
methyl 2-{5-[2-(3,4-dichlorobenzamido)phenyl]pentyl}benzoate as
an orange oil (53 mg; 85%). Saponification of this intermediate
with sodium hydroxide yielded the product as a white solid (47 mg;
1
5
7%). LCMS method 2: 1.961 min; 438.0 (M + H). H NMR (700
MHz, DMSO-d ): δ 10.06 (s, 1H), 8.17 (d, J = 2.1 Hz, 1H), 7.90 (m,
6
1
1
H), 7.78 (t, J = 8.5 Hz, 1H), 7.73 (dd, J = 7.7, 1.5 Hz, 1H), 7.37 (m,
H), 7.31−7.26 (m, 2H), 7.25 (dd, J = 7.5, 1.3 Hz, 1H), 7.24−7.20
1
solid. LC−MS 5: 9.10 min; 410.10 (M + H). H NMR (400 MHz,
(
m, 2H), 7.16 (dd, J = 7.7, 1.3 Hz, 1H), 2.86−2.81 (m, 2H), 2.60−
DMSO): δ 10.17 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 8.3
Hz, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.84 (d, J = 1.4 Hz, 1H), 7.82−
7.76 (m, 2H), 7.65−7.48 (m, 5H), 7.41 (d, J = 7.6 Hz, 1H), 7.35 (d, J
= 7.5 Hz, 1H), 7.30 (d, J = 7.1 Hz, 1H), 7.24 (d, J = 7.1 Hz, 1H), 3.80
2
.55 (m, 2H), 1.57−1.46 (m, 4H), 1.30 (p, J = 7.5 Hz, 2H). HRMS
calcd for C H Cl NO , 456.1128; found, 456.1116.
25
23
2
3
2
-{5-[2-(2,6-Dichloro-benzoylamino)-phenyl]-pentyl}-benzoic
Acid (9m). The synthesis was carried out as described for 9a. 2-[5-(2-
Amino-phenyl)-pentyl]-benzoic acid methyl ester (57 mg; 0.18
mmol) and 2,6-dichloro-benzoic acid (33 mg; 0.18 mmol) gave
methyl 2-{5-[2-(2,6-dichlorobenzamido)phenyl]pentyl}benzoate as
an orange oil (52 mg; 85%). Saponification of this intermediate
(
s, 3H), 2.99 (s, 4H).
To a solution of 3-(2-{2-[(naphthalene-1-carbonyl)-amino]-
phenyl}-ethyl)-benzoic acid methyl ester (40 mg; 0.10 mmol) in
methanol (3 mL) and water (3 mL), sodium hydroxide (1 M in water,
0
4
was acidified with 2 M HCl. The solution was extracted with ethyl
acetate. The organic layers were combined, washed with a small
amount of water and brine, dried over anhydrous Na SO , and
.49 mL; 0.97 mmol) was added. The resulting mixture was heated at
5 °C for 3 h. Methanol was evaporated, and the aqueous solution
with sodium hydroxide yielded the product as a white solid (45 mg;
1
5
7%). LCMS method 2: 1.854 min; 438.1 (M + H). H NMR (700
MHz, DMSO-d ): δ 10.16 (s, 1H), 7.76 (dd, J = 8.1, 1.5 Hz, 1H),
6
7
7
7
2
.56 (d, J = 8.1 Hz, 2H), 7.49 (dd, J = 8.7, 7.5 Hz, 1H), 7.43 (td, J =
.4, 1.5 Hz, 1H), 7.40 (dd, J = 7.8, 1.5 Hz, 1H), 7.29−7.25 (m, 3H),
.23 (td, J = 7.5, 1.8 Hz, 1H), 7.20 (td, J = 7.3, 1.5 Hz, 1H), 2.92−
.88 (m, 2H), 2.67−2.62 (m, 2H), 1.56 (tq, J = 14.8, 7.6, 6.8 Hz, 4H),
.36 (p, J = 7.7 Hz, 2H). HRMS calcd for C H Cl NO , 456.1128;
2
4
evaporated to dryness under reduced pressure. The residue was
redissolved in a small volume of DCM, which was once again
evaporated to dryness under reduced pressure affording the product
16a (37 mg; 0.09 mmol; 89% yield) as a white solid. LC−MS 5: 7.80
1
2
5
23
2
3
1
found, 456.1124.
min; 394.10 (M − H). H NMR (400 MHz, DMSO): δ 12.85 (s,
3
-(2-{2-[(Naphthalene-1-carbonyl)-amino]-phenyl}-ethyl)-ben-
1
8
7
2
H), 10.17 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 8.2 Hz, 1H),
.02 (dd, J = 7.6, 1.7 Hz, 1H), 7.87 (d, J = 1.4 Hz, 1H), 7.80 (d, J =
.0 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.62 (m, 1H), 7.59−7.53 (m,
H), 7.51 (d, J = 7.5 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.41−7.34
zoic Acid (16a). Thionyl chloride (0.47 mL; 6.48 mmol) was dropped
into the solution of 3-vinyl-benzoic acid 10 (0.80 g; 5.40 mmol) in
methanol (10 mL) over 10 min at 0 °C. After 3 h, the second portion
of thionyl chloride (0.47 mL; 6.48 mmol) was added at RT, and the
reaction mixture was stirred overnight. Methanol was evaporated, and
the residue was poured in water. The pH was adjusted to 7 with 1 M
NaOH and the resulting mixture was extracted with diethyl ether (3 ×
(m, 2H), 7.30 (d, J = 7.0 Hz, 1H), 7.24 (d, J = 7.0 Hz, 1H), 3.02−
2
.98 (m, 4H). HRMS calcd for C H NO , 396.1594; found,
26 21 3
3
96.1583.
-{2-[2-(Naphthalene-1-sulfonylamino)-phenyl]-ethyl}-benzoic
3
2
0 mL). Organic extracts were combined and washed with water and
Acid (16b). The compound was prepared as described for 16a with
the exception that the respective sulfonylchloride was used instead of
the carboxylchoride. 3-{(E)-2-[2-(Naphthalene-1-sulfonylamino)-
phenyl]-vinyl}-benzoic acid methyl ester (38 mg; 0.09 mmol) yielded
brine, dried over MgSO , and evaporated to give 3-vinyl-benzoic acid
4
methyl ester 11 (720 mg; 82% yield) as a light brown oil.
2-Bromo-phenylamine 12 (0.15 g; 0.87 mmol), 3-vinyl-benzoic
acid methyl ester 11 (0.17 g; 1.05 mmol), palladium(II) acetate (4
mg; 0.02 mmol), tri-o-tolyl-phosphane (21 mg; 0.07 mmol), and
triethylamine (1 mL) were placed in a reacting vial. The resulting
mixture was argonated for 15 min and after capping the vial, the
reaction mixture was stirred overnight at 125 °C. After dissolving in
DCM and filtration, all solvents were removed under reduced
pressure. The brown-orange semisolid product was purified by
chromatography giving the product 13 as a yellow-brown oil, which is
crystallized after a few minutes. (0.165 g, 75% yield). LC−MS 4: 3.56
the final product in 76% (25 mg) as beige foam. LC−MS 4: 3.722
1
min; 432.1 (M + H). H NMR (400 MHz, DMSO-d ): δ 12.90 (s,
6
1
8
H), 10.02 (s, 1H), 8.74 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.2 Hz, 1H),
.04 (dd, J = 8.0, 1.6 Hz, 1H), 8.01 (dd, J = 7.3, 1.2 Hz, 1H), 7.75 (dt,
J = 7.7, 1.4 Hz, 1H), 7.71 (s, 1H), 7.67−7.53 (m, 3H), 7.35 (t, J = 7.6
Hz, 1H), 7.20−7.13 (m, 2H), 7.09 (td, J = 7.4, 1.5 Hz, 1H), 7.03 (td,
J = 7.6, 1.8 Hz, 1H), 6.86 (dd, J = 7.9, 1.4 Hz, 1H), 2.59 (s, 4H).
HRMS calcd for C H NO S, 432.1264; found, 432.1261.
2
5
21
4
4
-{2-[2-(Naphthalene-1-sulfonylamino)-phenyl]-ethyl}-benzoic
1
min; 254.00 (M + H). H NMR (400 MHz, DMSO): δ 8.18 (d, J =
Acid (16c). The compound was prepared as described for 16b. Methyl
4-[2-[2-(naphthalene-1-sulfonamido)phenyl]ethenyl]benzoate (30
mg; 0.07 mmol) yielded the final product in 83% (24 mg) as white
1
7
.4 Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.81 (dd, J = 7.8, 1.4 Hz, 1H),
.50 (dd, J = 16.2, 7.8 Hz, 2H), 7.47 (dd, J = 7.8, 1.2 Hz, 1H), 7.08
1
(
1
d, J = 16.1 Hz, 1H), 6.98 (dd, J = 6.8, 1.4 Hz, 1H), 6.66 (dd, J = 8.1,
.0 Hz, 1H), 6.55 (d, J = 7.4 Hz, 1H), 5.42 (s, 2H), 3.88 (s, 3H).
-[(E)-2-(2-Amino-phenyl)-vinyl]-benzoic acid methyl ester 13
60 mg; 0.24 mmol) was dissolved in anhydrous DCM (3 mL),
foam. LC−MS 3: 1.76 min; 432.00 (M + H). H NMR (400 MHz,
DMSO-d ): δ 12.77 (s, 1H), 9.96 (s, 2H), 8.80−8.72 (m, 3H), 8.22
6
3
(dd, J = 8.4, 1.3 Hz, 3H), 8.13−8.04 (m, 3H), 8.01 (dd, J = 7.4, 1.2
Hz, 3H), 7.82−7.75 (m, 6H), 7.72−7.61 (m, 6H), 7.57 (dd, J = 8.2,
7.3 Hz, 3H), 7.17−6.99 (m, 15H), 6.87 (dd, J = 7.9, 1.4 Hz, 3H),
2.66−2.53 (m, 12H). HRMS calcd for C H NO S, 432.1264; found,
(
followed by triethylamine (0.09 mL; 0.71 mmol). The reaction
mixture was stirred at RT for 20 min, and then naphthalene-1-
carbonyl chloride (39 μL; 0.25 mmol) was added in one portion.
After 6 h at RT, the reaction mixture was diluted with ethyl acetate
and the organic layer was washed with 2 M NaOH and water, 2 M
2
5
21
4
432.1263.
4-{2-[2-(Naphthalene-2-sulfonylamino)-phenyl]-ethyl}-benzoic
Acid (16d). The compound was prepared as described for 16b.
Methyl 4-[2-[2-(naphthalene-2-sulfonamido)phenyl]ethenyl]-
benzoate (31 mg; 0.07 mmol) yielded the final product in 79% (23
HCl and water, saturated NaHCO , and water and brine. After drying
3
over anhydrous Na SO and concentrated under reduced pressure,
2
4
1
the crude product was treated with a small volume of diethyl ether,
mg) as white foam. LC−MS 3: 1.78 min; 432.00 (M + H). H NMR
1
1918
https://doi.org/10.1021/acs.jmedchem.1c00448
J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
400 MHz, DMSO-d ): δ 12.75 (s, 1H), 9.75 (s, 2H), 8.32 (d, J = 1.8
(dd, J = 7.7, 1.5 Hz, 1H), 2.78−2.66 (m, 4H). HRMS calcd for
6
Hz, 3H), 8.14−8.07 (m, 6H), 8.06−8.00 (m, 3H), 7.81−7.74 (m,
H), 7.70 (m, 3H), 7.65 (m, 3H), 7.20 (dd, J = 7.6, 1.7 Hz, 3H), 7.12
dd, J = 8.7, 7.3 Hz, 9H), 7.06 (m, 3H), 6.91 (dd, J = 7.9, 1.4 Hz,
H), 2.82−2.74 (m, 6H), 2.74−2.65 (m, 6H). HRMS calcd for
C H NO S, 382.1108; found, 382.1099.
2
1
19
4
9
(
3
3-{2-[2-(4-Fluoro-benzenesulfonylamino)-phenyl]-ethyl}-benzoic
Acid (16h). The compound was prepared as described for 16f and
1
obtained as a white solid. LCMS 3: 1.69 min; 400.00 (M + H). H
C H NO S, 432.1264; found, 432.1264.
NMR (400 MHz, DMSO-d ): δ 12.93−12.77 (m, 1H), 9.85−9.73
6
25
21
4
2
-{2-[2-(Naphthalene-1-sulfonylamino)-phenyl]-ethyl}-benzoic
(m, 1H), 7.82−7.80 (m, 1H), 7.80−7.75 (m, 1H), 7.71−7.67 (m,
2H), 7.64−7.60 (m, 2H), 7.44−7.38 (m, 2H), 7.27 (dd, J = 7.6, 1.8
Hz, 1H), 7.16 (td, J = 7.4, 1.6 Hz, 1H), 7.12 (td, J = 7.6, 1.8 Hz, 1H),
6.89 (dd, J = 7.7, 1.6 Hz, 1H), 2.82−2.69 (m, 4H). HRMS calcd for
C H FNO S, 400.1013; found, 400.1008.
Acid (16e). The compound was prepared as described for 16b.
Methyl 4-[2-[2-(naphthalene-2-sulfonamido)phenyl]ethenyl]-
benzoate (30 mg; 0.07 mmol) yielded the final product in 78% (22
1
mg) as white foam. LC−MS 3: 1.894 min; 432.0 (M + H). H NMR
2
1
18
4
(
8
400 MHz, DMSO-d ): δ 12.92−12.84 (m, 1H), 9.84−9.78 (m, 1H),
3-{2-[2-(5-Fluoro-2-methyl-benzenesulfonylamino)-phenyl]-
6
.77−8.71 (m, 1H), 8.22−8.18 (m, 1H), 8.08−8.03 (m, 1H), 8.01
ethyl}-benzoic Acid (16i). The compound was prepared as described
for 16f and obtained as an amorphous colorless solid.
(
dd, J = 7.3, 1.2 Hz, 1H), 7.80 (dd, J = 7.8, 1.5 Hz, 1H), 7.66−7.60
1
(
m, 2H), 7.59−7.54 (m, 1H), 7.40 (td, J = 7.5, 1.5 Hz, 1H), 7.28 (td,
LCMS 3: 1.75 min; 414.00 (M + H). H NMR (400 MHz, DMSO-
J = 7.5, 1.3 Hz, 1H), 7.14 (dd, J = 7.6, 1.9 Hz, 1H), 7.09 (td, J = 7.3,
d ): δ 13.12−12.52 (m, 1H), 10.17−9.54 (m, 1H), 7.83−7.80 (m,
6
1
.5 Hz, 1H), 7.04 (td, J = 7.5, 2.0 Hz, 1H), 6.93−6.89 (m, 1H), 6.90−
.86 (m, 1H), 2.88−2.81 (m, 2H), 2.58−2.52 (m, 2H). HRMS calcd
1H), 7.80−7.77 (m, 1H), 7.46−7.35 (m, 5H), 7.27 (dd, J = 7.6, 1.8
Hz, 1H), 7.18 (td, J = 7.5, 1.5 Hz, 1H), 7.11 (td, J = 7.6, 1.7 Hz, 1H),
6.85 (dd, J = 7.9, 1.4 Hz, 1H), 2.81−2.74 (m, 4H), 2.50−2.48 (m,
6
for C H NO S, 432.1264; found, 432.1264.
25
21
4
2
-{5-[2-(Naphthalene-1-sulfonylamino)-phenyl]-pentyl}-benzoic
3H). HRMS calcd for C22H20FNO S, 414.1170; found, 414.1160.
4
Acid (10). Synthesis was carried out as described for 9a with the
exception that the respective sulfonylchloride was used and not the
carboxylchloride. Methyl 2-{5-[2-(naphthalene-1-sulfonamido)-
phenyl]pentyl}benzoate yielded the product (25 mg) as a colorless
3-{2-[2-(4-Methoxy-2,3-dimethyl-benzenesulfonylamino)-phe-
nyl]-ethyl}-benzoic Acid (16j). The compound was prepared as
described for 16f. 3-[2-(2-Amino-phenyl)-ethyl]-benzoic acid methyl
ester (60 mg; 0.22 mmol) and 4-methoxy-2,3-dimethylbenzene-1-
sulfonyl chloride (58 mg; 0.25 mmol) yielded a product as a white
1
solid. LC−MS 3: 1.937 min; 456.0 (M-OH); 496.0 (M + Na). H
NMR (400 MHz, DMSO-d ): δ 12.86−12.69 (m, 1H), 9.91−9.79
solid in 36% yield (37 mg) over the last two steps. LC−MS 2: 1.79
6
1
(m, 1H), 8.76−8.71 (m, 1H), 8.18 (d, J = 8.2 Hz, 1H), 8.09−8.04 (m,
min; 440.0 (M + H). H NMR (400 MHz, DMSO-d
): δ 12.86 (s,
6
1
7
1
2
0
4
H), 7.96 (dd, J = 7.4, 1.3 Hz, 1H), 7.77 (dd, J = 7.8, 1.5 Hz, 1H),
.69−7.61 (m, 2H), 7.53 (t, J = 7.8 Hz, 1H), 7.47 (td, J = 7.5, 1.5 Hz,
H), 7.31−7.23 (m, 2H), 7.10−6.99 (m, 3H), 6.98−6.93 (m, 1H),
.82−2.74 (m, 2H), 2.23−2.13 (m, 2H), 1.32−1.22 (m, 2H), 1.00−
.90 (m, 2H), 0.87−0.77 (m, 2H). HRMS calcd for C H NO S,
1H), 9.52 (s, 1H), 7.83−7.81 (m, 1H), 7.81−7.75 (m, 1H), 7.58 (d, J
= 8.9 Hz, 1H), 7.42−7.39 (m, 2H), 7.22 (dd, J = 7.4, 2.0 Hz, 1H),
7.14−7.05 (m, 2H), 6.92 (dd, J = 7.6, 1.7 Hz, 1H), 6.88 (d, J = 8.9
Hz, 1H), 3.80 (s, 3H), 2.82−2.71 (m, 4H), 2.47 (s, 3H), 2.03 (s, 3H).
HRMS calcd for C H NO S, 440.1526; found, 440.1520.
2
8
27
4
24 25
5
74.1734; found, 474.1721.
-{2-[2-(Naphthalene-2-sulfonylamino)-phenyl]-ethyl}-benzoic
3-{2-[2-(2,5-Dichloro-benzenesulfonylamino)-phenyl]-ethyl}-
3
benzoic Acid (16k). The compound was prepared as described for
1
Acid (16f). 3-{(E)-2-[2-(Naphthalene-2-sulfonylamino)-phenyl]-
vinyl}-benzoic acid methyl ester (42 mg; 0.08 mmol) was dissolved
in ethanol (10 mL) under argon in a three-necked flask, which was
equipped with a hydrogen baloon and argon and vacuum pins. After
palladium on carbon (10%, 40 mg; 0.04 mmol) was added, the flask
was evacuated and back filled with argon three times. Then, the
hydrogen valve was opened and the reaction mixture was stirred
overnight at RT vigorously. Then, the catalyst was filtered off and the
filtrate was evaporated to give 3-{2-[2-(naphthalene-2-sulfonylami-
no)-phenyl]-ethyl}-benzoic acid methyl ester as a crude product (41
mg; 0.09 mmol). Without further purification, the crude was dissolved
in ethanol (4 mL), and 5 N NaOH (0.34 mL; 1.69 mmol; 20.00
equiv) was added under stirring. The reaction mixture was stirred
overnight and then diluted with water, and acidified with 2 M HCl.
The precipitate was taken up in ethyl acetate and extracted with water.
After repeated re-extraction with ethyl acetate, the combined organic
phases were washed with water and brine, dried over Na SO , and
16f. LCMS 3: 1.80 min; 452.00 (M + H). H NMR (400 MHz,
DMSO-d ): δ 13.05−12.57 (m, 1H), 10.43−9.90 (m, 1H), 7.86−7.83
6
(m, 1H), 7.81−7.77 (m, 2H), 7.73−7.71 (m, 1H), 7.47−7.37 (m,
3H), 7.29 (dd, J = 7.6, 1.7 Hz, 1H), 7.19 (td, J = 7.5, 1.4 Hz, 1H),
7.12 (td, J = 7.6, 1.7 Hz, 1H), 6.88 (dd, J = 7.8, 1.4 Hz, 1H), 2.91−
2.79 (m, 4H). HRMS calcd for C H Cl NO S, 450.0328; found,
2
1
17
2
4
450.0315.
