Demonstrating Ligandability of the LC3A and LC3B Adapter Interface

Article abstract of DOI:10.1021/acs.jmedchem.0c01564

Autophagy is the common name for a number of lysosome-based degradation pathways of cytosolic cargos. The key components of autophagy are members of Atg8 family proteins involved in almost all steps of the process, from autophagosome formation to their selective fusion with lysosomes. In this study, we show that the homologous members of the human Atg8 family proteins, LC3A and LC3B, are druggable by a small molecule inhibitor novobiocin. Structure-activity relationship (SAR) studies of the 4-hydroxy coumarin core scaffold were performed, supported by a crystal structure of the LC3A dihydronovobiocin complex. The study reports the first nonpeptide inhibitors for these protein interaction targets and will lay the foundation for the development of more potent chemical probes for the Atg8 protein family which may also find applications for the development of autophagy-mediated degraders (AUTACs).

Full text of DOI:10.1021/acs.jmedchem.0c01564

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Article
Demonstrating Ligandability of the LC3A and LC3B Adapter
Interface
Markus Hartmann, Jessica Huber, Jan S. Kramer, Jan Heering, Larissa Pietsch, Holger Stark,
̈
Dalibor Odadzic, Iris Bischoff, Robert Furst, Martin Schröder, Masato Akutsu, Apirat Chaikuad,
Volker Dötsch, Stefan Knapp, Ricardo M. Biondi, Vladimir V. Rogov, and Ewgenij Proschak*
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ABSTRACT: Autophagy is the common name for a number of
lysosome-based degradation pathways of cytosolic cargos. The key
components of autophagy are members of Atg8 family proteins
involved in almost all steps of the process, from autophagosome
formation to their selective fusion with lysosomes. In this study, we
show that the homologous members of the human Atg8 family
proteins, LC3A and LC3B, are druggable by a small molecule
inhibitor novobiocin. Structure−activity relationship (SAR) studies of
the 4-hydroxy coumarin core scaffold were performed, supported by a
crystal structure of the LC3A dihydronovobiocin complex. The study
reports the first nonpeptide inhibitors for these protein interaction targets and will lay the foundation for the development of more
potent chemical probes for the Atg8 protein family which may also find applications for the development of autophagy-mediated
degraders (AUTACs).
they have two additional, characteristic α-helices located N-
terminally to their UBL core. This N-terminal subdomain
varies significantly among the different members of the Atg8
family and packs onto the UBL core, forming a deep
hydrophobic pocket (HP1). Another hydrophobic pocket
(HP2) is formed by hydrophobic residues of the central α-
helix and β-strand 2 of the UBL core. HP1, HP2, and β-strand
2 of Atg8/LC3/GABARAP engage in interaction networks
associated with autophagy and membrane trafficking (reviewed
in refs 4 and 13). The majority of selective autophagy receptors
interact with Atg8/LC3/GABARAP through a so-called LIR
(LC3-interacting region), also known as AIM (Atg8-interact-
ing motif)). AIMs are short linear motifs with a central
consensus sequence of Θ−X−X−Γ, where Θ is an aromatic
amino acid (W/F/Y), Γ is a hydrophobic one (L/I/V), and X
can be any amino acid. The aromatic residue usually interacts
with the HP1 pocket, while Γ is placed into HP2 pockets.
Negatively charged residues located N-terminally to Θ (but
also within and after the core LIR) increase affinity of LIR-
containing proteins for Atg8/LC3/GABARAP. Amino-acid
variations of LIR affect specificity for