3-{(E)-2-[2-(4-Methoxy-2,3-dimethyl-benzenesulfonylamino)-
phenyl]-vinyl}-benzoic Acid (15a). To a solution of 3-[(E)-2-(2-
amino-phenyl)-vinyl]-benzoic acid methyl ester (60 mg; 0.22 mmol)
in acetonitrile (5 mL) were added 4-methoxy-2,3-dimethyl-
benzenesulfonyl chloride (61 mg; 0.25 mmol) and sodium hydrogen
carbonate (23 mg; 0.27 mmol). The reaction was stirred for 7 days at
RT. Methanol (5 mL) and sodium hydroxide solution c(NaOH) = 2
mol/l (2 N) (2.3 mL) were added and the mixture was stirred for 6 h
at RT. The reaction mixture was evaporated to dryness. The residue
was purified by chromatography giving the product in 53% (53 mg)
2
4
evaporated. The residue was purified by chromatography giving 3-{2-
2-(naphthalene-2-sulfonylamino)-phenyl]-ethyl}-benzoic acid (29
mg; 0.06 mmol; 75% yield) as a white solid. LCMS 5: 8.30 min;
yield as an amorphous white solid. LCMS 2: 1.78 min; 438.00 (M +
1
[
H). H NMR (500 MHz, DMSO-d
6
): δ 13.10−12.99 (m, 1H), 9.81
(s, 1H), 8.10−8.08 (m, 1H), 7.84 (dt, J = 7.6, 1.4 Hz, 1H), 7.68−7.64
(m, 1H), 7.61−7.58 (m, 1H), 7.52−7.48 (m, 2H), 7.34 (d, J = 16.2
Hz, 1H), 7.26−7.18 (m, 3H), 6.99 (d, J = 16.2 Hz, 1H), 6.75 (d, J =
8.9 Hz, 1H), 3.67 (s, 3H), 2.40 (s, 3H), 1.85 (s, 3H). HRMS calcd for
1
4
32.4 (M + H); 430.30 (M − H). H NMR (400 MHz, DMSO-d ): δ
6
9
.83 (s, 1H), 8.31 (d, J = 1.9 Hz, 1H), 8.14−8.06 (m, 1H), 8.05−7.99
(
(
m, 1H), 7.82−7.78 (m, 2H), 7.76 (dt, J = 7.6, 1.5 Hz, 1H), 7.69
ddd, J = 8.2, 6.9, 1.4 Hz, 1H), 7.64 (ddd, J = 8.2, 6.8, 1.4 Hz, 1H),
C
H
23NO
S, 438.1370; found, 438.1366.
24
5
7
1
2
.34 (t, J = 7.6 Hz, 1H), 7.30−7.22 (m, 3H), 7.13 (td, J = 7.4, 1.5 Hz,
H), 7.06 (td, J = 7.6, 1.7 Hz, 1H), 6.90 (dd, J = 7.9, 1.4 Hz, 1H),
.83−2.73 (m, 2H), 2.74−2.64 (m, 2H). HRMS calcd for
3-[(E)-2-[2-(naphthalene-1-sulfonamido)phenyl]ethenyl]benzoic
Acid (15b). Step 1. Thionyl chloride (0.59 mL; 8.10 mmol) was
dropped into the mixture of 3-vinyl-benzoic acid 10 (1.00 g; 6.75
mmol) in anhydrous methanol (10 mL) over 10 min at 0 °C and
warmed up to RT over 3 h. Then, a second portion of thionyl chloride
(0.59 mL; 8.10 mmol) was added at RT. After 48 h, methanol was
evaporated, and the residue was poured in water. The pH was
adjusted to 7 with 1 M NaOH and the resulting mixture was extracted
with diethyl ether (3 × 20 mL). Organic extracts were combined and
C H NO S, 432.1264; found, 432.1264.
25
21
4
3
-[2-(2-Benzenesulfonylamino-phenyl)-ethyl]-benzoic Acid
(
16g). The compound was prepared as described for 16f and
obtained as a colorless solid.
1
LCMS 3: 1.664 min; 382.00 (M + H). H NMR (400 MHz,
DMSO-d ): δ 12.88−12.81 (m, 1H), 9.68 (s, 1H), 7.81−7.79 (m,
6
1
7
1
H), 7.79−7.75 (m, 1H), 7.72−7.68 (m, 2H), 7.65−7.60 (m, 1H),
.57−7.51 (m, 2H), 7.43−7.38 (m, 2H), 7.24 (dd, J = 7.5, 1.8 Hz,
H), 7.14 (td, J = 7.4, 1.6 Hz, 1H), 7.09 (td, J = 7.6, 1.9 Hz, 1H), 6.92
washed with water and brine, dried over MgSO , and evaporated to
4
give 3-vinyl-benzoic acid methyl ester 11 (1.05 g; 6.44 mmol; 95.5%)
as a light brown oil. LCMS 2: 3.56 min.
1
1919
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J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Step 2. 3-Vinyl-benzoic acid methyl ester (0.95 g; 5.86 mmol),
triethylamine (0.30 mL), and 2-bromo-phenylamine 12 (0.50 g; 2.91
mmol) were placed in a reacting vial. The mixture was purged with
argon for 15 min and trans-{bis(acetato)}bis[o-{(di-{o-
tolylphosphino)}benzyl]}dipalladium(II) (Herrmann’s palladacycle)
for 10 min, 1,8-diazabicyclo[5.4.0]undec-7-ene and DBU (1.47 mL;
9.82 mmol) were added, the vessel was capped, and the mixture was
heated with stirring for 3 h at 85−90 °C. The reaction mixture was
evaporated, the residue was dissolved in DCM, and the product was
adsorbed on silica gel. The solvent was evaporated and the residue
(81.75 mg; 0.09 mmol) was added and vial was capped. The reaction
was placed in a filtering funnel. The product was eluted with Et O.
2
mixture was stirred overnight at 120 °C, dissolved in DCM, and
filtered. The filtrate was concentrated and residual triethylamine was
coevaporated with diethyl ether. The brown-orange gummy residue
was purified by chromatography to give 3-[(E)-2-(2-amino-phenyl)-
vinyl]-benzoic acid methyl ester 13 (0.21 g; 28%) as a light brown
semisolid. LCMS 4: 3.58 min.
Step 3. 3-[(E)-2-(2-Amino-phenyl)-vinyl]-benzoic acid methyl
ester (0.05 g; 0.20 mmol) was dissolved in acetonitrile (5 mL) and
NaHCO3 (19.90 mg; 0.24 mmol) was added, followed by
naphthalene-1-sulfonyl chloride (49.22 mg; 0.22 mmol). The reaction
The filtrate was evaporated to give crude methyl 3-[2-(2-
nitrophenyl)vinyl]benzoate (2.66 g; 86%) as a yellow solid. LCMS
4: 3.74 min, 3.84 min; 40.4%; 57.1% of E and Z isomers (not specified
which is which).
Step 2. Methyl 3-[(E)-2-(2-nitrophenyl)vinyl]benzoate (0.56 g;
1.00 mmol) was dissolved in ethanol (25 mL). Iron dust (0.58 g; 10
mmol) and ammonium chloride (1.10 g; 20 mmol) were added. The
mixture was stirred and heated to reflux for 3 h. The inorganic
precipitate was filtered off, the filtrate was diluted with ethyl acetate,
and extracted with water. The organic layer was dried over Na SO
2
4
mixture was stirred at RT for 48 h, then it was diluted with Et O and
2
and evaporated to give crude methyl 3-[(E/Z)-2-(2-aminophenyl)-
vinyl]benzoate (0.531 g; 82.7%) as a light brown foam. LCMS 4: 3.43
min.
Step 3. The mixture of methyl 3-[(E)-2-(2-aminophenyl)vinyl]-
benzoate and methyl 3-[(Z)-2-(2-aminophenyl)vinyl]benzoate (531
mg; 0.82 mmol) was dissolved in acetonitrile (25 mL) and NaHCO3
extracted with water and brine. Organic extract was dried over
Na SO , evaporated, and residue was purified by chromatography to
2
4
give 3-{(E)-2-[2-(naphthalene-1-sulfonylamino)-phenyl]-vinyl}-ben-
zoic acid methyl ester (60 mg; 68%) as a beige film. LCMS 4: 4.15
min; 422.1 [M − H].
Step 4. 3-{(E)-2-[2-(Naphthalene-1-sulfonylamino)-phenyl]-
vinyl}-benzoic acid methyl ester (20.00 mg; 0.04 mmol) was stirred
overnight in 5 M NaOH (2 mL) and MeOH (1 mL). The reaction
mixture was poured into 5.5 mL of 2 M HCl. The acidic suspension of
crude product was extracted twice with ethyl acetate. Combined
organic extracts were washed with water and brine, then dried over
Na SO , and filtered. The filtrate was evaporated in vacuo to obtain
(
206 mg; 2.45 mmol) was added, followed by naphthalene-2-sulfonyl
chloride (445 mg; 1.96 mmol). The mixture was stirred overnight,
diluted with ethyl acetate, and extracted with water to remove
inorganics. The organic extract was washed with brine and dried over
Na SO , then evaporated to give 700 mg of brown oil, which was
purified by chromatography. The obtained foam (543 mg) was
crystallized from 8 mL of MeOH. The precipitate was filtered off,
washed with cold methanol, and dried in vacuo to give 3-{(E)-2-[2-
2
4
2
4
the product (19 mg; 96%) as a beige solid. LCMS 5: 8.2 min; 428.2
1
[
1
(
7
7
M − H]. H NMR (400 MHz, DMSO): δ: 13.04 (s, 1H), 10.36 (s,
(naphthalene-2-sulfonylamino)-phenyl]-vinyl}-benzoic acid methyl
H), 8.81 (d, J = 8.7 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.98−7.90
1
ester (175 mg; 46%) as an off-white solid. H NMR (400 MHz,
DMSO-d ): δ 10.10 (s, 1H), 8.23 (d, J = 1.9 Hz, 1H), 7.96 (dd, J =
m, 3H), 7.81 (d, J = 7.7 Hz, 1H), 7.71−7.64 (m, 1H), 7.58 (dd, J =
6
.7, 1.4 Hz, 1H), 7.55−7.44 (m, 2H), 7.40 (t, J = 7.7 Hz, 1H), 7.30−
8
7
6
2
1
.0, 1.3 Hz, 1H), 7.94−7.88 (m, 2H), 7.81 (dt, J = 6.9, 1.6 Hz, 2H),
.69 (dd, J = 8.7, 1.9 Hz, 1H), 7.67−7.64 (m, 1H), 7.56 (ddd, J = 8.2,
.9, 1.5 Hz, 1H), 7.51 (ddd, J = 8.1, 6.9, 1.4 Hz, 1H), 7.46−7.37 (m,
H), 7.30−7.24 (m, 2H), 7.24−7.16 (m, 2H), 6.89 (d, J = 16.2 Hz,
H), 3.93 (s, 3H).
.13 (m, 4H), 7.06 (d, J = 16.3 Hz, 1H), 6.87 (d, J = 16.2 Hz, 1H).
−
HRMS calcd for C H NO S , 428.0962; found, 428.0956.
25
18
4
3
-[(E)-2-[2-(Naphthalene-2-sulfonamido)phenyl]ethenyl]benzoic
Acid (15c). Step 1. 3-[(E)-2-(2-Amino-phenyl)-vinyl]-benzoic acid
methyl ester (0.05 g; 0.20 mmol), naphthalene-2-sulfonyl chloride
The filtrate from crystallization was evaporated to dryness to give a
(
49.22 mg; 0.22 mmol), and NaHCO (19.90 mg; 0.24 mmol) were
3
brown oil, which was triturated with 4 mL of cold MeOH to
precipitate residual E isomer, which was filtered off and washed with a
small amount of cold MeOH. The filtrate was evaporated to give 3-
stirred at RT for 18 h in acetonitrile (5 mL). The reaction mixture
was diluted with ethyl acetate and washed with water. The organic
layer was washed with brine and dried over Na SO . The solvent was
2
4
{
(Z)-2-[2-(naphthalene-2-sulfonylamino)-phenyl]-vinyl}-benzoic acid
evaporated and 3-{(E)-2-[2-(naphthalene-2-sulfonylamino)-phenyl]-
vinyl}-benzoic acid methyl ester was isolated by chromatography
1
methyl ester (248 mg; 61%) as a brown gum. H NMR (400 MHz,
DMSO-d ): δ 8.31 (d, J = 1.8 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.98
(
7
7
6
6
(
62.90 mg 64%) as a beige solid. LCMS 4: 4.12 min.
Step 2. 3-{(E)-2-[2-(Naphthalene-2-sulfonylamino)-phenyl]-
d, J = 8.7 Hz, H), 7.90−7.85 (m, 1H), 7.80 (dd, J = 8.6, 1.9 Hz, 2H),
.64−7.52 (m, 4H), 7.51 (d, J = 1.9 Hz, 1H), 7.17−7.11 (m, 2H),
.06 (t, J = 7.7 Hz, 1H), 6.98−6.94 (m, 2H), 6.89−6.84 (m, 1H),
.70 (d, J = 12.1 Hz, 1H), 6.52 (d, J = 12.2 Hz, 1H), 3.75 (s, 3H).
Step. 4: 3-{(Z)-2-[2-(Naphthalene-2-sulfonylamino)-phenyl]-
vinyl}-benzoic acid methyl ester (20 mg; 0.04 mmol) was stirred
overnight in a mixture of 5 M NaOH (2 mL) and MeOH (1 mL).
Then, it was acidified with 5.5 mL of 2 M HCl. The resulting foggy
mixture was extracted twice with ethyl acetate. The organic extract
was washed with water, brine, and dried over Na SO . Evaporation
vinyl}-benzoic acid methyl ester (50 mg; 0.10 mmol) was dissolved
in methanol (2 mL), and 5 N NaOH (0.40 mL; 2.01 mmol) was
added. The reaction mixture was stirred for 1 h, then it was diluted
with water and acidified to pH 3−4 with 1 M HCl. The precipitate
was taken up with ethyl acetate and the water layer was extracted with
ethyl acetate. Combined organic extracts were washed with brine and
dried over Na SO . The solvent was evaporated and the resulting oil
was purified by chromatography giving 36 mg of a mixture of E & Z
isomers (12:88%). The sample was repurified by chromatography to
give 3-{(Z)-2-[2-(naphthalene-2-sulfonylamino)-phenyl]-vinyl}-ben-
2
4
gave the product (15.30 mg; 85%) as a beige gassy solid. LCMS 5: 8.2
1
min; 428.1 [M − H]; H NMR (400 MHz, DMSO): δ: 13.07 (s, 1H),
1
0.12 (s, 1H), 8.23 (d, J = 1.4 Hz, 1H), 8.01−7.90 (m, 3H), 7.86−
.77 (m, 2H), 7.71 (dd, J = 8.7, 1.8 Hz, 1H), 7.68−7.63 (m, 1H), 7.54
7
(
4
dtd, J = 16.2, 7.0, 1.2 Hz, 2H), 7.43−7.34 (m, 2H), 7.27−7.18 (m,
−
H), 6.89 (d, J = 16.3 Hz, 1H). HRMS calcd for C H NO S ,
2
4
2
5
18
4
4
28.0962; found, 428.0956.
3
-[(Z)-2-[2-(Naphthalene-2-sulfonamido)phenyl]ethenyl]-
benzoic Acid (15d). Step 1. Triphenylphosphine (2.58 g; 9.82 mmol)
was added to the solution of methyl 3-bromomethyl-benzoate (2.50 g;
zoic acid (16.30 mg; 38%) as an off-white solid. LCMS 6: 14.6
min; 428.4 [M − H]; H NMR (400 MHz, DMSO-d
1
1
0.91 mmol) in anhydrous acetonitrile (50 mL). The mixture was
6
): δ 12.85 (s,
argonated for 5 min, and the vessel was capped and heated with
8H), 9.92 (s, 9H), 8.32 (d, J = 1.9 Hz, 10H), 8.23 (d, J = 1.8 Hz, 1H),
8.08−8.00 (m, 20H), 8.00−7.93 (m, 4H), 7.93 ? 7.88 (m, 10H),
7.84−7.77 (m, 13H), 7.71 (dd, J = 8.7, 1.9 Hz, 2H), 7.67−7.48 (m,
44H), 7.39 (d, J = 6.4 Hz, 3H), 7.27−7.16 (m, 6H), 7.13 (ddd, = 8.5,
7.0, 1.7 Hz, 11H), 7.09−7.01 (m, 20H), 6.99−6.84 (m, 32H), 6.67
(d, J = 12.2 Hz, 10H), 6.52 (d, J = 12.3 Hz, 10H). HRMS calcd for
stirring for 3 h at 85−90 °C. Then, the reaction mixture was cooled to
RT, and 50 mL of Et O was added. The mixture was stirred at RT for
2
2
h. During stirring, a white precipitate forms. After that, the reaction
mixture was filtered and the white solid was transferred into a 150 mL
reactor. Anhydrous acetonitrile (50 mL) was added, followed by 2-
nitro-benzaldehyde (1.48 g; 9.82 mmol). The mixture was argonated
−
C H NO S , 428.0962; found, 428.0955.
2
5
18
4
1
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J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
4
-[(E)-2-[2-(Naphthalene-2-sulfonamido)phenyl]ethenyl]benzoic
yl ester (56 mg; 48.9%) obtained as a white powder was used in the
next step without purification. LCMS 4: 3.48 min; 390.00 [M − H].
Step 3. 4-(2-Benzenesulfonylamino-phenylethynyl)-benzoic acid
methyl ester (56 mg; 0.10 mmol) was dissolved in THF (3 mL) and
water (1 mL). Then, lithium hydroxide monohydrate (25 mg; 0.60
mmol) was added, and the reaction mixture was stirred at RT
overnight. Then, THF was evaporated, the remainder was acidified to
pH = 5, and extracted with ethyl acetate. The organic layer was dried
over sodium sulfate and evaporated under reduced pressure. The
residue was purified by chromatography to give the product (30 mg;
Acid (15e). Step 1. Thionyl chloride (0.82 mL; 11.18 mmol) was
dropped into 4-vinyl-benzoic acid (1.50 g; 9.31 mmol) in anhydrous
methanol (15 mL) over 10 min at 0 °C. After 3 h, the second portion
of thionyl chloride (0.61 mL; 8.38 mmol) was added at RT, and the
reaction mixture was stirred overnight. Methanol was evaporated, and
the residue was poured in water. The pH was adjusted to 7 with 1 M
NaOH and the mixture was extracted with diethyl ether (3 × 20 mL).
Organic extracts were combined and washed with water and brine,
dried over MgSO , and evaporated to give methyl 4-vinyl-benzoate
1.53 g; 93%) as a brown semi solid. LCMS 4:3.53 min.
4
1
(
38%) as a white powder. LCMS 5: 6.3 min; 376.5; H NMR (400
Step 2. 2-Bromo-phenylamine (0.43 g; 2.50 mmol), 4-vinyl-benzoic
MHz, DMSO-d ): δ 13.13 (s, 1H), 10.08 (s, 1H), 8.00 (d, J = 8.4 Hz,
6
acid methyl ester (0.40 g; 2.27 mmol), palladium(II) acetate (10.74
mg; 0.05 mmol), tri-o-tolyl-phosphane (55.31 mg; 0.18 mmol), and
triethylamine (4 mL) were argonated for 15 min and the vial was
capped. The reaction mixture was stirred overnight at 125 °C,
dissolved in DCM, and filtered. The filtrate was concentrated, and the
residual triethylamine was coevaporated with diethyl ether. The
brown-orange semisolid product was purified by chromatography to
give methyl 4-[(E)-2-(2-amino-phenyl)-vinyl]-benzoate (385 mg;
2H), 7.72−7.70 (m, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.56−7.52 (m,
1H), 7.48 (dd, J = 7.7, 1.3 Hz, 1H), 7.44−7.39 (m, 3H), 7.33 (dd,
−
1H), 7.25 (td, J = 7.6, 1.2 Hz, 1H). HRMS calcd for C H NO S ,
2
1
14
4
376.0649; found, 376.0646.
4-[2-(3-Methoxy-benzenesulfonylamino)-phenylethynyl]-ben-
zoic Acid (17b). 4-(2-Amino-phenylethynyl)-benzoic acid methyl
ester (51 mg; 0.20 mmol) was dissolved with sodium bicarbonate (25
mg; 0.30 mmol) in acetonitrile (5 mL), 3-methoxy-benzenesulfonyl
chloride (0.04 mL; 0.27 mmol) was added, and the reaction mixture
was stirred at RT for 72 h. After evaporating, the crude product 4-[2-
6
+
7%) as yellow crystals. LCMS 4: 3.57 min; 254 [M + H]; 295 [M
H + ACN].
Step 3. 4-[(E)-2-(2-Amino-phenyl)-vinyl]-benzoic acid methyl
(
3-methoxy-benzenesulfonylamino)-phenylethynyl]-benzoic acid
methyl ester was obtained (94 mg; 0.15 mmol) as a brown solid.
The residue was suspended in methanol (2 mL) and treated with
sodium hydroxide solution (5 M, 4 mL) to pH 12 and stirred at RT
for 2 h. Then, the formed solution was acidified with hydrochloric
acid (10% wt) to pH 3 and extracted three times by ethyl acetate.
ester (100 mg; 0.39 mmol), naphthalene-2-sulfonyl chloride (107
mg; 0.47 mmol), and NaHCO (49.75 mg; 0.59 mmol) were stirred
3
overnight in acetonitrile (2 mL) at RT. Crude methyl 4-[(E)-2-[2-
(naphthalene-2-sulfonamido)phenyl]ethenyl]benzoate (product A)
was not isolated and already subjected to the saponification step.
Crude RM was diluted with methanol, and 5 N NaOH was added.
The resulting clear mixture was stirred for 2 h and RM was diluted
with water and acidified with 2 M HCl. Formed precipitate of crude
Combined organics extracts were dried over anhydrous Na SO . After
2
4
evaporating, the crude was purified by chromatography giving the
product in 27% (22 mg) yield as a white solid. LCMS 5: 7.90 min;
1
4
1
7
2
06.00 (M − H). H NMR (400 MHz, DMSO-d ): δ 13.16 (s, 1H),
4
-{(E)-2-[2-(naphthalene-2-sulfonylamino)-phenyl]-vinyl}-benzoic
6
0.06 (s, 1H), 8.01−7.95 (m, 2H), 7.63−7.57 (m, 2H), 7.49 (dd, J =
.7, 1.5 Hz, 1H), 7.42 (ddd, J = 8.2, 7.3, 1.6 Hz, 1H), 7.38−7.32 (m,
H), 7.29−7.20 (m, 2H), 7.18 (dd, J = 2.6, 1.7 Hz, 1H), 7.09 (ddd, J
acid (product B) was taken up to ethyl acetate. The water layer was
extracted twice with ethyl acetate. Combined organic extracts were
washed with water and brine, then dried over Na SO , and
2
4
=
8.2, 2.6, 1.0 Hz, 1H), 3.60 (s, 3H). HRMS calcd for C H NO S,
evaporated. The residue was purified by FCC (DCM → 95:5
DCM/MeOH) to obtain 4-{(E)-2-[2-(naphthalene-2-sulfonylami-
no)-phenyl]-vinyl}-benzoic acid (117.90 mg; 0.26 mmol; 65.4%) as
22 17 5
4
08.0900; found, 408.0889.
4
-[2-(4-Methoxy-benzenesulfonylamino)-phenylethynyl]-ben-
zoic Acid (17c). The compound was prepared as described for 17b. 4-
[2-(4-Methoxy-benzenesulfonylamino)-phenylethynyl]-benzoic acid
methyl ester (164 mg; 0.24 mmol) yielded 35% (36 mg) of the
a yellow solid. LC−MS 5: 8.2 min; 428.2 [M − H], 430.3 [M + H]
1
H NMR (400 MHz, DMSO-d ) d: 12.89 (s, 1H), 10.07 (s, 1H), 8.03
6
(
d, J = 8.7 Hz, 1H), 7.98 (d, J = 8.7 Hz, 1H), 7.87 (d, J = 7.4 Hz, 1H),
.85−7.81 (m, 2H), 7.73 ? 7.64 (m, 2H), 7.61 (ddd, J = 8.2, 6.9, 1.4
Hz, 1H), 7.56 (ddd, J = 8.2, 6.8, 1.4 Hz, 1H), 7.34−7.28 (m, 2H),
.28−7.24 (m, 2H), 7.23−7.17 (m, 2H), 6.92 (d, J = 16.3 Hz, 1H).
1
product as a rose solid. LCMS 2: 1.72 min; 408.00 (M + H). H
7
NMR (400 MHz, DMSO-d ): δ 13.30−12.96 (m, 1H), 9.85 (s, 1H),
6
8
.01−7.97 (m, 2H), 7.64−7.62 (m, 2H), 7.62−7.60 (m, 2H), 7.47
7
(
dd, J = 7.7, 1.6 Hz, 1H), 7.42−7.37 (m, 1H), 7.32 (dd, J = 8.2, 1.3
Hz, 1H), 7.23 (td, J = 7.5, 1.4 Hz, 1H), 6.93−6.89 (m, 2H), 3.69 (s,
H). HRMS calcd for C H NO S, 408.0900; found, 408.0890.
−
HRMS calcd for C H NO S , 428.0962; found, 428.09553.
25
18
4
4
-[2-(2-Benzenesulfonamidophenyl)ethynyl]benzoic Acid (17a).
3
2
2
17
5
Step 1. A screw cap vessel was charged with 4-bromo-benzoic acid
methyl ester (15.55 g; 0.07 mol), 2-ethynylphenylamine (7 mL; 0.06
mol), and diisopropylamine (13 mL; 0.090 mol). The resulting
mixture was purged with argon for 10 min. Then, copper(I) iodide
4
-[2-(4-Methoxy-2,3-dimethyl-benzenesulfonylamino)-phenyle-
thynyl]-benzoic Acid (17d). The compound was prepared as
described for 17b. 4-(2-Amino-phenylethynyl)-benzoic acid methyl
ester (80 mg; 0.23 mmol) yielded 27% (32 mg) of the product as a
(569 mg; 3.00 mmol) and tetrakis(triphenylphosphine)palladium(0)
(3.5 g; 3 mmol) were added and the reaction mixture was stirred at 70
°C overnight, diluted with ethyl acetate, and washed with water.
1
white solid. LC−MS 2: 1.86 min; 436.00 (M + H). H NMR (400
MHz, DMSO-d ): δ 13.10 (s, 1H), 9.85 (s, 1H), 8.00−7.95 (m, 2H),
6
7
.65 (d, J = 8.9 Hz, 1H), 7.63−7.58 (m, 2H), 7.49−7.44 (m, 1H),
.39−7.32 (m, 2H), 7.20−7.15 (m, 1H), 6.83 (d, J = 9.0 Hz, 1H),
Combined organic phases were washed with brine, dried over
Na SO , filtered, and evaporated in vacuo. The crude product was
7
2
4
3.76 (s, 3H), 2.45 (s, 3H), 1.93 (s, 3H). HRMS calcd for
C H NO S, 436.1213; found, 436.1206.
crystallized from toluene to give 4-(2-amino-phenylethynyl)-benzoic
acid methyl ester (9.42 g; 58%) as a yellow solid. The filtrate after
crystallization was concentrated in vacuo, and an additional amount of
product was isolated by chromatography to give: 4-(2-amino-
phenylethynyl)-benzoic acid methyl ester (4.40 g; 28%) as a yellow
solid. LCMS 4: 3.68 min; 252.0 [M + H].
2
4
21
5
4
-[2-(2,5-Dichloro-benzenesulfonylamino)-phenylethynyl]-ben-
zoic Acid (17e). 4-(2-Amino-phenylethynyl)-benzoic acid methyl
ester (140 mg; 0.56 mmol), sodium bicarbonate (52 mg; 0.61 mmol),
and 2,5-dichlorobenzenesulfonyl chloride (151 mg; 0.61 mmol) were
mixed in CH CN (4.20 mL), and the reaction mixture was stirred at
3
Step 2. 4-(2-Amino-phenylethynyl)-benzoic acid methyl ester (50
mg; 0.20 mmol), benzenesulfonyl chloride (52.72 mg; 0.30 mmol),
and sodium bicarbonate (25.07 mg; 0.30 mmol) were dissolved in
anhydrous acetonitrile (5 mL). The reaction mixture was stirred at
RT for 48 h. The reaction mixture was diluted with ethyl acetate and
washed with water twice. The organic layer was washed with brine,
dried over sodium sulfate, and evaporated under reduced pressure.
Crude 4-(2-benzenesulfonylamino-phenylethynyl)-benzoic acid meth-
60 °C for one night. DCM was added followed by HCl 1 M. The
aqueous layer was extracted once again with DCM. Combined organic
layers were dried over Na SO . Solvents were removed under vacuum
to afford the ester intermediate. It was dissolved in THF (5 mL) and
MeOH (5 mL), and NaOH (500 μL; 5 M; 2.50 mmol) was added.
The reaction was stirred at 45 °C for one night and the solvents were
removed under vacuum. Aqueous workup was performed: DCM (5
mL) and water (3 mL) were added; HCl 5 M (>500 μL) was added
2
4
1
1921
https://doi.org/10.1021/acs.jmedchem.1c00448
J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
to reach pH = 1. The aqueous phase was extracted once again with
[2-(naphthalene-2-sulfonylamino)-phenylethynyl]-benzoic acid meth-
DCM. The solvent was removed under vacuum to afford the desired
yl ester (92 mg; 83%), which was saponified to the white solid
1
1
product in 54% (133 mg) yield as a pink solid. H NMR (300 MHz,
product (19 mg; 22%). H NMR (400 MHz, DMSO-d ): δ 13.15 (s,
6
DMSO): δ 13.20 (br s, 1H), 10.53 (s, 1H), 8.02−7.92 (m, 2H), 7.86
1H), 10.20 (s, 1H), 8.36 (d, J = 1.8 Hz, 1H), 8.00−7.96 (m, 1H),
7.93 (d, J = 1.9 Hz, 1H), 7.91 (d, J = 1.6 Hz, 2H), 7.90 (d, J = 1.8 Hz,
1H), 7.71 (dd, J = 8.7, 1.9 Hz, 1H), 7.63 (ddd, J = 8.2, 6.8, 1.3 Hz,
1H), 7.55 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 7.46−7.43 (m, 3H), 7.42−
7.38 (m, 2H), 7.24 (ddd, J = 7.6, 6.4, 2.3 Hz, 1H). LC−MS 5: 8.5
(
d, J = 2.0 Hz, 1H), 7.64−7.51 (m, 5H), 7.42 (dd, J = 7.3, 1.5 Hz,
1
4
H), 7.39−7.23 (m, 2H). HRMS calcd for C H Cl NO S,
2
1
13
2
4
46.0015; found, 445.9993.
-{2-[2-(Naphthalene-1-sulfonamido)phenyl]ethynyl}benzoic
4
−
Acid (17f). 4-(2-Amino-phenylethynyl)-benzoic acid methyl ester (80
mg; 0.31 mmol) was dissolved in anhydrous pyridine (1 mL) and
naphthalene-1-sulfonyl chloride (95 mg; 0.42 mmol) was added.
Then reaction was stirred at RT for overnight, 15 mL of water was
added and acidified with 2 M HCl solution to pH 3. The mixture was
extracted with ethyl acetate, dried over anhydrous Na SO , and
min; 428.0 [M + H]; HRMS calcd for C H NO S , 426.0806;
2
5
16
4
found, 426.0804.
3-{2-[2-(Naphthalene-2-sulfonamido)phenyl]ethynyl}benzoic
Acid (17i). 3-(2-Amino-phenylethynyl)-benzoic acid methyl ester (37
mg; 0.15 mmol), naphthalene-2-sulfonyl chloride (50 mg; 0.22
mmol), and NaHCO (19 mg; 0.22 mmol) were stirred overnight at
2
4
3
evaporated. The residue was purified by chromatography to yield 4-
2-(naphthalene-1-sulfonylamino)-phenylethynyl]-benzoic acid meth-
RT in acetonitrile (2 mL). Second portion of naphthalene-2-sulfonyl
[
chloride (17 mg; 0.07 mmol) and NaHCO (12 mg; 0.15 mmol) were
3
yl ester (127 mg; 87%) as a white solid. LCMS 4: 4.44 min; 440 [M −
added and stirring was continued for additional 24 h. The reaction
mixture was diluted with ethyl acetate, and washed with water and
brine. The organic extract was dried over Na SO and evaporated.
H].
The ester was dissolved in THF (5 mL), then water (2 mL) and
lithium hydroxide monohydrate (34 mg; 0.41 mmol) was added, and
the mixture was stirred at RT overnight. The THF was evaporated
and 15 mL of water was added to the residue. The mixture was
acidified with 2 M hydrochloric acid to pH 3 and extracted three
times with ethyl acetate. The combined organics extracts were dried
over anhydrous Na SO . Evaporation resulted in the product (119
2
4
The oily residue was purified by chromatography to give 3-[2-
(naphthalene-2-sulfonylamino)-phenylethynyl]-benzoic acid methyl
1
ester (28 mg; 43%) as a white solid. H NMR (400 MHz, DMSO-
d ): δ 10.24 (s, 1H), 8.32 (d, J = 1.9 Hz, 1H), 8.01 (td, J = 1.7, 0.6
6
Hz, 1H), 7.98−7.93 (m, 1H), 7.93−7.83 (m, 3H), 7.68 (dd, J = 8.7,
1.9 Hz, 1H), 7.59 (ddd, J = 8.2, 6.9, 1.3 Hz, 1H), 7.55 (dt, J = 7.7, 1.5
Hz, 1H), 7.52−7.47 (m, 2H), 7.44−7.39 (m, 3H), 7.23 (dt, J = 7.5,
2
4
1
mg; 88%) as a white solid. H NMR (400 MHz, DMSO-d ): δ: 13.15
6
(
s, 1H), 10.37 (s, 1H), 8.67 (d, J = 8.6 Hz, 1H), 8.15 (d, J = 8.2 Hz,
4.4 Hz, 1H), 3.93 (s, 3H). LCMS 5: 7.7 min; 440.1 [M − H]; HRMS
−
1
7
7
4
4
H), 8.09 (dd, J = 7.4, 1.3 Hz, 1H), 7.98 (dd, J = 8.0, 1.5 Hz, 1H),
.96−7.91 (m, 2H), 7.58−7.44 (m, 3H), 7.43−7.39 (m, 3H), 7.38−
.33 (m, 2H), 7.19 (dq, J = 8.2, 4.4 Hz, 1H). LC−MS 5: 6.7 min;
calcd for C25
H
16NO
4
S , 426.0806; found, 426.0802.
The ester (21 mg; 0.05 mmol) was added to a mixture of 5 N
NaOH (0.2 mL; 0.95 mmol) and methanol (2 mL). The suspension
became a solution within 10 min of stirring at RT. After 1 h, the
reaction mixture was diluted with water and neutralized with 1 M
HCl. The formed precipitate was taken up to AcOEt. The organic
layer was extracted with brine, dried over Na SO , and evaporated to
−
28.0 [M + H]; HRMS calcd for C H NO S , 426.0806; found,
2
5
16
4
26.0805.
2
-Fluoro-4-[2-(naphthalene-1-sulfonylamino)-phenylethynyl]-
benzoic Acid (17g). Step 1. 2-Ethynyl-phenylamine (0.10 mL; 0.84
mmol) and 4-bromo-2-fluoro-benzoic acid methyl ester (0.19 mL;
2
4
give 40 mg of oily residue, which was purified by chromatography.
1
.25 mmol) were added to the vessel containing acetonitrile (6 mL)
Proper fractions were combined and evaporated to give the product
1
and diisopropyl-amine (0.18 mL; 1.25 mmol). The resulting mixture
was argonated for 10 min. Then, copper(I) iodide (7.97 mg; 0.04
mmol) and tetrakis(triphenylphosphine)palladium(0) (48.33 mg;
(15 mg; 73%) as a beige solid. H NMR (400 MHz, DMSO-d ): δ:
6
13.24 (s, 1H), 10.25 (s, 1H), 8.33 (d, J = 1.8 Hz, 1H), 8.08 (t, J = 1.7
Hz, 1H), 7.96−7.86 (m, 4H), 7.71 (dd, J = 8.7, 1.9 Hz, 1H), 7.60
(ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 7.55−7.46 (m, 3H), 7.44−7.37 (m,
3H), 7.26−7.18 (m, 1H). LC−MS 5: 8.5 min; 428.0 [M + H];
0.04 mmol) were added. The reaction mixture was stirred at 100 °C
for 1 h and then at 70 °C for 3 h. The reaction mixture was diluted
with ethyl acetate and filtrated. The filtrate was evaporated and the
residue was purified by chromatography giving 4-(2-aminophenyle-
thynyl)-2-fluoro-benzoic acid methyl ester (159 mg; 71%) as a yellow
solid. LCMS 4: 3.78 min; 270.30 [M + H].
−
HRMS calcd for C H NO S , 426.0806; found, 426.0802.
2
5
16
4
4-{2-[2-(Quinoline-8-sulfonamido)phenyl]ethynyl}benzoic Acid
(17j). Quinoline-8-sulfonyl chloride (95 mg; 0.42 mmol) was added
to a mixture of 4-(2-amino-phenylethynyl)-benzoic acid methyl ester
Step 2. Naphthalene-1-sulfonyl chloride (37 mg; 0.16 mmol) was
added to a mixture of 4-(2-aminophenylethynyl)-2-fluoro-benzoic
acid methyl ester (40 mg; 0.15 mmol) in pyridine (0.5 mL). The
reaction was carried out for 5h at 70 °C, quenched with water, and
acidified with 2 M HCl. The crude product was extracted with ethyl
acetate. The solvent was evaporated to give 2-fluoro-4-[2-
(70 mg; 0.28 mmol) and NaHCO (35.10 mg; 0.42 mmol) in
3
acetonitrile (5 mL). The reaction was carried out at RT. After 72 h,
the reaction was charged with an additional amount of quinoline-8-
sulfonyl chloride (44 mg; 0.20 mmol) and another one after 6 days
with quinoline-8-sulfonyl chloride (19 mg; 0.08 mmol). The reaction
was quenched with water after stirring for 8 days at RT and the
product was extracted with ethyl acetate from the water phase, which
was acidified with 2 M HCl. The combined organic phase was dried
(
naphthalene-1-sulfonylamino)-phenylethynyl]-benzoic acid methyl
ester (74 mg; 99%) as a brown solid. LCMS 4: = 4.37 min; 458.0
[
M − H].
over anhydrous Na SO . The solvent was evaporated and the residue
2 4
Step 3. The ester (74 mg; 0.15 mmol) was dissolved in methanol
5 mL) and NaOH (1.11 mL; 2.21 mmol) was added. The reaction
was purified by chromatography to yield 4-[2-(quinoline-8-sulfony-
lamino)-phenylethynyl]-benzoic acid methyl ester (116 mg; 92%) as a
nude solid. LC−MS 4: 4.09 min; 443.3 [M + H]; 441.1 [M − H].
The ester (60 mg; 0.13 mmol) was dissolved in methanol (3 mL), and
5 M NaOH (0.5 mL; 2.66 mmol) was added. The reaction mixture
was stirred for 2.5 h at 60 °C, diluted with ethyl acetate, washed with
water in the presence of 2 M HCl, and extracted with ethyl acetate.
(
mixture was stirred overnight at RT, diluted with ethyl acetate,
washed with acidified water (pH = 5), and extracted with ethyl
acetate. The combined organic layers were dried over Na SO and
2
4
evaporated. The crude product was purified by chromatography to
1
yield the product (35 mg; 53%) as a white solid. H NMR (400 MHz,
DMSO-d ): δ 13.48 (s, 1H), 10.48 (s, 1H), 8.71−8.64 (m, 1H),
The combined organic layers were dried over Na SO and evaporated.
2 4
6
8
1
3
.17−8.10 (m, 1H), 8.04 (dd, J = 7.4, 1.2 Hz, 1H), 7.99−7.93 (m,
H), 7.84 (t, J = 8.1 Hz, 1H), 7.54−7.33 (m, 6H), 7.22−7.13 (m,
The crude product was purified by chromatography to give the
1
product (30 mg; 53%) as a white solid. H NMR (400 MHz, DMSO-
H). LC−MS 5: 6.7 min; 444.0 [M − H]; HRMS calcd for
d ): δ 13.21 (s, 1H), 9.26 (s, 1H), 8.79 (dd, J = 4.3, 1.8 Hz, 1H), 8.47
6
−
C H FNO S , 444.0711; found, 444.0706.
(dd, J = 8.4, 1.8 Hz, 1H), 8.39 (dd, J = 7.3, 1.5 Hz, 1H), 8.28 (dd, J =
8.3, 1.5 Hz, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.74 (dd, J = 8.3, 7.3 Hz,
1H), 7.64−7.58 (m, 2H), 7.55 (dd, J = 8.3, 4.3 Hz, 1H), 7.43 (ddd, J
= 18.1, 8.0, 1.3 Hz, 2H), 7.30 (ddd, J = 8.5, 7.5, 1.6 Hz, 1H), 7.07 (td,
J = 7.6, 1.2 Hz, 1H). LC−MS 5: 6.6 min; 426.7 [M − H]; HRMS
25
15
4
4
-{2-[2-(naphthalene-2-sulfonamido)phenyl]ethynyl}benzoic
Acid (17h). The compound was prepared as descried for 17f. 4-(2-
Amino-phenylethynyl)-benzoic acid methyl ester (51 mg; 0.20 mmol)
and naphthalene-2-sulfonyl chloride (50 mg; 0.22 mmol) yielded 4-
1
1922
https://doi.org/10.1021/acs.jmedchem.1c00448
J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+
calcd for C H N2O S , 429.0909; found, 429.0901, calcd for
151.93, 143.83, 138.96, 133.91, 132.96, 131.95, 131.78, 131.40,
130.67, 130.11, 126.86, 126.51, 124.72, 122.37, 120.77, 119.84,
113.99, 104.35, 94.68, 87.72, 57.15. LC−MS 5: 6.8 min; 459.4 [M +
2
4
17
4
−
C H N O S , 427.0758; found, 427.0750.
-(Methylamino)-4-{2-[2-(7-methylquinoline-8-sulfonamido)-
phenyl]ethynyl}benzoic Acid (17k). Step 1. 2-Ethynyl-phenylamine
24
15
2
4
2
+
H]; HRMS calcd for C H N O S , 459.1015; found, 459.1006;
2
5
19
2
5
−
(
0.1 mL; 0.58 mmol) and 4-bromo-2-methylamino-benzoic acid
calcd for C H N O S , 457.0864; found, 457.0860.
25 17 2 5
methyl ester (120 mmol) were dissolved in anhydrous acetonitrile (4
mL) and the mixture was argonated for 10 min. Then, copper(I)
iodide (3 mg; 0.01 mmol) and tetrakis(triphenylphosphine)-
palladium(0) (17 mg; 0.01 mmol) were added and the reaction
mixture was stirred at 65 °C overnight. After dilution with ethyl
acetate and filtration, the residue was purified by chromatography to
afford 4-(2-amino-phenylethynyl)-2-methylamino-benzoic acid meth-
yl ester (100 mg; 70%) as a yellow solid. LCMS 4: 4.17 min; m/z =
4-{2-[2-(7-Methylquinoline-8-sulfonamido)phenyl]ethynyl}-
benzoic Acid (17m). 4-(2-Amino-phenylethynyl)-benzoic acid methyl
ester (100 mg; 0.39 mmol) was dissolved in anhydrous pyridine (0.5
mL) and 7-methyl-quinoline-8-sulfonyl chloride (103 mg; 0.42
mmol) was added in one portion. The reaction mixture was stirred
overnight. An additional portion of 7-methyl-quinoline-8-sulfonyl
chloride (50 mg; 0.21 mmol) was added, and the reaction mixture was
stirred overnight. A third portion of 7-ethyl-quinoline-8-sulfonyl
chloride (20 mg; 0.08 mmol) was added, and stirring continued for
overnight. The reaction mixture was diluted with toluene, evaporated,
and the crude product was purified by chromatography to give 4-[2-
(7-methyl-quinoline-8-sulfonylamino)-phenylethynyl]-benzoic acid
methyl ester (96 mg; 53%) as a white solid. LCMS 4: 4.496 min;
457.20 [M + H]; 455.10 [M − H]. The ester (96 mg; 0.20 mmol)
was dissolved in THF (3 mL) and water (1 mL), and lithium
hydroxide monohydrate (49 mg; 1.16 mmol) was added. The reaction
mixture was stirred overnight at RT and partially evaporated, diluted
with water, and neutralized with 2 M HCl. The product was extracted
with ethyl acetate, washed with water and brine, dried over Na SO ,
2
81.15 [M + H].
Step 2. 4-(2-Amino-phenylethynyl)-2-methylamino-benzoic acid
methyl ester (100 mg; 0.35 mmol) was dissolved in pyridine (2 mL),
and 7-methyl-quinoline-8-sulfonyl chloride (100 mg; 0.42 mmol) was
added. The reaction mixture was stirred at RT overnight. The solvent
was coevaporated with toluene, and the crude was purified by
chromatography to afford 2-methylamino-4-[2-(7-methyl-quinoline-8-
sulfonylamino)-phenylethynyl]-benzoic acid methyl ester (150 mg;
0
4
.31 mmol) as a yellow solid. LCMS 4: 4.68 min; 486.25 [M + H];
84.25 [M − H].
Step 3. To a stirred solution of 2-methylamino-4-[2-(7-methyl-
2
4
quinoline-8-sulfonylamino)-phenylethynyl]-benzoic acid methyl ester
150 mg; 0.31 mmol) in water (0.5 mL) and THF (0.5 mL), lithium
and evaporated under reduced pressure. Crude was purified by
(
chromatography to give the product (70 mg; 40%) as a white solid.
1
hydroxide (37 mg; 1.53 mmol) was added. The reaction mixture was
stirred at RT for 6 h and NaOH (61 mg; 1.53 mmol) was added. The
reaction mixture was stirred at RT overnight. A second portion of
NaOH (245 mg; 6.12 mmol) and MeOH (3 mL) was added, and the
reaction mixture was stirred at 50 °C for 6 h. The reaction mixture
was neutralized with HCl 1 N, and the aqueous layer was extracted
with DCM, dried with Na SO , and evaporated. The crude was
H NMR (300 MHz, DMSO-d ): δ 13.22 (s, 1H), 9.67 (s, 1H), 8.77
6
(dd, J = 4.3, 1.8 Hz, 1H), 8.41 (dd, J = 8.4, 1.8 Hz, 1H), 8.14 (d, J =
8.5 Hz, 1H), 8.07−8.02 (m, 2H), 7.69−7.65 (m, 2H), 7.61 (d, J = 8.5
Hz, 1H), 7.53−7.47 (m, 2H), 7.43 (dd, J = 7.7, 1.6 Hz, 1H), 7.33−
7.27 (m, 1H), 7.03 (td, J = 7.6, 1.2 Hz, 1H), 2.90 (s, 3H). LC−MS 4:
+
6.8 min; 443.4 [M + H]; HRMS calcd for C H N O S , 443.1066;
2
5
19
2
4
−
2
4
found, 443.1060; calcd for C H N O S , 441.0914; found,
25 17
2
4
purified by chromatography to yield the product (101 mg; 70%) as a
441.0913.
1
yellow solid. H NMR (300 MHz, DMSO-d ): δ 9.68 (s, 1H), 8.80
2-Methoxy-4-{2-[2-(7-methylquinoline-8-sulfonamido)phenyl]-
ethynyl}benzoic Acid (17n). Step 1. 2-Ethynyl-phenylamine (0.15
mL; 1.28 mmol), 4-bromo-2-methoxy-benzoic acid methyl ester (345
mg; 1.41 mmol), and diisopropylamine (0.28 mL; 1.92 mmol) were
added to the pressure vessel. Then anhydrous acetonitrile (5 mL) was
added, and the reaction mixture was bubbled with argon for 10 min.
Then, copper(I) iodide (12 mg; 0.06 mmol) and tetrakis-
(triphenylphosphine)palladium(0) (74 mg; 0.06 mmol) were added.
The reaction mixture was stirred at 60 °C overnight, diluted with
ethyl acetate, and filtrated. The filtrate was washed with water and
brine, dried over Na SO , and evaporated under reduced pressure.
6
(
dd, J = 4.3, 1.8 Hz, 1H), 8.43 (dd, J = 8.4, 1.8 Hz, 1H), 8.15 (d, J =
8
7
1
6
.5 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.57−
.48 (m, 2H), 7.42 (dd, J = 7.7, 1.5 Hz, 1H), 7.30 (ddd, J = 8.6, 7.5,
.6 Hz, 1H), 7.02 (td, J = 7.6, 1.1 Hz, 1H), 6.87 (d, J = 1.5 Hz, 1H),
.77 (dd, J = 8.1, 1.5 Hz, 1H), 2.92 (s, 3H), 2.90 (s, 3H); not visible
signals of the carboxylic protons. LC−MS 5: 7.2 min; 472.1 [M + H];
+
HRMS calcd for C H N O S , 472.1326; found, 472.1324; calcd for
26
22
3
4
−
C H N O S , 470.1180; found, 470.1174.
26
20
3
4
4
-{2-[2-(5-Methoxyquinoline-8-sulfonamido)phenyl]ethynyl}-
benzoic Acid (17l). 4-(2-Amino-phenylethynyl)-benzoic acid methyl
ester (9 g; 33.09 mmol) was suspended in anhydrous pyridine (40
mL) under argon. 5-Methoxy-quinoline-8-sulfonyl chloride (9.38 g;
2
4
The crude was purified by chromatography to give 4-(2-amino-
phenylethynyl)-2-methoxy-benzoic acid methyl ester (326 mg; 87%)
as a yellow solid. LCMS 4: RT = 1.45 min; 282.7 [M + H].
Step 2. A mixture of 4-(2-amino-phenylethynyl)-2-methoxy-
benzoic acid methyl ester (100 mg; 0.34 mmol), 7-methyl-
quinoline-8-sulfonyl chloride (99 mg; 0.41 mmol), and anhydrous
pyridine (0.5 mL) was stirred overnight at RT. The reaction mixture
was diluted with toluene and evaporated under reduced pressure. The
crude was purified by chromatography provide to: 2-methoxy-4-[2-(7-
methyl-quinoline-8-sulfonylamino)-phenylethynyl]-benzoic acid
methyl ester (145 mg; 86%) as a white solid. LCMS 4: 1.60 min;
487.7 [M + H].
3
6.40 mmol) was added in three portions. The resulting mixture was
stirred at RT overnight. The reaction mixture was evaporated in vacuo
to dryness with toluene. The residue was suspended in water and
extracted to ethyl acetate. Combined organic layers were washed with
brine, dried over anhydrous Na SO , and evaporated in vacuo. The
2
4
crude product was purified by chromatography to give: 4-[2-(5-
methoxy-quinoline-8-sulfonylamino)-phenylethynyl]-benzoic acid
methyl ester (6.50 g; 38.2%) as a white solid. LCMS 4: 3.89 min;
4
.73.1 [M + H]. The ester (6.40 g; 12.46 mmol) was suspended in
THF (210 mL). Water (70 mL) and lithium hydroxide monohydrate
2.09 g; 49.84 mmol) were added. The resulting mixture was stirred at
5 °C overnight and THF was removed in vacuo. The remainder was
(
4
Step 3. 2-Methoxy-4-[2-(7-methyl-quinoline-8-sulfonylamino)-phe-
nylethynyl]-benzoic acid methyl ester (145 mg; 0.30 mmol) was
dissolved in THF (6 mL) and water (2 mL); then, lithium hydroxide
monohydrate (43 mg; 1.03 mmol) was added, and the reaction
mixture was stirred at rt overnight. THF was removed in vacuo, the
residue was diluted with additional portion of water, and neutralized
with 1 M HCl. The product was extracted to ethyl acetate, the organic
layer was washed with brine, dried over Na SO , and evaporated to
diluted with 200 mL water and adjusted to pH = 6 (2 M HCl). The
formed precipitate product was filtered off, washed with water, and
dried on air. The product was suspended in 200 mL of ethanol and
refluxed for 30 min, then solid was collected upon filtration, rinsed
with cold ethanol, and dried in vacuo overnight to obtain the product
1
(
5.91 g; 100%) as a white solid. H NMR (300 MHz, DMSO-d ): δ
6
2
4
1
3.23 (s, 1H), 9.05 (s, 1H), 8.76 (dd, J = 4.3, 1.8 Hz, 1H), 8.53 (dd, J
8.5, 1.8 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.06−8.02 (m, 2H),
dryness. The crude product was purified by chromatography to
provide the product (75 mg; 46%) as a white solid. H NMR (400
1
=
7
7
4
.66−7.63 (m, 2H), 7.54−7.47 (m, 2H), 7.42−7.38 (m, 1H), 7.34−
MHz, DMSO-d ): δ 12.87 (s, 1H), 9.68 (s, 1H), 8.80−8.76 (m, 1H),
6
.27 (m, 1H), 7.15 (d, J = 8.5 Hz, 1H), 7.05 (td, J = 7.5, 1.1 Hz, 1H),
8.43−8.39 (m, 1H), 8.16−8.13 (m, 1H), 7.74 (d, 1H), 7.61 (d, 1H),
7.53−7.49 (m, 2H), 7.42 (dd, J = 7.7, 1.5 Hz, 1H), 7.33−7.27 (m,
.04 (s, 3H). 13C NMR (75 MHz, DMSO-d ): δ 167.18, 159.75,
6
1
1923
https://doi.org/10.1021/acs.jmedchem.1c00448
J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
2
2
H), 7.19 (dd, J = 7.9, 1.4 Hz, 1H), 7.05−7.01 (m, 1H), 3.89 (s, 3H),
Na SO , and evaporated. The residue was purified by chromatography
2 4
to yield the product (49 mg; 100%) as a yellow-brown glass. H NMR
+
1
.90 (s, 3H). LCMS 5: 6.7 min; 473.1 [M + H] ; HRMS calcd for
+
−
C H N O S , 473.1171; found, 473.1166; calcd for C H N O S ,
(400 MHz, DMSO-d ): δ 13.37 (s, 1H), 10.28 (s, 1H), 8.39 (d, J =
26
21
2
5
26 19
2
5
6
4
71.1020; found, 471.1016.
1.8 Hz, 1H), 8.03−7.92 (m, 4H), 7.91−7.86 (m, 1H), 7.77 (dd, J =
5
-{2-[2-(Naphthalene-2-sulfonamido)phenyl]ethynyl}pyridine-2-
8.7, 1.9 Hz, 1H), 7.71 (dd, J = 5.9, 3.0 Hz, 1H), 7.60 (ddd, J = 8.2,
carboxylic Acid (18a). Step 1. 2-Ethynyl-phenylamine (1 mL; 8.37
mmol) and 5-bromo-pyridine-2-carboxylic acid methyl ester (3 mg;
6
=
.8, 1.3 Hz, 1H), 7.55−7.46 (m, 2H), 7.48−7.35 (m, 2H), 7.25 (td, J
7.4, 1.6 Hz, 1H). LCMS 2: 7.7 min; 429.1 [M + H], 427.00 [M −
1
2.55 mmol) were added to the vessel containing acetonitrile (50
−
+
H] ; HRMS calcd for C H N O S , 429.0909; found, 429.0902;
calcd for C H N O S , 427.0758; found, 427.0752.
2
4
17
2
4
mL). Diisopropylamine (1.8 mL; 12.55 mmol) was added, and the
resulting mixture was argonated for 10 min. Then, copper(I) iodide
−
2
4
15
2
4
2
-{2-[2-(Naphthalene-2-sulfonamido)phenyl]ethynyl}pyridine-4-
(
(
80 mg; 0.42 mmol) and tetrakis(triphenylphosphine)palladium(0)
483 mg; 0.42 mmol) were added, and the reaction mixture was
carboxylic Acid (20). Step 1. 2-Ethynyl-phenylamine (0.1 mL; 0.84
mmol) and 2-bromoisonicotinic acid methyl ester (271 mg; 1.25
mmol) were dissolved in acetonitrile (6 mL), diisopropylamine (0.2
mL; 1.25 mmol) was added, and the mixture was argonated for 10
min. Then, copper(I) iodide (8 mg; 0.04 mmol) and tetrakis-
stirred overnight at 70 °C. After cooling, the yellow precipitate was
filtered and washed with cold ethyl acetate and hexane. The solid
material was dissolved in DCM (250 mL) and washed with water and
brine to remove diisopropylamine hydrobromide. The organic layer
was dried over Na SO and evaporated to give 5-(2-amino-
phenylethynyl)-pyridine-2-carboxylic acid methyl ester (2.06 g;
9
(triphenylphosphine)palladium(0) (48 mg; 0.04 mmol) were added
2
4
and the reaction mixture was stirred overnight at 70 °C. The reaction
mixture was diluted with ethyl acetate and filtered. The filtrate was
evaporated and the residue was purified by chromatography to give 2-
5%) as a yellow solid. LCMS 1: 3.22 min; 253.2 [M + H].
Step 2. Naphthalene-2-sulfonyl chloride (98 mg; 0.43 mmol) was
(2-amino-phenylethynyl)isonicotinic acid methyl ester (34 mg; 16%)
added to a mixture of 5-(2-amino-phenylethynyl)-pyridine-2-carbox-
ylic acid methyl ester (100 mg; 0.39 mmol) in pyridine (0.70 mL).
The reaction was carried out at RT overnight and quenched with
water. The product was extracted with ethyl acetate from the water
phase after acidification with 2 M HCl. The solvent was evaporated to
yield 5-[2-(naphthalene-2-sulfonylamino)-phenylethynyl]-pyridine-2-
carboxylic acid methyl ester (181 mg; 99%) as a light yellow solid.
LCMS 1: 3.85 min; 443.05 [M + H]; 441.05 [M − H].
Step 3. 5-[2-(Naphthalene-2-sulfonylamino)-phenylethynyl]-pyri-
dine-2-carboxylic acid methyl ester (181 mg; 0.39 mmol) was
dissolved in methanol (4 mL). Water (2 mL) and lithium hydroxide
hydrate (408 mg; 9.71 mmol) were added. RM was stirred overnight
at RT. The mixture was acidified with hydrochloric acid and extracted
as a yellow solid. LCMS 4: 3.25 min; 253.0 [M + H].
Step 2. 2-(2-Amino-phenylethynyl)-isonicotinic acid methyl ester
(34 mg; 0.13 mmol) was suspended with sodium bicarbonate (17 mg;
0.20 mmol) in acetonitrile (4 mL), and naphthalene-2-sulfonyl
chloride (41 mg; 0.18 mmol) was added. The reaction was stirred at
rt for 72 h and after evaporating, crude 2-[2-(naphthalene-2-
sulfonylamino)-phenylethynyl]-isonicotinic acid methyl ester (67
mg; 92%) was obtained as a brown solid. LCMS 4: 3.89 min; 441.0
[
M − H].
Step 3. The ester (67 mg; 0.12 mmol) was suspended in 3 mL
methanol and treated with 5 M solution NaOH to pH 12. The
mixture was stirred at RT for 2 h when everything was dissolved. After
acidification with 10% solution of HCl to pH 5 and extraction three
times with ethyl acetate, the combined organic extracts were dried
with ethyl acetate. Combined organic layers were dried over Na SO4
2
and evaporated. The product was isolated by chromatography as a
1
under anhydrous Na SO and evaporated. After chromatography, the
2 4
white solid (94 mg; 56%). H NMR (400 MHz, DMSO-d ): δ 13.37
6
product was obtained (7 mg; 12% calculated for two steps) as a bright
(
s, 1H), 10.23 (s, 1H), 8.64 (d, J = 2.0 Hz, 1H), 8.33 (d, J = 1.8 Hz,
1
brown solid. H NMR (400 MHz, DMSO-d ): δ 13.98 (s, 1H), 10.39
6
1
1
7
H), 8.00 (d, J = 8.1 Hz, 1H), 7.96−7.82 (m, 4H), 7.67 (dd, J = 8.6,
(s, 1H), 8.67 (dd, J = 5.0, 0.9 Hz, 1H), 8.35 (d, J = 1.8 Hz, 1H), 8.14
.9 Hz, 1H), 7.60 (ddd, J = 8.2, 6.7, 1.2 Hz, 1H), 7.54−7.38 (m, 4H),
+
(dd, J = 1.6, 0.9 Hz, 1H), 7.93−7.84 (m, 3H), 7.77 (dd, J = 5.0, 1.7
Hz, 1H), 7.73 (dd, J = 8.6, 1.9 Hz, 1H), 7.59 (ddd, J = 8.2, 6.8, 1.3
Hz, 1H), 7.53−7.49 (m, 1H), 7.48 (d, J = 1.4 Hz, 1H), 7.47−7.43 (m,
.26 (td, J = 7.1, 2.0 Hz, 1H). LC−MS 2: 6.2 min; 429.1 [M + H] ,
+
HRMS calcd for C H N O S , 429.0909; found, 429.0901; calcd for
24
17
2
4
−
C H N O S , 427.0758; found, 427.0754.
24
15
2
4
1
H), 7.43 (t, J = 1.5 Hz, 1H), 7.29−7.22 (m, 1H). LCMS 5: 7.5 min;
6
-{2-[2-(Naphthalene-2-sulfonamido)phenyl]ethynyl}pyridine-2-
+
429.0 [M + H], 427.00 [M − H]; HRMS calcd for C H N O S ,
carboxylic Acid (19). Step 1. 6-Bromo-pyridine-2-carboxylic acid
24 17
2
4
−
4
29.0909; found, 429.0903; calcd for C H N O S , 427.0758;
methyl ester (250 mg; 1.16 mmol), 2-ethynyl-phenylamine (0.2 mL;
24 15
2
4
found, 427.0752.
1.74 mmol), and diisopropylamine (0.3 mL; 2.31 mmol; 2.00 equiv)
5
-{2-[2-(Naphthalene-2-sulfonamido)phenyl]ethynyl}pyridine-3-
were dissolved in acetonitrile (2.5 mL) and purged with argon for 10
min. Then, tetrakis(triphenylphosphine)palladium(0) (67 mg; 0.06
mmol) and copper(I) iodide (11 mg; 0.06 mmol) were added and the
reaction mixture was heated to 65−70 °C for 18 h. After dilution with
ethyl acetate and filtration, 6-(2-amino-phenylethynyl)-pyridine-2-
carboxylic acid methyl ester was obtained after chromatography (346
mg; 100%) as a brow-green glue. LCMS 1: 3.29 min; 253.7 [M + H].
Step 2. 6-(2-Amino-phenylethynyl)-pyridine-2-carboxylic acid
methyl ester (70 mg; 0.24 mmol), naphthalene-2-sulfonyl chloride
carboxylic Acid (21). Step 1. 2-Ethynyl-phenylamine (0.1 mL; 0.84
mmol) and 5-bromo-nicotinic acid methyl ester (272 mg; 1.25 mmol)
were dissolved in acetonitrile (6 mL). Diisopropylamine (0.2 mL;
1
.25 mmol) was added, and the mixture was argonated for 10 min.
Then, copper(I) iodide (8 mg; 0.04 mmol) and tetrakis-
triphenylphosphine)palladium(0) (48 mg; 0.04 mmol) were added,
(
and the mixture was stirred at 70 °C overnight. After dilution with
ethyl acetate and filtration, the residue was purified by chromatog-
raphy giving 5-(2-amino-phenylethynyl)-nicotinic acid methyl ester
(
60 mg; 0.26 mmol), and NaHCO3 (24 mg; 0.29 mmol) were
(
174 mg; 83%) as a yellow solid. LCMS 4: 3.32 min; 253.3 [M + H].
Step 2. Naphthalene-2-sulfonyl chloride (61 mg; 0.27 mmol) was
suspended in acetonitrile (2 mL) and the mixture was stirred
overnight at RT. The reaction mixture was portioned by water and
ethyl acetate. The organic layer was washed with water and brine,
dried over Na SO , and evaporated. The residue was purified by
added to a mixture of 5-(2-aminophenylethynyl)-nicotinic acid
methyl ester (50 mg; 0.20 mmol; 1.00 equiv) and NaHCO (25
3
2
4
chromatography to give 6-[2-(naphthalene-2-sulfonylamino)-phenyl-
ethynyl]-pyridine-2-carboxylic acid methyl ester (49 mg; 46%), which
was used directly for saponification. LCMS 1: 3.84 min; 441.1 [M −
H].
mg; 0.30 mmol) in acetonitrile (5 mL). The reaction was carried out
at rt for 48 h. Then, additional amount of naphthalene-2-sulfonyl
chloride (22 mg; 0.10 mmol) was added, and the mixture was stirred
at RT for 24 h. After quenching with water and acidification with 2 M
HCl, the product was extracted with ethyl acetate. Combined organic
Step 3. The ester (49 mg; 0.11 mmol) was dissolved in methanol
(
3 mL) and 5 N NaOH (1 mL; 4.77 mmol) was added. The resulting
layers were washed with brine and dried over anhydrous Na SO . The
2 4
slurry was refluxed for 2 h to complete ester hydrolysis. The reaction
mixture was diluted with water and carefully acidified with 2 M HCl
to adjust pH 5. Formed precipitate was taken up in ethyl acetate and
the organic layer was washed with water and brine, dried over
solvent was evaporated and residue was purified by chromatography
to yield 5-[2-(naphthalene-2-sulfonylamino)-phenylethynyl]-nicotinic
acid methyl ester (76 mg; 86%) as white solid. LCMS 4: 3.86 min;
441.1 [M − H].
1
1924
https://doi.org/10.1021/acs.jmedchem.1c00448
J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Step 3. The ester (76 mg; 0.17 mmol) was dissolved in methanol
3 mL), and 5 M NaOH (0.7 mL; 3.40 mmol) was added. The
reaction mixture was stirred for 3 h, diluted with water, and acidified
Step 3. The ester (36 mg; 0.08 mmol) was dissolved in THF (3
mL) and water (1 mL), then lithium hydroxide monohydrate (29 mg;
0.70 mmol) was added. The reaction mixture was stirred at RT
overnight. Then, THF was removed, the remainder was acidified to
pH 5, and extracted to ethyl acetate. The organic phase was dried over
anhydrous sodium sulfate, filtered, and evaporated in vacuo. The
product was purified by chromatography and obtained as a yellow
(
with hydrochloric acid. After extraction and chromatography, the
1
product was isolated (60 mg; 82%) as a white solid. H NMR (400
MHz, DMSO-d ): δ 13.70 (s, 1H), 10.25 (s, 1H), 8.98 (d, J = 2.1 Hz,
6
1
8
6
7
H), 8.61 (d, J = 2.1 Hz, 1H), 8.31 (q, J = 2.0 Hz, 2H), 7.85 (dd, J =
.7, 3.2 Hz, 3H), 7.62 (dd, J = 8.7, 1.9 Hz, 1H), 7.57 (ddd, J = 8.3,
.9, 1.3 Hz, 1H), 7.51−7.40 (m, 4H), 7.29−7.22 (m, 1H). LCMS 5:
.6 min; 429.0 [M + H], 427.2 [M − H]; HRMS calcd for
1
powder (29 mg; 29% counted for steps 2 and 3). H NMR (400
MHz, DMSO-d ): δ 13.25 (s, 1H), 10.25 (s, 1H), 8.99 (d, J = 2.1 Hz,
6
1H), 8.39 (d, J = 1.8 Hz, 1H), 8.26 (dd, J = 8.1, 2.2 Hz, 1H), 7.98 (d,
J = 8.2 Hz, 1H), 7.92 (t, J = 8.0 Hz, 2H), 7.75 (dd, J = 8.7, 1.9 Hz,
+
−
C H N O S , 429.0909; found, 429.0904; calcd for C H N O S ,
2
4
17
2
4
24 15
2
4
1
H), 7.64−7.59 (m, 2H), 7.56−7.36 (m, 4H), 7.25 (td, J = 7.4, 1.4
4
27.0758; found, 427.0752.
Hz, 1H). LCMS 5: 6.2 min; 428.9 [M + H], HRMS calcd for
C
4
-{2-[2-(Naphthalene-2-sulfonamido)phenyl]ethynyl}pyridine-2-
+
−
carboxylic Acid (22). Step 1. 2-Ethynyl-phenylamine (0.1 mL; 0.84
H
24
N
17
2
O
4
S , 429.0909; found, 429.0902; calcd for C24
H N O S ,
15 2 4
mmol) and 4-bromo-pyridine-2-carboxylic acid methyl ester (277 mg;
.25 mmol) were dissolved in acetonitrile (5 mL). Diisopropylamine
0.2 mL; 1.25 mmol) was added, and the resulting mixture was
427.0758; found, 427.0752.
5-{2-[2-(4-Methoxynaphthalene-1-sulfonamido)phenyl]ethynyl}-
pyrimidine-2-carboxylic Acid (24). Step 1. 2-Ethynyl-phenylamine
1
(
(
2
0.1 mL; 0.84 mmol) was added to a solution of 5-bromo-pyrimidine-
-carboxylic acid methyl ester (272 mg; 1.25 mmol) in acetonitrile (3
argonated for 10 min. Then, copper(I) iodide (8 mg; 0.04 mmol) and
tetrakis(triphenylphosphine)palladium(0) (48 mg; 0.04 mmol) were
added, and the reaction mixture was heated for 3 h at 60 °C. After
dilution with ethyl acetate and filtration, the residue was purified by
chromatography to give 4-(2-aminophenylethynyl)-pyridine-2-carbox-
ylic acid methyl ester (176 mg; 83%) as a yellow solid. LCMS 4: 3.16
min; 253.0 [M + H].
Step 2. Naphthalene-2-sulfonyl chloride (78 mg; 0.35 mmol) was
added to 4-(2-aminophenylethynyl)-pyridine-2-carboxylic acid methyl
ester (80 mg; 0.31 mmol) dissolved in pyridine (0.5 mL). After
reaction at RT overnight, water was added, acidified with 2 M HCl,
and the product was extracted with ethyl acetate. The solvent was
evaporated and the residue was purified by chromatography to yield
mL) and diisopropylamine (0.2 mL; 1.25 mmol). The mixture was
argonated for 10 min, and then copper(I) iodide (8 mg; 0.04 mmol)
and tetrakis(triphenylphosphine)palladium(0) (48 mg; 0.04 mmol)
were added. The reaction mixture was stirred for 4.5 h at 80 °C. Then,
it was diluted with ethyl acetate and filtered. The filtrate was
evaporated and was purified by chromatography to give 5-(2-
aminophenylethynyl)-pyrimidine-2-carboxylic acid methyl ester (210
mg; 97%) as a yellow solid. LCMS 4: 2.79 min; 254.3 [M + H].
Step 2. 4-Methoxy-naphthalene-1-sulfonyl chloride (89 mg; 0.34
mmol) was added to a mixture of 5-(2-aminophenylethynyl)-
pyrimidine-2-carboxylic acid methyl ester (60 mg; 0.23 mmol) and
NaHCO (29 mg; 0.34 mmol) in acetonitrile (5 mL). After 12 h at
3
4
-[2-(naphthalene-2-sulfonylamino)-phenylethynyl]-pyridine-2-car-
RT, an additional amount of 4-methoxy-naphthalene-1-sulfonyl
boxylic acid methyl ester (118 mg; 84%) as a light yellow solid.
LCMS 4: 3.80 min; 443.1 [M + H]; 441.0 [M − H].
Step 3. The ester (37 mg; 0.08 mmol) was dissolved in methanol
chloride (56 mg; 0.23 mmol) and NaHCO (19 mg; 0.23 mmol)
3
was added, also after 48 h and after 5 days (12 mg; 0.05 mmol). The
reaction mixture was quenched with water after 11 days, acidified with
(
5 mL), and 5 M NaOH (0.32 mL; 1.62 mmol) was added. The
2
M HCl, and the product was extracted with ethyl acetate from the
reaction mixture was stirred for 3 h at 50 °C. After acidification with
hydrochloric acid and extraction with ethyl acetate, the combined
organic layers were dried over Na SO and evaporated. The product
water phase. The combined organic phase was dried over anhydrous
Na SO . The solvent was evaporated and residue was purified by
2
4
2
4
chromatography to yield 5-[2-(4-methoxynaphthalene-1-sulfonylami-
no)-phenylethynyl]-pyrimidine-2-carboxylic acid methyl ester (18
mg; 15%) as a cream-colored solid. LCMS 4: 3.57 min; 472.2 [M +
H].
was purified by chromatography to obtain 4-[2-(naphthalene-2-
sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid (7 mg;
1
1
(
1
5
8%) as a white solid. H NMR (400 MHz, DMSO-d ): δ 10.34
6
br, 1H), 8.64 (d, J = 5.0 Hz, 1H), 8.31 (d, J = 1.8 Hz, 1H), 8.08 (s,
H), 7.87 (t, J = 7.8 Hz, 3H), 7.68−7.52 (m, 2H), 7.52−7.36 (m,
H), 7.25 (td, J = 7.5, 7.0, 2.4 Hz, 1H); not visible signal of the acidic
Step 3. The ester (18 mg; 0.04 mmol) was dissolved in methanol
2 mL), and 5 M NaOH (0.14 mL; 0.71 mmol) was added. The
(
reaction mixture was stirred for 2.5 h at 50 °C, diluted with water in
the presence of 2 M HCl, and extracted with ethyl acetate. Combined
proton. LCMS 5: 5.7 min; 429.0 [M + H], 427.2 [M − H]; RMS
+
calcd for C H N O S , 429.0909; found, 429.0901; calcd for
2
4
17
2
4
organic layers were dried over Na SO and evaporated. The crude
−
2
4
C H N O S , 427.0758; found, 427.0751.
2
4
15
2
4
product was purified by chromatography to give the product (8 mg;
6
-{2-[2-(Naphthalene-2-sulfonamido)phenyl]ethynyl}pyridine-3-
1
4
9%) as a white solid. H NMR (400 MHz, DMSO-d ): δ 10.21 (br,
6
carboxylic Acid (23). Step 1. A mixture of 2-ethynylphenylamine (0.1
mL; 0.68 mmol), 6-bromo-nicotinic acid methyl ester (221 mg; 1.02
mmol), and diisopropylamine (0.15 mL; 1.02 mmol) in acetonitrile
1
H), 8.75 (s, 2H), 8.63−8.55 (m, 1H), 8.12−8.04 (m, 1H), 7.97 (d, J
=
8.4 Hz, 1H), 7.55−7.41 (m, 2H), 7.41−7.28 (m, 3H), 7.20 (td, J =
7.5, 1.3 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H). LC−MS 5:
6.0 min; 459.8 [M + H], 457.8 [M − H]; HRMS calcd for
(
4 mL) was argonated for 10 min, and then heated to 65 °C
overnight. The reaction mixture was diluted with ethyl acetate and
filtered. The residue was purified by chromatography to give 6-(2-
amino-phenylethynyl)-nicotinic acid methyl ester (157 mg; 88%) as a
yellow solid. LCMS 4: RT = 3.20 min; 256.60 [M + H].
−
C H N O S , 458.0816; found, 458.0817.
2
4
16
3
5
5
-{2-[2-(Naphthalene-2-sulfonamido)phenyl]ethynyl}pyrazine-
2
-carboxylic Acid (25). Step 1. 5-bromo-pyrazine-2-carboxylic acid
methyl ester (150 mg; 0.69 mmol), 2-ethynyl-phenylamine (81 mg;
.69 mmol), and diisopropylamine (0.2 mL; 1.04 mmol) were
dissolved in and acetonitrile (2 mL) and argonated. Copper(I) iodide
7 mg; 0.03 mmol) and tetrakis(triphenylphosphine)palladium(0)
Step 2. 6-(2-Amino-phenylethynyl)-nicotinic acid methyl ester (60
mg; 0.23 mmol), naphthalene-2-sulfonyl chloride (58 mg; 0.26
mmol), and sodium bicarbonate (29 mg; 0.35 mmol) were suspended
in acetonitrile (3 mL). The reaction mixture was stirred at RT for 48
h. A second portion of naphthalene-2-sulfonyl chloride (58 mg; 0.26
mmol) was added. After 24 h, the reaction mixture was evaporated
and the residue was dissolved in water and twice extracted with ethyl
acetate. Combined organic phases were washed with brine, dried over
anhydrous sodium sulfate, filtered, and evaporated in vacuo. The
crude product was purified by chromatography to obtain 6-[2-
0
(
(40 mg; 0.03 mmol) were added. The resulting mixture was heated
for 6 h at 60 °C, diluted with ethyl acetate and DCM, filtered, and
evaporated under reduced pressure. The residue was purified by
chromatography affording 5-(2-aminophenylethynyl)-pyrazine-2-car-
boxylic acid methyl ester (95 mg; 54%) as a yellow solid. LCMS 4:
2.95 min; 254.0 [M + H]; 252.1 [M − H].
Step 2. 5-(2-Amino-phenylethynyl)-pyrazine-2-carboxylic acid
methyl ester (50 mg; 0.20 mmol), naphthalene-2-sulfonyl chloride
(54 mg; 0.24 mmol), and sodium bicarbonate (83 mg; 0.99 mmol)
were suspended in acetonitrile (2 mL) for 4 days at RT. An additional
(
naphthalene-2-sulfonylamino)-phenylethynyl]-nicotinic acid methyl
ester (36 mg; 34%) as a yellow powder. LCMS 4: 3.91 min; 443.2 [M
H].
+
1
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https://doi.org/10.1021/acs.jmedchem.1c00448
J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
amount of sodium bicarbonate (41 mg; 0.49 mmol) and naphthalene-
8.1 Hz, 1H), 8.01 (dd, J = 8.1, 2.1 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H),
7.52 (dd, J = 8.4, 4.2 Hz, 1H), 7.45 (ddd, J = 7.7, 3.6, 1.3 Hz, 2H),
7.35 (td, J = 8.3, 7.9, 1.6 Hz, 1H), 7.12 (td, J = 7.5, 1.2 Hz, 1H).
HRMS calcd for C H N O S, 430.0856; found, 430.0847.
2
-sulfonyl chloride (22 mg; 0.10 mmol) was added for additional 24
h. The reaction mixture was evaporated, and the residue was
portioned by ethyl acetate and water. The organic layer was washed
with water and brine, dried over anhydrous sodium sulfate, and
filtered. The filtrate was evaporated and purified by chromatography
to give 5-[2-(naphthalene-2-sulfonylamino)-phenylethynyl]-pyrazine-
2
3
15
3
4
5-[2-(2-Methyl-quinoline-8-sulfonylamino)-phenylethynyl]-pyri-
dine-2-carboxylic Acid (18c). To a solution of 5-[2-(2-methyl-
quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid
methyl ester (61 mg; 0.11 mmol) in methanol (10 mL) was added
sodium hydroxide solution c(NaOH) = 2 mol/l (2 N) (1.1 mL; 2.21
mmol) and stirred for 16 h at RT. The product was isolated by
2
4
-carboxylic acid methyl ester (22 mg; 25%). LCMS 4: 3.41 min;
44.1 [M + H].
Step 3. The ester (22 mg; 0.05 mmol) was dissolved in methanol
1
(
2
5 mL). Water (2 mL) and lithium hydroxide monohydrate (116 mg;
.76 mmol) were added and the resulting mixture was heated for 2 h
chromatography in 32% (17 mg) yield as an off-white solid. H NMR
(500 MHz, DMSO-d ): δ 14.49−12.20 (m, 1H), 9.28 (s, 1H), 8.60−
6
at 55 °C. The mixture was diluted with water and acidified with a
solution of HCl. The product was extracted to ethyl acetate and the
organic layer was washed with water and brine, dried over anhydrous
sodium sulfate, and filtered. The filtrate was evaporated and purified
by chromatography yielding the product as a yellow solid (18 mg;
8.57 (m, 1H), 8.36 (dd, J = 7.3, 1.4 Hz, 1H), 8.28 (d, J = 8.5 Hz, 1H),
8.19 (dd, J = 8.2, 1.4 Hz, 1H), 8.07−8.03 (m, 1H), 7.88 (dd, J = 8.1,
2.1 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.47 (dd, J = 7.7, 1.5 Hz, 1H),
7.43−7.39 (m, 2H), 7.35 (td, J = 7.6, 1.6 Hz, 1H), 7.13 (td, J = 7.5,
1.2 Hz, 1H), 2.45 (s, 3H). HRMS calcd for C H N O S, 444.1013;
2
4
17
3
4
0
4
1
3
7
.04 mmol; 20% calculated for steps 2 and 3). LCMS 5: 6.1 min;
found, 444.1006.
+
1
29.8 [M + H] . H NMR (400 MHz, DMSO-d ) d: 13.83 (s, 1H),
5-[2-(3-Methyl-quinoline-8-sulfonylamino)-phenylethynyl]-pyri-
dine-2-carboxylic Acid (18d). To a solution of 5-[2-(3-methyl-
quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid
methyl ester (126 mg; 0.24 mmol) in methanol (10 mL) was added
sodium hydroxide solution c(NaOH) = 2 mol/l (2 N) (2.4 mL; 4.71
mmol) and stirred for 16 h at RT. The product was isolated by
6
0.33 (s, 1H), 9.06 (s, 1H), 8.82 (s, 1H), 8.36 (s, 1H), 7.99−7.84 (m,
H), 7.70 (dd, J = 8.7, 1.6 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.56−
.45 (m, 3H), 7.42 (d, J = 7.9 Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H).
−
HRMS calcd for C H N O S , 428.0711; found, 428.0704.
23
14
3
4
6
-{2-[2-(5-Methoxyquinoline-8-sulfonamido)phenyl]ethynyl}-
pyridazine-3-carboxylic Acid 26. Step 1. 6-Bromo-pyridazine-3-
carboxylic acid methyl ester (222 mg; 1.02 mmol), 2-ethynylphenyl-
amine (0.1 mL; 0.68 mmol), and diisopropylamine (0.15 mL; 1.02
mmol) were dissolved in acetonitrile (4.8 mL). The mixture was
argonated for 15 min. Tetrakis(triphenylphosphine)palladium(0) (39
mg; 0.03 mmol) and copper(I) iodide (6.5 mg; 0.03 mmol) were
added and the reaction mixture was stirred at RT overnight. After
dilution with ethyl acetate and filtration, the filtrate was washed with
water twice, washed with brine, dried over sodium sulfate, and
evaporated. The crude product was purified by chromatography to
obtain 6-(2-amino-phenylethynyl)-pyridazine-3-carboxylic acid meth-
yl ester (153 mg; 88%). LCMS 4: 3.12 min; 254.0 [M + H].
chromatography in 35% (37 mg) yield as a white solid. LC−MS 2:
1
1.64 min; 444.00 [M + H]. H NMR (400 MHz, DMSO-d ): δ
6
13.99−12.78 (m, 1H), 9.39 (s, 1H), 8.68−8.64 (m, 1H), 8.61 (d, J =
2.2 Hz, 1H), 8.21 (dd, J = 7.4, 1.4 Hz, 1H), 8.18−8.13 (m, 2H),
8.12−8.08 (m, 1H), 7.96 (dd, J = 8.1, 2.1 Hz, 1H), 7.65 (t, J = 7.8 Hz,
1H), 7.50−7.45 (m, 1H), 7.42 (dd, J = 7.8, 1.6 Hz, 1H), 7.39−7.33
(m, 1H), 7.16−7.10 (m, 1H), 2.27 (s, 3H). HRMS calcd for
C H N O S, 444.1013; found, 444.1006.
2
4
17
3
4
5-[2-(4-Methyl-quinoline-8-sulfonylamino)-phenylethynyl]-pyri-
dine-2-carboxylic Acid (18e). To a solution of 5-[2-(4-methyl-
quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid
methyl ester (103 mg; 0.21 mmol) in methanol (10 mL) was added
sodium hydroxide solution c(NaOH) = 2 mol/l (2 N) (2.1 mL; 4.12
mmol) and stirred for 16 h at RT. The product was isolated by
Step 2. 6-(2-Amino-phenylethynyl)-pyridazine-3-carboxylic acid
methyl ester (40 mg; 0.15 mmol) and 5-methoxy-quinoline-8-sulfonyl
chloride (47 mg; 0.18 mmol) in pyridine (0.5 mL) were reacted
overnight under argon at RT. Then, the reaction mixture was diluted
with ethyl acetate, washed with 1 N HCl, washed with water and
brine, dried over sodium sulfate, and evaporated. The crude product
was purified by chromatography to give 6-[2-(5-methoxy-quinoline-8-
sulfonylamino)-phenylethynyl]-pyridazine-3-carboxylic acid methyl
ester (63 mg; 84%) as a yellow solid. LCMS 4: 3.59 min; 475.0 [M
chromatography in 17% (16 mg) yield as a light yellow solid. LC−MS
1
2: 1.63 min; 444.00 [M + H]. H NMR (400 MHz, DMSO-d ): δ
6
9.38 (s, 1H), 8.66 (dd, J = 2.2, 0.7 Hz, 1H), 8.65 (d, J = 4.4 Hz, 1H),
8.38−8.33 (m, 2H), 8.10 (dd, J = 8.1, 0.9 Hz, 1H), 7.96 (dd, J = 8.1,
2.1 Hz, 1H), 7.72 (dd, J = 8.4, 7.4 Hz, 1H), 7.48−7.45 (m, 1H), 7.43
(dd, J = 7.7, 1.6 Hz, 1H), 7.37−7.31 (m, 2H), 7.11 (td, J = 7.5, 1.2
Hz, 1H), 2.65−2.61 (m, 3H). HRMS calcd for C H N O S,
2
4
17
3
4
+
H].
444.1013; found, 444.1005.
Step 3. The ester (63 mg; 0.13 mmol) was dissolved in THF (5
5-{2-[2-(5-Methylquinoline-8-sulfonamido)phenyl]ethynyl}-
pyridine-2-carboxylic Acid (18f). To a solution of 5-[2-(5-methyl-
quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid
methyl ester (80 mg; 0.15 mmol) in methanol (10 mL) was added
sodium hydroxide solution c(NaOH) = 2 mol/l (2 N) (1.5 mL; 3.08
mmol) and stirred for 6 h at RT. The product was isolated by
mL) and water (2 mL), then lithium hydroxide monohydrate (19 mg;
.45 mmol) was added. The reaction mixture was stirred at RT
0
overnight. THF was removed, the remainder was acidified to pH 5,
and extracted with ethyl acetate. The organic layer was dried over
sodium sulfate and evaporated. The crude was purified by
chromatography giving the product (18 mg; 25%) as a brown solid.
chromatography in 65% (47 mg) yield as a beige solid. LC−MS 2:
1
1
H NMR (400 MHz, DMSO-d ): δ 14.07 (s, 1H), 9.30 (s, 1H), 8.90
1.64 min; 444.00 [M + H]. H NMR (500 MHz, DMSO-d ): δ 9.10
6
6
(
1
(
dd, J = 4.3, 1.8 Hz, 1H), 8.52 (dd, J = 8.6, 1.8 Hz, 1H), 8.33 (dd, J =
3.8, 8.6 Hz, 2H), 8.07 (d, J = 8.7 Hz, 1H), 7.55−7.48 (m, 3H), 7.39
td, J = 7.9, 1.6 Hz, 1H), 7.16−7.09 (m, 2H), 4.04 (s, 3H). LCMS 5:
(dd, J = 4.2, 1.7 Hz, 1H), 8.58 (d, J = 1.5 Hz, 1H), 8.46 (dd, J = 8.5,
1.8 Hz, 1H), 8.33 (d, J = 7.4 Hz, 1H), 7.84 (s, 2H), 7.58 (dd, J = 8.5,
4.3 Hz, 1H), 7.50−7.45 (m, 1H), 7.17 (dd, J = 7.6, 1.7 Hz, 1H), 7.12
(dd, J = 8.5, 1.1 Hz, 1H), 6.87 (m, 1H), 6.43 (m, 1H), 2.89 (s, 1H),
+
6
.4 min; 461.0 [M + H]; HRMS calcd for C H N O S , 461.0920;
23 17 4 5
−
found, 461.0914; calcd for C H N O S , 459.0769; found,
2.73 (s, 1H), 2.65 (s, 3H). HRMS calcd for C24
H N O S, 444.1013;
17 3 4
2
3
15
4
5
4
59.0767.
found, 444.1008.
5
-[2-(Quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-
5-[2-(6-Methyl-quinoline-8-sulfonylamino)-phenylethynyl]-pyri-
dine-2-carboxylic Acid (18g). To a solution of 5-[2-(6-methyl-
quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid
methyl ester (151 mg; 0.25 mmol) in methanol (10 mL) was added
sodium hydroxide solution c(NaOH) = 2 mol/l (2 N) (2.5 mL; 5.08
mmol) and stirred for 4 days at RT. The product was isolated by
carboxylic Acid (18b). 5-[2-(Quinoline-8-sulfonylamino)-phenyl-
ethynyl]-pyridine-2-carboxylic acid methyl ester (60 mg; 0.13
mmol) was dissolved in methanol (3 mL), and lithium hydroxide
hydrate (139 mg; 3.31 mmol) and water (1 mL) were added. The
product was obtained by chromatography in 91% (53 mg) as a light
1
yellow solid. LCMS 4: 3.27 min; 430.0 [M + H]. H NMR (400
MHz, DMSO-d ): δ 13.41 (s, 1H), 9.43 (s, 1H), 8.81 (dd, J = 4.3, 1.8
Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.45 (dd, J = 8.4, 1.8 Hz, 1H), 8.37
dd, J = 7.3, 1.4 Hz, 1H), 8.27 (dd, J = 8.3, 1.5 Hz, 1H), 8.12 (d, J =
chromatography in 31% yield (35 mg) as a white solid. LCMS 2: 1.68
1
min.; 444.00 [M + H]. H NMR (400 MHz, DMSO-d ): δ 14.26−
6
6
12.32 (m, 1H), 9.38 (s, 1H), 8.74−8.68 (m, 2H), 8.32 (dd, J = 8.5,
1.7 Hz, 1H), 8.20 (d, J = 1.9 Hz, 1H), 8.13−8.08 (m, 1H), 8.02−7.97
(
1
1926
https://doi.org/10.1021/acs.jmedchem.1c00448
J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
m, 2H), 7.49−7.41 (m, 3H), 7.35 (td, J = 7.9, 1.6 Hz, 1H), 7.12 (td,
isolated by chromatography (37 mg; 90%) as a light yellow solid.
J = 7.6, 1.2 Hz, 1H), 2.53−2.50 (m, 3H). HRMS calcd for
C H N O S, 444.1013; found, 444.1003.
LCMS 4: 6.10 min; 457.70 [M − H]; 459.80 [M + H].
1
H NMR (400 MHz, DMSO-d ): δ 13.45 (s, 1H), 9.19 (s, 1H),
24
17
3
4
6
5
-[2-(7-Methyl-quinoline-8-sulfonylamino)-phenylethynyl]-pyri-
8.79−8.71 (m, 2H), 8.50 (dd, J = 8.6, 1.8 Hz, 1H), 8.32 (d, J = 8.4
Hz, 1H), 8.12 (d, J = 8.1 Hz, 1H), 8.04 (dd, J = 8.1, 2.1 Hz, 1H),
7.51−7.43 (m, 2H), 7.42 (dd, J = 7.7, 1.6 Hz, 1H), 7.34 (td, J = 8.4,
8.0, 1.6 Hz, 1H), 7.16−7.04 (m, 2H), 4.03 (s, 3H). HRMS calcd for
C H N O S, 460.0962; found, 460.0959.
dine-2-carboxylic Acid (18h). To a solution of 5-[2-(7-methyl-
quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid
methyl ester (73 mg; 0.12 mmol) in methanol (10 mL) was added
sodium hydroxide solution c(NaOH) = 2 mol/l (2 N) (1.2 mL; 2.44
mmol) and stirred for 16 h at RT. The product was isolated by
2
4
17
3
5
5-[2-(5-Methoxy-quinoline-8-sulfonylamino)-phenylethynyl]-3-
chromatography in 29% yield (16 mg) as a rose solid. LC−MS 2: 1.66
methyl-pyridine-2-carboxylic Acid (18k). Methyl-5-[2-(2-
Aminophenyl)ethynyl]-4-methoxypyridine-2-carboxylate (3.50 g;
13.01 mmol) was dissolved in pyridine (20 mL), and 7-methylquino-
line-8-sulfonyl chloride (3.77 g; 115.61 mmol) were added. The
reaction mixture was stirred at room temperature overnight. The
solvent was coevaporated with toluene, and crude was purified by
FCC (silica, hexane 100% to DCM 100% to DCM/EtOAc 85:15
gradient) to afford methyl 4-methoxy-5-{2-[2-(7-methylquinoline-8-
sulfonamido)phenyl]ethynyl}pyridine-2-carboxylate (5.60 g; 88.0%)
as a pale yellow solid. LCMS 4: 3.92 min; 472.2 [M + H], 470.2 [M −
H]. To a stirred solution of 3-methyl-5-[2-(7-methylquinoline-8-
sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid methyl
ester (5.60 g; 10.33 mmol) in water (15 mL) and THF (15 mL)
was added lithium hydroxide (1.24 g; 51.66 mmol). The reaction
mixture was stirred for 4 h, concentrated in vacuo, and neutralized
with 1 N HCl. The aqueous layer was extracted with DCM, dried over
1
min; 444.00 [M + H]. H NMR (700 MHz, DMSO-d ): δ 9.74 (s,
6
1
H), 8.82−8.78 (m, 2H), 8.38 (dd, J = 8.2, 1.3 Hz, 1H), 8.16−8.11
(m, 2H), 8.09 (dd, J = 8.0, 2.0 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H),
7
3
.51−7.45 (m, 3H), 7.37−7.32 (m, 1H), 7.10−7.06 (m, 1H), 2.87 (s,
H). HRMS calcd for C H N O S, 444.1013; found, 444.1011.
2
4
17
3
4
3
-Methyl-5-[2-(7-methyl-quinoline-8-sulfonylamino)-phenyle-
thynyl]-pyridine-2-carboxylic Acid (18i). Step 1. A mixture of 5-
Bromo-3-methyl-pyridine-2-carboxylic acid methyl ester (8 g; 34.77
mmol) and 2-ethynylphenylamine (4.35 mL; 38.25 mmol) in AcCN
(
50 mL) was argonated. Diisopropylamine (6.84 mL; 52.16 mmol)
was added, followed by tetrakis(triphenylphosphine)palladium(0)
2.01 g; 1.74 mmol) and copper(I)iodide (0.33 g; 1.74 mmol) under
(
argon. The resulting mixture was heated for 12 h at 65 °C and cooled
down to RT. The formed precipitate was isolated by filtration, washed
with n-hexane, and dissolved in DCM. The organic layer was washed
twice with water and brine, dried over anhydrous Na SO , and
Na SO , and evaporated. The solid was washed with pentane and
2
4
2
4
EtOH to afford the product (4.70 g; 99%) as a white solid. LCMS 10:
filtered. The filtrate was concentrated and loaded on a long pad of
silica covered with a small layer of Celite. Impurities were eluted using
DCM (darker yellow fraction), and then the product was eluted by a
mixture of DCM/AcOEt 10% v/v. The fraction was concentrated and
the precipitating solid was collected by filtration, washed with n-
hexane, and dried to give 5-(2-amino-phenylethynyl)-3-methyl-
pyridine-2-carboxylic acid methyl ester (7.72 g; 82%) as a yellow
solid. LCMS 4: 1.44 min; 267.30 [M + H].
1
6
6
.8 min; 458.0 [M + H]; 456 [M − H]. H NMR (300 MHz, DMSO-
d ): δ: 9.73 (s, 1H), 8.80 (dd, J = 4.3, 1.8 Hz, 1H), 8.57 (d, J = 1.9
Hz, 1H), 8.39 (dd, J = 8.3, 1.8 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.86
(
7
d, J = 2.0 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.53−7.40 (m, 3H),
.34 (ddd, J = 8.6, 7.5, 1.6 Hz, 1H), 7.08 (td, J = 7.6, 1.2 Hz, 1H),
+
2
.87 (s, 3H), 2.53 (s, 3H). HRMS calcd for C H N O S , 458.1169;
25 20
3
4
−
found, 458.1165; calcd for C H N O S , 456.1024; found,
2
5
18
3
4
4
Step 2. 5-(2-aminophenylethynyl)-3-methyl-pyridine-2-carboxylic
acid methyl ester (70 mg; 0.24 mmol) was dissolved in anhydrous
pyridine (0.5 mL), and 5-methoxyquinoline-8-sulfonyl chloride
pyridine-2-carboxylic Acid (18l). To a solution of 5-[2-(5-ethoxy-
quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid
methyl ester (163 mg; 0.20 mmol) in methanol (10 mL) was added
sodium hydroxide solution c(NaOH) = 2 mol/l (2 N) (2 mL; 4
mmol) and stirred for 16 h at RT. The product was isolated by
(
69.30 mg;0.27 mmol) was added in one portion. The reaction
mixture was stirred overnight till full consumption of starting material
was confirmed by TLC. The reaction mixture was diluted with
toluene and evaporated in vacuo. The crude product was purified by
FCC (SiHP column, EtOAc gradient in hexane) to give: 5-[2-(5-
methoxyquinoline-8-sulfonylamino)-phenylethynyl]-3-methyl-pyri-
dine-2-carboxylic acid methyl ester (103 mg; 85%) as a white solid.
LCMS 4: 3.97 min; 488.1 [M + H].
Step 3. 5-[2-(5-Methoxyquinoline-8-sulfonylamino)-phenylethyn-
yl]-3-methyl-pyridine-2-carboxylic acid methyl ester (103 mg; 0.21
mmol eq) was dissolved in THF (5 mL) and water (2 mL). Lithium
hydroxide monohydrate (30.77 mg; 0.73 mmol) was added, and the
resulting mixture was stirred overnight at RT. Consumption of the
starting material was confirmed on TLC. The reaction mixture was
partially evaporated and neutralized with 2 M HCl. The precipitate
was extracted to ethyl acetate. The organic layer was washed with
chromatography (33%; 88 mg) as a yellow solid. LC−MS 2: 1.71−
1
1
1
8
.72 min; 474.00 [M + H]. H NMR (400 MHz, DMSO-d ): δ
6
4.31−12.26 (m, 1H), 9.17 (s, 1H), 8.76 (dd, J = 4.3, 1.8 Hz, 1H),
.73 (dd, J = 2.1, 0.9 Hz, 1H), 8.49 (dd, J = 8.6, 1.8 Hz, 1H), 8.29 (d,
J = 8.4 Hz, 1H), 8.11 (dd, J = 8.1, 0.9 Hz, 1H), 8.02 (dd, J = 8.1, 2.1
Hz, 1H), 7.50−7.44 (m, 2H), 7.41 (dd, J = 7.7, 1.5 Hz, 1H), 7.36−
7
=
.30 (m, 1H), 7.11−7.05 (m, 2H), 4.27 (q, J = 7.0 Hz, 2H), 1.44 (t, J
7.0 Hz, 3H). HRMS calcd for C H N O S, 474.1118; found,
25 19 3 5
4
74.1102.
5
-{2-[2-(5-Ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-4-
methoxypyridine-2-carboxylic Acid (18m). To a solution of 5-[2-(5-
ethoxy-quinoline-8-sulfonylamino)-phenylethynyl]-4-methoxy-pyri-
dine-2-carboxylic acid methyl ester (160 mg; 0.19 mmol) in methanol
(10 mL) was added sodium hydroxide solution c(NaOH) = 2 mol/l
brine, dried over Na SO , and evaporated under reduced pressure.
2
4
Crude was purified by FCC (SiHP column, 0−10% methanol gradient
(
2 N) (1.9 mL; 3.85 mmol) and stirred for 4 h at RT. The product
in DCM) to give the product (78 mg; 67%) as a white solid. LCMS
was isolated by chromatography (19%; 19 mg) as a light yellow solid.
LC−MS 7: 3.76 min; 504.00 [M + H]. H NMR (400 MHz, DMSO-
d ): δ 9.06 (s, 1H), 8.65 (dd, J = 4.3, 1.8 Hz, 1H), 8.60 (s, 1H), 8.49
1
1
1
0: 6.5 min; 474.5 [M + H]. H NMR (300 MHz, DMSO-d ): δ:
6
1
3.34 (s, 1H), 9.15 (s, 1H), 8.76 (dd, J = 4.3, 1.8 Hz, 1H), 8.52−8.48
6
(
2
4
m, 2H), 8.31 (d, J = 8.4 Hz, 1H), 7.79−7.77 (m, 1H), 7.50−7.45 (m,
(dd, J = 8.5, 1.8 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.77 (s, 1H), 7.54
(d, J = 8.3 Hz, 1H), 7.48 (dd, J = 8.6, 4.3 Hz, 1H), 7.35 (dd, J = 7.7,
1.5 Hz, 1H), 7.28 (td, J = 8.5, 8.0, 1.6 Hz, 1H), 7.14 (d, J = 8.5 Hz,
1H), 6.98 (t, J = 7.5 Hz, 1H), 4.29 (q, J = 6.9 Hz, 2H), 4.13 (s, 3H),
1.45 (t, J = 6.9 Hz, 3H). HRMS calcd for C H N O S, 504.1224;
H), 7.42−7.38 (m, 1H), 7.36−7.30 (m, 1H), 7.14−7.06 (m, 2H),
.03 (s, 3H), CH partially covered by DMSO-d signal). HRMS for
3
6
+
−
C H N O S , 474.1118; found, 474.1116; calcd for C H N O S ,
25
20
3
5
25 18
3
5
4
72.0973; found, 472.0976.
2
6
21
3
6
5
-[2-(5-Methoxy-quinoline-8-sulfonylamino)-phenylethynyl]-
found, 504.1215.
pyridine-2-carboxylic Acid (18j). 5-[2-(5-Methoxy-quinoline-8-sulfo-
nylamino)-phenylethynyl]-pyridine-2-carboxylic acid methyl ester (42
mg; 0.09 mmol) was dissolved in methanol (2 mL). Water and
lithium hydroxide hydrate (92 mg; 2.20 mmol) were added. The
reaction mixture was stirred overnight at RT. The product was
5
-{2-[5-Chloro-2-(5-ethoxyquinoline-8-sulfonamido)phenyl]-
ethynyl}-4-methoxypyridine-2-carboxylic Acid (18n). Step 1:
Methyl-5-bromo-4-meothoxypicolinate (1.5 g; 6.1 mmol) was
dissolved in ACN (15 mL) under a nitrogen atmosphere, and
trimethylsilylacetylene (1.7 mL, 12.19 mmol) was added, followed by
1
1927
https://doi.org/10.1021/acs.jmedchem.1c00448
J. Med. Chem. 2021, 64, 11904−11933

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
diisopropylamine (1.3 mL, 9.14 mmol), copper(I)iodide (116 mg, 0.6
mmol), and tetrakis(triphenylphosphine)-palladium(0) (704 mg, 0.61
mmol). The reaction was stirred for 16 h at 80 °C. After dilution with
ethyl acetate and three extractions with water, the organic phase was
dried over Na SO and evaporated to dryness after filtration. The
(dd, J = 8.4, 1.5 Hz, 1H), 8.31 (dd, J = 7.4, 1.5 Hz, 1H), 8.10 (dd, J =
8.1, 0.6 Hz, 1H), 7.98 (dd, J = 8.1, 2.1 Hz, 1H), 7.62 (dd, J = 8.3, 7.4
Hz, 1H), 7.45−7.42 (m, 2H), 7.36−7.32 (m, 1H), 7.26−7.21 (m,
2H), 7.11 (td, J = 7.6, 1.1 Hz, 1H), 7.00 (d, J = 5.4 Hz, 1H), 6.92−
6.87 (m, 3H), 4.36−4.32 (m, 2H), 4.13−4.10 (m, 2H), 3.96−3.93
(m, 2H), 3.88−3.85 (m, 2H). HRMS calcd for C H N O S,
2
4
residue was purified by chromatography, giving methyl 4-methoxy-5-
3
3
27
3
7
[
(
2-(trimethylsilyl)ethynyl]pyridine-2-carboxylate as a yellow solid
1.5 g, 90%).
610.1642; found, 610.1633.
5-[2-(2-{4-[(1-Phenyl-2,9,12,15-tetraoxaheptadecan-17-yl)oxy]-
quinoline-8-sulfonamido}phenyl)ethynyl]pyridine-2-carboxylic
Acid (18q). To a solution of 5-[2-(4-chloro-quinoline-8-sulfonylami-
no)-phenylethynyl]-pyridine-2-carboxylic acid methyl ester (40 mg;
Step 2: Silylacetylene (1.5 g, 5.46 mmol) from step 1 was dissolved
in methanol (40 mL), and K CO (75 mg, 0.55 mmol) was added.
2
3
The mixture was stirred for 16 h at RT and diluted with ethyl acetate
and water. After phase separation, the aqueous phase was extracted
three times with ethyl acetate, and the combined organic phase was
washed with brine, dried over Na SO , filtered, and evaporated to
0
.05 mmol) in DMF (3 mL) were added 2-{2-[2-(6-benzyloxy-
hexyloxy)-ethoxy]-ethoxy}-ethanol (187 mg; 0.54 mmol) and
potassium tert-butylate (36 mg; 0.32 mmol). The reaction was stirred
for 16 h at RT. The reactions were evaporated to dryness and the
residue was purified by chromatography giving the product in 63%
2
4
dryness giving methyl 5-ethynyl-4-methoxypyridine-2-carboxylate as a
crude brown solid (851 mg, 72%).
Step 3: 4-Cl-2-I-Aniline (614 mg, 2.33 mmol) and acetylene (753
mg, 3.49 mmol) from step-2 were dissolved in ACN (10 mL) under
nitrogen, and diisopropylamine (0.5 m, 3.49 mmol), copper(I)iodide
27 mg) yield as a light yellow solid. LC−MS 2: 2.05 min; 768.00 [M
1
H]. H NMR (700 MHz, DMSO-d ): δ 8.69−8.68 (m, 1H), 8.60
6
1
7
7
7
.3 Hz, 1H), 8.11−8.09 (m, 1H), 7.98 (dd, J = 8.0, 2.1 Hz, 1H),
.67−7.64 (m, 1H), 7.44−7.42 (m, 2H), 7.35−7.32 (m, 1H), 7.32−
.29 (m, 2H), 7.29−7.26 (m, 2H), 7.26−7.23 (m, 1H), 7.11 (td, J =
.6, 0.9 Hz, 1H), 6.98 (d, J = 5.4 Hz, 1H), 4.40 (s, 2H), 4.31−4.29
(
(
44 mg, 0.23 mmol), and tetrakis(triphenylphosphine)-palladium(0)
269 mg, 0.23 mmol) were added. The reaction was stirred for 5 days
at 80 °C, diluted with ethyl acetate, and washed three times with
water, dried over Na SO , and evaporated to dryness after filtration.
2
4
(
2
2
2
m, 2H), 3.87−3.85 (m, 2H), 3.63−3.61 (m, 2H), 3.54−3.52 (m,
The crude was purified by chromatography, giving methyl 5-[2-(2-
amino-5-chlorophenyl)ethynyl]-4-methoxypyridine-2-carboxylate as a
yellow solid (390 mg, 27%).
H), 3.49−3.47 (m, 2H), 3.40−3.39 (m, 2H), 3.35 (t, J = 6.5 Hz,
H), 3.29 (t, J = 6.6 Hz, 2H), 1.55−1.44 (m, 2H), 1.42−1.37 (m,
H), 1.33−1.19 (m, 4H). HRMS calcd for C H N O S, 768.2949;
Step 4: To a solution of the aniline product from step 3 (50 mg,
42 45
3
9
found, 768.2931.
0
.08 mmol) in pyridine (3 mL) was added 5-ethoxyquinoline-8-
sulfonylchloride (65 mg, 0.24 mmol), and the reaction was stirred for
6 h at RT. After evaporation, the product was isolated by
5
-[2-(2-{4-[(1-Phenyl-2,9,12,15-tetraoxaheptadecan-17-yl)oxy]-
quinoline-8-sulfonamido}phenyl)ethynyl]pyridine-2-carboxylic
Acid (18r). To a solution of 5-[2-(4-chloro-quinoline-8-sulfonylami-
no)-phenylethynyl]-pyridine-2-carboxylic acid methyl ester (60 mg;
1
chromatography, giving 5-[5-chloro-2-(5-ethoxy-quinoline-8-sulfony-
lamino)-phenylethynyl]-4-methoxy-pyridine-2-carboxylic acid methyl
ester as a brown oil (71 mg, 99%).
Step 5: To a solution of the ester from step 4 (71 mg; 0.08 mmol)
in methanol (5 mL) was added sodium hydroxide solution c(NaOH)
0.08 mmol) in DMF (4 mL) were added 2-[2-(4-nitro-phenoxy)-
ethoxy]-ethanol (198 mg; 0.81 mmol) and potassium tert-butylate (55
mg; 0.49 mmol). The reaction was stirred for 16 h at RT. The
reactions were evaporated to dryness and the residue was purified by
chromatography giving the product in 16% (9 mg) yield as a yellow
=
2 mol/l (2 N) (0.8 mL; 1.67 mmol) and stirred for 4 h at RT. The
product was isolated by chromatography (33%; 15 mg) as a yellow
1
solid. LC−MS 2: 1.82 min; 655.00 [M + H]. H NMR (500 MHz,
1
solid. LC−MS 7: 4.27−4.34 min; 538.00−541.00 [M + H]. H NMR
DMSO-d ): δ 13.75−12.90 (m, 1H), 9.47−9.33 (m, 1H), 8.67 (dd, J
6
(500 MHz, DMSO-d ): δ 9.13 (s, 1H), 8.66 (dd, J = 4.3, 1.8 Hz, 1H),
6
=
2.0, 0.7 Hz, 1H), 8.61 (d, J = 5.3 Hz, 1H), 8.33−8.29 (m, 2H),
8.61 (s, 1H), 8.52 (dd, J = 8.5, 1.8 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H),
7.79 (s, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.49 (dd, J = 8.5, 4.3 Hz, 1H),
7.44 (d, J = 2.5 Hz, 1H), 7.37 (dd, J = 9.0, 2.5 Hz, 1H), 7.13 (d, J =
8.5 Hz, 1H), 4.30 (q, J = 7.0 Hz, 2H), 4.13 (s, 3H), 1.46 (t, J = 7.0
8
2
7
4
3
6
.18−8.14 (m, 2H), 8.10 (dd, J = 8.1, 0.7 Hz, 1H), 7.97 (dd, J = 8.1,
.1 Hz, 1H), 7.61 (dd, J = 8.3, 7.4 Hz, 1H), 7.45−7.42 (m, 2H),
.36−7.32 (m, 1H), 7.14−7.09 (m, 3H), 7.00 (d, J = 5.4 Hz, 1H),
.36−4.32 (m, 2H), 4.30−4.27 (m, 2H), 3.96−3.93 (m, 2H), 3.92−
.89 (m, 2H). HRMS calcd for C H N O S, 655.1493; found,
55.1485.
-(2-{2-[4-(2-{2-[2-(2-{[(tert-Butoxy)carbonyl]amino}ethoxy)-
Hz, 3H). HRMS calcd for C H ClN O S, 538.0834; found,
2
6
20
3
6
3
3
26
4
9
5
38.0825.
-{2-[2-(5-Ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-3-
methylamino)pyridine-2-carboxylic Acid (18o). To a solution of 5-
2-(5-ethoxy-quinoline-8-sulfonylamino)-phenylethynyl]-3-methyla-
5
5
(
[
ethoxy]ethoxy}ethoxy)quinoline-8-sulfonamido]phenyl}ethynyl)-
pyridine-2-carboxylic Acid (18s). To a solution of 5-[2-(4-chloro-
quinoline-8-sulfonylamino)-phenylethynyl]-pyridine-2-carboxylic acid
methyl ester (40 mg; 0.05 mmol) in DMF (3 mL) were added 1-Boc-
amino-3,6,9-trioxaundecanyl-11-ol (167 mg; 0.54 mmol) and
potassium tert-butylate (36 mg; 0.32 mmol). The reaction was stirred
for 16 h at RT. The reactions were evaporated to dryness, and the
residue was purified by chromatography giving the product in 37%
mino-pyridine-2-carboxylic acid methyl ester (209 mg; 0.34 mmol) in
methanol (10 mL) was added sodium hydroxide solution c(NaOH) =
2
mol/l (2 N) (3.4 mL; 6.87 mmol) and stirred for 16 h at RT. The
product was isolated by chromatography (29%; 50 mg) as a yellow
solid. LC−MS 2: 1.71−1.74 min; 503.00 [M + H].
1
H NMR (500 MHz, DMSO-d ): δ 9.13 (s, 1H), 8.78 (dd, J = 4.3,
6
1.8 Hz, 1H), 8.51 (dd, J = 8.6, 2.1 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H),
7.89 (d, J = 1.7 Hz, 1H), 7.52−7.44 (m, 2H), 7.41 (d, J = 7.3 Hz,
1H), 7.33 (m, 1H), 7.22 (d, J = 1.7 Hz, 1H), 7.09 (dd, J = 8.3, 6.0 Hz,
2H), 4.28 (q, J = 7.0 Hz, 2H), 2.90 (s, 3H), 1.45 (t, J = 7.0 Hz, 3H).
(
16 mg) yield as an orange solid. LC−MS 2: 1.72 min; 721.00 [M +
1
H]. H NMR (700 MHz, DMSO-d ): δ 13.77−13.12 (m, 1H), 9.44−
6
9
.37 (m, 1H), 8.70−8.69 (m, 1H), 8.61 (d, J = 5.3 Hz, 1H), 8.37 (dd,
J = 8.3, 1.5 Hz, 1H), 8.33 (dd, J = 7.3, 1.4 Hz, 1H), 8.11−8.09 (m,
HRMS calcd for C H N O S, 503.1384; found, 503.1371.
26
22
4
5
1H), 7.99 (dd, J = 8.0, 2.1 Hz, 1H), 7.68−7.65 (m, 1H), 7.45−7.42
5
-[2-(2-{4-[2-(2-Phenoxyethoxy)ethoxy]quinoline-8-
(m, 2H), 7.36−7.33 (m, 1H), 7.13−7.10 (m, 1H), 7.00 (d, J = 5.4 Hz,
sulfonamido}phenyl)ethynyl]pyridine-2-carboxylic Acid (18p). To a
solution of 5-[2-(4-chloro-quinoline-8-sulfonylamino)-phenylethyn-
yl]-pyridine-2-carboxylic acid methyl ester (60 mg; 0.09 mmol) in
DMF (4 mL) were added 2-(2-phenoxyethoxy)ethan-1-ol (156 mg;
1
3
3
9
H), 6.75−6.70 (m, 1H), 4.33−4.30 (m, 2H), 3.88−3.86 (m, 2H),
.65−3.62 (m, 2H), 3.56−3.53 (m, 2H), 3.51−3.48 (m, 2H), 3.46−
.44 (m, 2H), 3.34 (t, J = 6.1 Hz, 2H), 3.05−3.01 (m, 2H), 1.34 (s,
H). HRMS calcd for C H N O S, 721.2538; found, 721.2519.
3
6
40
4
10
0.85 mmol) and potassium tert-butylate (58 mg; 0.51 mmol). The
5
-(2-{2-[4-(2-{2-[2-(2-Aminoethoxy)ethoxy]ethoxy}ethoxy)-
reaction was stirred for 16 h at RT, evaporated to dryness, and the
residue was purified by chromatography giving the product in 19%
quinoline-8-sulfonamido]phenyl}ethynyl)pyridine-2-carboxylic
Acid (18t). To a solution of 5-{2-[4-(2-{2-[2-(2-tert-butoxycarbony-
lamino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-quinoline-8-sulfonylamino]-
phenylethynyl}-pyridine-2-carboxylic acid (371 mg; 0.20 mmol) in
1,4-dioxane (40 mL) was added 4 N HCl in dioxane (5 mL) and
(
10 mg) yield as a yellow solid. LCMS 2: 1.85 min; 610.00 [M + H].
1
H NMR (500 MHz, DMSO-d ): δ 13.94−12.87 (m, 1H), 9.66−9.12
(
6
m, 1H), 8.68 (dd, J = 2.0, 0.7 Hz, 1H), 8.61 (d, J = 5.3 Hz, 1H), 8.34
1
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stirred for 16 h at RT. The reaction was evaporated to dryness, and
the residue was purified by chromatography giving the product in 39%
GA) or MCT4 (TGATGATAAAACAGCCAAAG). Three
days after nucleofection, cells were trypsinized and plated as
single cells in 96 well plates. Colonies were picked and
screened with next generation sequencing (NGS) to detect
possible knock-out (KO) clones. The target region in MCT1
(
49 mg) yield as a white solid. LC−MS 2: 1.31 min; 621.00 [M + H].
1
H NMR (500 MHz, DMSO-d ): δ 13.89−12.88 (m, 1H), 9.56−9.23
6
(
8
m, 1H), 8.69−8.66 (m, 1H), 8.61 (d, J = 5.3 Hz, 1H), 8.36 (dd, J =
.4, 1.4 Hz, 1H), 8.33 (dd, J = 7.3, 1.4 Hz, 1H), 8.10 (d, J = 8.1 Hz,
(
Fw: CAGGCTTGTGAACCATGGTG, Rev: AGTAGC-
1
H), 7.99 (dd, J = 8.1, 2.1 Hz, 1H), 7.80−7.69 (m, 2H), 7.69−7.65
CACTCACCTCCAAT) or MCT4 (Fw: CTATGG-
GAGGGGCTGTGG, Rev: GCCATAGAGCATAGCCAG-
CA) was amplified, cleaned, and sequenced (Illumina, FC-
420-1004). MCT1 and MCT4 KO clones were identified,
expanded, and frozen. MiaPaCa2 cells: CRISPR KO clones
were generated and provided by Horizon Discovery, UK.
Lactate Efflux and Intracellular Accumulation. For
lactate efflux measurement, 10,000 cells/well MDA-MB-231,
10,000 cells/well SNU-398, 5000 cells/well MiaPaca2, and
15,000 cells/well RT-4 cells were seeded onto a 384-well plate
in complete media and allowed for overnight recovery at 37 °C
(
m, 1H), 7.47−7.42 (m, 2H), 7.37−7.32 (m, 1H), 7.15−7.10 (m,
1
H), 7.00 (d, J = 5.4 Hz, 1H), 4.34−4.30 (m, 2H), 3.89−3.85 (m,
H), 3.67−3.63 (m, 4H), 3.57−3.52 (m, 6H), 2.97−2.91 (m, 2H).
2
HRMS calcd for C H N O S, 621.2014; found, 621.1999.
31
32
4
8
3
-{5-[(2-{2-[2-(2-{[8-({2-[2-(6-Carboxypyridin-3-yl)ethynyl]-
phenyl}sulfamoyl)quinolin-4-yl]oxy}ethoxy)ethoxy]ethoxy}ethyl)-
carbamoyl]pentyl}-2-[(1E,3E)-5-[(2Z)-3,3-dimethyl-5-sulfo-1-(3-sul-
fopropyl)-2,3-dihydro-1H-indol-2-ylidene]penta-1,3-dien-1-yl]-3-
methyl-5-sulfo-1-(3-sulfonatopropyl)-3H-indol-1-ium (18u). To a
solution of 5-{2-[4-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-
ethoxy)-quinoline-8-sulfonylamino]-phenylethynyl}-pyridine-2-car-
boxylic acid (10 mg; 16 mmol) in THF (2 mL) were added Alexa
Fluor 647 (20 mg; 16 mmol) and triethylamine (2.5 μL; 16 mmol).
The reaction was stirred for 16 h at RT. The reaction was evaporated
to dryness, and the residue was purified by chromatography giving the
crude product in 29% (10 mg) yield.
in the atmosphere of 5% CO . On the following day, cells were
2
washed with LCIS buffer (Life Tech, Cat. #A14291DJ) with
final wash leaving 24 μL of LCIS buffer above the cells. Test
compounds dissolved in DMSO were added to the assay plate
using a D300 digital dispenser, and the plate was preincubated
for 30 min at RT, protecting from light. D-(+)-Glucose in LCIS
was added to an assay plate using a MultiFlo FX dispenser, to
obtain a final concentration of 25 mM glucose in every well.
Assay plates were incubated for 4 h at 37 °C. Next, 20 μL of
medium from above the cells was collected and transferred into
a new clear 384-well flat-bottom plate. A reconstituted lactate
reagent (P000024, M Dialysis AB) was added to readout well,
keeping a sample/reagent volume ratio of 1:1. Plates were
briefly centrifuged at 4 °C, and the absorbance at 530 nm at 30
°C was measured using a Spark 20 M Tecan multimode plate
BIOLOGY
■
Cell Culture Conditions. HEK293 cells were obtained
from CLS and cultured in MEM with L-glutamine (31095-029,
Gibco) supplemented with 10% fetal bovine serum (FBS) heat
inactivated (h.i.) (S0115, Biochrom), 1% non-essential amino
acids (NEAA) (11140035, Life Technologies), and 1 mM
sodium pyruvate (11360088, Life Technologies). MDA-MB-
2
31 cells were obtained from ATCC and were cultured in
DMEM with 1 g/L glucose (31885-023, Gibco) and 1 mM
sodium pyruvate ((11360088, Life Technologies) supple-
mented with 10% FBS h.i, (10270-106, Gibco). MiaPaca2
cells were obtained from ATCC and were cultured in DMEM
with 4.5 g/L glucose (41965-039, Gibco) supplemented with
6
reader. For intracellular lactate accumulation, 0.5 × 10 cells/
6
6
well MDA-MB-231, 1 × 10 cells/well SNU-398, 0.8 × 10
6
cells/well MiaPaca2, and 0.75 × 10 cells/well RT-4 cells were
seeded onto 6-well plates in complete growth media and
allowed for overnight recovery at 37 °C in the atmosphere of
1
0% FBS h.i. (10270-106, Gibco), 2.5% horse serum h.i.
(
(
Gibco, Cat. #16050-122), and 1 mM sodium pyruvate
11360088, Life Technologies). SNU-398 (obtained from
5% CO . On the following day, cells were washed twice with
2
ATCC) and RT-4 (obtained from ATCC) cells were cultured
in RPMI1640 with 2 g/L glucose (21875-034, Gibco)
supplemented with 10% FBS h.i. (10270−106, Gibco) and 1
mM sodium pyruvate (11360088, Life Technologies). Cells
LCIS and 1.35 mL LCIS was added onto the cells. Then, test
compounds diluted in DMSO were added into assay plates and
preincubated for 30 min at RT, protecting from light. D-
(+)-Glucose solution in LCIS was added into assay plates, to
obtain a final concentration of 25 mM glucose in every well.
Plates were incubated for 4 h at 37 °C. For metabolite
collection, cells were washed twice with PBS, 180 μL of
methanol was added to the culture dish for 30 s, and then
gradually diluted with 125 μL of water for another 30 s. The
extracted solution was transferred to a 96-well V-shaped
bottom plate and centrifuged at 2300g at 4 °C for 5 min.
Determination of lactate was carried out using the UHPLC-
ESI-Q-Orbitrap-MS technique.
were grown at 5% CO and 37 °C in a humidified incubator.
2
NCI-H358 and NCI-H441 cells were obtained from ATCC
and cultured in RPMI-1640 (21875-034, Gibco) supplemented
with 10% FBS (Corning, 35015CV). MC38 cells were
obtained from Scripps Research Institute. For in vitro assays,
MC38 cells were cultured in DMEM (41966, Gibco)
supplemented with 10% FBS (S0615, Biochrom), 1% NEAA
(
M7145, Sigma), Pen/Strep (P4333, Sigma), and 0.25 μg/mL
Plasmocin (ant-mpp, Invivogene) at 10% CO and 37 °C in a
2
humidified incubator, and harvested with 0.05% Trypsin
25,000 MC38 cells/well were seeded onto a 96-well plate
(353075, Falcon) in 200 μL complete growth media and
allowed for overnight recovery at 37 °C in the atmosphere of
(
25300054, Gibco). For in vivo studies, MC38 cells were
cultured in DMEM (Gibco 11865-092), supplemented with
0% FBS (Corning, 35015CV) at 5% CO2 and 37 °C,
1
10% CO . On the following day, cells were washed once with
2
passaged 2−21 times before in vivo implantation, and
phosphate buffered saline (PBS) (14190-094, Gibco) and 200
μL of fresh media were added. Test compounds dissolved in
DMSO were added into the assay plate using a Tecan D300e
Digital Dispenser, and the plate was incubated at 37 °C in the
harvested with TrypLE Express (Gibco 12605-010).
Generation of MCT1 and MCT4 CRISPR KO. MC38
6
cells: 1 × 10 MC38 cells were nucleofected using the
Nucleofector 2b device (Lonza) and nucleofection reagents Kit
V (Lonza, VACA-1003) in combination with Cas9 Alt-R S.p.
Cas9 Nuclease V3 (IDT), Alt-R CRISPR-Cas9 tracrRNA
atmosphere of 10% CO for 4 or 24 h for lactate intracellular
2
accumulation or lactate efflux measurement, respectively. For
intracellular lactate measurement, the supernatant was
discarded, cells were washed once with cold PBS, and
(
IDT), and crRNA MCT1 (CACCAGCGATCATTACTG-
1
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resuspended in 200 μL of cold Lactate Assay Buffer (ab65331,
AbCam), homogenized, and transferred to a fresh 96-well
round bottom plate (735-0016, VWR). The plate was briefly
centrifuged, and the supernatants were transferred into a fresh
harvest, the cells were washed twice with PBS, pelleted for 5
min at 1100 g, and then frozen at −80 °C.
Membrane Preparation and Receptor Solubilization.
Membrane preparation from HEK293 cells expressing MCT4-
3
5
9
6-well plate (353075, Falcon). For lactate efflux measure-
eGFP was performed as described in the literature.
ment, supernatants were transferred to a fresh 96-well round
bottom plate (735-0016, VWR), briefly centrifuged, and
supernatants were transferred into a fresh 96-well plate
Membrane preparation was then diluted with TBS (50 mM
Tris/HCl pH 7.4, 150 mM NaCl) supplemented with protease
inhibitor cocktail (Roche #04906845001) (henceforth termed
TBS/PI) to a concentration of 1 mg/mL (total membrane
protein), and 0.25% Lauryl Maltose Neopentyl Glycol
(
353075, Falcon). Lactate efflux and intracellular accumulation
measurements were performed following manufacturer’s
instructions (ab65331, AbCam) and the absorbance was
measured at 450 nm using the Varioscan Lux (Thermo
Scientific) plate reader.
Viability Assay. 180 cells/well MDA-MB-231, 1000 cells/
well SNU-398, 80 cells/well MiaPaca2, and 2000 cells/well
RT-4 cells were seeded onto a 384-well plate in complete
growth media and allowed for overnight recovery at 37 °C in
(
LMNG) (Anatrace #NG310) was added at a final
concentration of 0.25% (w/v). The membrane preparation
was solubilized for 1 h at 4 °C with end-over-end rotation.
Insoluble material was pelleted by centrifugation typically for 1
h at 100,000 g at 4 °C. The supernatant containing solubilized
MCT4-eGFP was used in FCCS experiments, which were
carried out at RT.
FCCS Measurements. All FCCS measurements were
the atmosphere of 5% CO . On the following day, cells were
2
35,37
performed as described previously.
Briefly, saturation
washed twice with PBS. After washing, medium supplemented
with 2.5% dialyzed FBS (SH3007903, HyClone) was added to
a final volume of 50 μL per well. Test compounds in DMSO
were added into the assay plate using a D300 digital dispenser.
Plates were incubated for 6 days (144 h) at 37 °C/5% CO2.
Following incubation 2× diluted MTS/PMS mix was added to
binding assays were first carried out to determine the
equilibrium dissociation constants (KD) of MCT4-EGFP
and 18u interaction. Then, IC -values were determined
5
0
from the inhibition binding data by using a previously
35
described four-parameter logistic function. All the experi-
ments were carried out after incubating the samples for 1 h at
RT while shaking. Data acquisition for samples in equilibrium
typically required 20−60 s per sample.
the plates and incubated for 3 h at 37 °C/5% CO . Absorbance
2
measurement at 490 nm was performed using a Spark 20 M
Tecan plate reader. Time 0 plates were used as a reference to
identify the difference between cytostatic and cytotoxic effects
of compounds.
Live Cell Imaging/Confocal Microscopy. Confocal laser
scanning microscopy was conducted on an LSM510 confocal
35
microscope as previously explained. Cells were washed once
1
000 MC38 cells/well were seeded onto a black/clear flat
with PBS, then compounds were added to the cells in a
solution of PBS with or without 0.05% Triton to allow the
permeabilization of the cell membrane. Images were acquired
after 5 min incubation at RT.
Mice. SCID beige, nude, and C57/Bl6 mice were obtained
from Charles River Laboratories. All mice used for experiments
were 7 to 12 week old females. Mice were housed with ad
libitum access to food and water in a pathogen-free facility. All
procedures were performed in accordance with institutional
protocols approved by the Institutional Animal Care and Use
Committee of EMD Serono Research and Development
Institute.
9
6-well plate (353219, Falcon) in 200 μL complete growth
media and allowed for overnight recovery at 37 °C in the
atmosphere of 10% CO . On the following day, cells were
2
washed once with PBS (14190-094, Gibco), and 200 μL of
fresh media was added. Test compounds dissolved in DMSO
were added into the assay plate using a Tecan D300e Digital
Dispenser, and the plate was incubated at 37 °C in the
atmosphere of 10% CO for 72 h. Viability was determined
2
following manufacturer’s instructions (CellTiter-Glo Lumines-
cent Cell Viability Assay; G7573, Promega), and the
luminescence was measured using the Varioscan Lux (Thermo
Scientific) plate reader.
Mouse Tumor Models. Cell lines were cultured as
described above, harvested during the log phase of growth
and injected subcutaneously above the right foreleg of each
animal within 1 h of collection. MDA-MB-231 cells were
Fluorescent Labeling of Molecule 18t to Generate
1
8u. Molecule 18t was dissolved and mixed in DMSO and
labeled via the reactive amino group to PEG4-DY-647
Dyomics). The mixture was incubated in the dark at RT for
(
harvested by trypsinization and resuspended to a concentration
2
h. The conjugate was purified by reversed-phase HPLC (HP-
6
5
× 10 /mouse in 200 μL of PBS: Matrigel to be implanted in
1
100, Agilent, Palo Alto, USA) using a linear gradient of water
female, Nu/Nu mice. About 14 days after cell implantation,
3
and acetonitrile. Resulting 18u was lyophilized, dissolved in
DMSO, and stored at −20 °C prior to the use in a binding
assay or in imaging experiments.
when the tumors reached 100−200 mm , tumor-bearing mice
were divided into the treatment groups using a rank sort
method that produces a starting mean tumor volume in each
6
Expression of MCT4-eGFP. The full-length protein of
MCT4 was cloned as C-terminal GFP fusion in a mammalian
expression vector pCGTO (a derivative of pcDNA3.1,
Invitrogen, Cat. no. V860-20) under the control of the TET-
promoter and expressed in HEK293 cells. Correct expression
of MCT4-eGFP was confirmed by fluorescence microscopy
group that is similar. 1 × 10 MC38 cells in PBS were
subcutaneously implanted into C57/BL6 mice. Mice were
randomized into treatment groups on the day of treatment
3
initiation, when tumor volumes reached 50−80 mm for the
3
efficacy study, and 300−400 mm for the biomarker (lactate)
study.
6
and western blotting. Typically, HEK293 cells (5 × 10 ) were
The tumor volume was calculated using tumor length (l)
2
transfected with 8 μg of plasmids encoding GFP-tagged
protein using the Nanofectin transfection kit (PAA, Cat. no.
Q051-005). Transfected cells were investigated microscopi-
cally or harvested 24 h after the transfection. During the
and width (w) measurements with the equation l × w /2.
Using electronic calipers, the length was measured along the
longest axis of the tumor, and the width was measured
perpendicular to the length.
1
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PHARMACOKINETICS AND
PHARMACODYNAMICS
Animal Treatment. To examine the PK and pharmacody-
namic (PD) relationships of 18i and 18k in MDA-MB-231 or
8n in MC38, tumors were established as described above. For
the analysis of compound concentrations in plasma and tumor
and intratumoral lactate accumulation, animals were treated
with vehicle or indicated concentrations of the compounds and
terminal plasma and tumor samples were collected upon
euthanasia at 2 h after the treatment. 1 was dissolved in vehicle
0.5% methocel, 0.25% tween20 in water) and administered at
50 mg/kg at a volume of 10 mL/kg for administration.
Stefano Minguzzi − Intana, Bioscience GmbH, 82152
Planegg, Germany
Andrzej Gondela − Ryvu Therapeutics, 30-394 Kraków,
Poland
Shivapriya Ramaswamy − EMD Serono Research &
Development Institute, Inc., Billerica, Massachusetts 01821,
United States
Anna Bartosik − Ryvu Therapeutics, 30-394 Kraków, Poland
Frank Czauderna − EMD Serono Research & Development
Institute, Inc., Billerica, Massachusetts 01821, United States
Lindsey Crowley − EMD Serono Research & Development
Institute, Inc., Billerica, Massachusetts 01821, United States
Pamela Wahra − EMD Serono Research & Development
Institute, Inc., Billerica, Massachusetts 01821, United States
Heike Schilke − Merck Healthcare KGaA, 64293 Darmstadt,
Germany
■
1
(
1
Determination of Compound Concentrations. 18i, k,
and n in plasma and tumor tissue were measured by HPLC-
MS/MS analysis. The compound concentration was deter-
mined using a standard curve (standard curve fitting was
accomplished with Analyst 1.7 software from AB Sciex).
Pharmacokinetic analyses were performed by non-compart-
mental methods using Phoenix WinNonlin version 6.4
Pharsight Corporation, Mountain view, CA).
Tumoral Intracellular Lactate Quantification. Tumor
samples (50−80 mg) were placed on ice in 400 μL of RIPA
buffer with benzonase, followed by homogenization by
homogenization stick and incubation for 20 min on ice.
Next, samples were sonicated and centrifuged for 20 min at 4
Pia Böpple − Merck Healthcare KGaA, 64293 Darmstadt,
Germany
Łukasz Dudek − Ryvu Therapeutics, 30-394 Kraków, Poland
Marcin Les − Ryvu Therapeutics, 30-394 Kraków, Poland
́
(
Paulina Niedziejko − Ryvu Therapeutics, 30-394 Kraków,
Poland
Kamila Olech − Ryvu Therapeutics, 30-394 Kraków, Poland
Henryk Pawlik − Ryvu Therapeutics, 30-394 Kraków, Poland
Łukasz Włoszczak − Ryvu Therapeutics, 30-394 Kraków,
Poland; Present Address: Institute of Organic Chemistry;
Polish Academy of Sciences; Kasprzaka 44/52; 01-224
Warsaw; Poland
°
C. Samples were diluted in RIPA buffer, and the lactate
concentration was measured with Lactate Reagent (M Dialysis
AB; ref#P000024) according to the manufacturer’s protocol.
Karol Zuchowicz − Ryvu Therapeutics, 30-394 Kraków,
Poland
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00448.
■
Jose Ramon Suarez Alvarez − Ryvu Therapeutics, 30-394
Kraków, Poland; Present Address: IU CINQUIMA/
*
́
Quimica Inorganica; Universidad de Valladolid; 47071
́
Valladolid (Spain).
HPLC traces of all target compounds; viability effect of
target compounds; SAR for acid isosteres; acid isosteres
revisited; bridge SAR revisited; SAR for middle ring B;
preferred conformation; off-target selectivity; MCT
isoform selectivity, structure of FFCS fluorescent probe
molecule 18u; binding of 18n to the mouse MCT4
protein; in vivo activity of 18k and 18i in xenograft
tumor models; inhibition of both MCT4 and MCT1
transporters; safety assessment; in silico field analysis;
supplement compounds; and HPLC traces for supple-
ment compounds (PDF)
Justyna Martyka − Ryvu Therapeutics, 30-394 Kraków,
Poland
Ewa Sitek − Ryvu Therapeutics, 30-394 Kraków, Poland
Maciej Mikulski − Ryvu Therapeutics, 30-394 Kraków,
Poland
Joanna Szczęsniak − Ryvu Therapeutics, 30-394 Kraków,
́
Poland
Sven Jäckel − Merck Healthcare KGaA, 64293 Darmstadt,
Germany
Mireille Krier − Merck Healthcare KGaA, 64293 Darmstadt,
Germany; orcid.org/0000-0003-4912-2925
Marcin Król − Ryvu Therapeutics, 30-394 Kraków, Poland
Ansgar Wegener − Merck Healthcare KGaA, 64293
Darmstadt, Germany
Compound smiles codes, MDA-MB-231 IC50 with
standard deviation, SNU-398, RT4, MiaPaCa-2 IC50,
FCCS Ki (CSV)
Michał Gałęzowski − Ryvu Therapeutics, 30-394 Kraków,
Poland
AUTHOR INFORMATION
Corresponding Author
Timo Heinrich − Merck Healthcare KGaA, 64293
Darmstadt, Germany; orcid.org/0000-0001-9120-8410;
Phone: +49 61 51 72 65 89; Email: timo.heinrich@
merckgroup.com
■
Mateusz Nowak − Ryvu Therapeutics, 30-394 Kraków,
Poland
Frank Becker − Intana, Bioscience GmbH, 82152 Planegg,
Germany
Christian Herhaus − Merck Healthcare KGaA, 64293
Darmstadt, Germany
Authors
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00448
Ada Sala-Hojman − Merck Healthcare KGaA, 64293
Darmstadt, Germany
Roberta Ferretti − EMD Serono Research & Development
Institute, Inc., Billerica, Massachusetts 01821, United States
Carl Petersson − Merck Healthcare KGaA, 64293
Darmstadt, Germany
Author Contributions
C.H. was supervising the project, together with M.N. and
M.G.; A.S.-H., S.R., A.B., J.S., J.M., E.S., M.M., and F.C. did all
1
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in vitro investigations, screening method development, and
data acquisition; R.F. designed and oversaw all in vivo studies;
L.C. and P.W. performed the in vivo studies; C.P. was
responsible for pharmacokinetics; A.W., S.M., and F.B. did all
biophysical considerations; A.G., H.S., P.B., L.D., M.L., P.N.,
K.O., H.P., L.W., K.Z., and J.R.S.A. synthesized all compounds;
and a plausible translocation cycle. J. Biol. Chem. 2009, 284, 20011−
0021.
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Davies, A. J.; Halestrap, A. P. Basigin (CD147) is the target for
organomercurial inhibition of monocarboxylate transporter isoforms 1
and 4: The ancillary protein for the insensitive MCT2 is embigin
2
(
(
gp70). J. Biol. Chem. 2005, 280, 27213−27221.
Mireille Krierr and Marcin Krol did the in silico work; and S.J.
́
was in charge of safety.
(
9) Gurrapu, S.; Jonnalagadda, S. K.; Alam, M. A.; Nelson, G. L.;
Sneve, M. G.; Drewes, L. R.; Mereddy, V. R. Monocarboxylate
transporter 1 inhibitors as potential anticancer agents. ACS Med.
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Notes
The authors declare the following competing financial
interest(s): The authors declare no competing interest. SM
and FB are current employees of Intana Bioscience GmbH.
Merck Healthcare KGaA sponsored the part of the study
performed at Intana Bioscience GmbH.
(10) Jonnalagadda, S.; Jonnalagadda, S. K.; Ronayne, C. T.; Nelson,
G. L.; Solano, L. N.; Rumbley, J.; Holy, J.; Mereddy, V. R.; Drewes, L.
R. Novel N,N-dialkyl cyanocinnamic acids as monocarboxylate
transporter 1 and 4 inhibitors. Oncotarget 2019, 10, 2355−2368.
(11) Benjamin, D.; Robay, D.; Hindupur, S. K.; Pohlmann, J.;
Colombi, M.; El-Shemerly, M. Y.; Maira, S.-M.; Moroni, C.; Lane, H.
A.; Hall, M. N. Dual inhibition of the lactate transporters MCT1 and
MCT4 is synthetic lethal with metformin due to NAD+ depletion in
cancer cells. Cell Rep. 2018, 25, 3047−3058.
ACKNOWLEDGMENTS
■
Birgitta Leuthner from Merck Healthcare KGaA was acknowl-
edged for overall project support with special contributions to
cellular screening activities. Friedrich Schuetze from Merck
Healthcare KGaA and Dimitry Kamen from EMD Serono were
acknowledged for contributing in the generation of in vitro
data. Oscar Ortiz and Petra Kerscher from Merck KGaA were
acknowledged for generating the MC38 MCT4 and MCT1
CRISPR K.O. clones. Charles Henry Fabritius from Ryvu
Therapeutics (currently at TOLREMO Therapeutics A.G.)
was acknowledged for his intellectual contribution and overall
project support. Melissa Derner and Molly Jenkins from EMD
Serono were acknowledged for their support in reviewing and
improving the manuscript and supporting information files.
(12) Renner, K.; Bruss, C.; Schnell, A.; Koehl, G.; Becker, H. M.;
Fante, M.; Menevse, A.-N.; Kauer, N.; Blazquez, R.; Hacker, L.;
Decking, S.-M.; Bohn, T.; Faerber, S.; Evert, K.; Aigle, L.; Amslinger,
S.; Landa, M.; Krijgsman, O.; Rozeman, E. A.; Brummer, C.; Siska, P.
J.; Singer, K.; Pektor, S.; Miederer, M.; Peter, K.; Gottfried, E.; Herr,
W.; Marchiq, I.; Pouyssegur, J.; Roush, W. R.; Ong, S.; Warren, S.;
Pukrop, T.; Beckhove, P.; Lang, S. A.; Bopp, T.; Blank, C. U.;
Cleveland, J. L.; Oefner, P. J.; Dettmer, K.; Selby, M.; Kreutz, M.
Restricting glycolysis preserves T cell effector functions and augments
checkpoint therapy. Cell Rep. 2019, 29, 135−150.
(13) Benítez-Rangel, E.; López-Méndez, M.; García, L.; Guerrero-
Hernández, A. DIDS (4,4’-Diisothiocyanatostilbene-2,2’-disulfonate)
directly inhibits caspase activity in HeLa cell lysates. Cell Death
Discovery 2015, 1, 15037.
ABBREVIATIONS
■
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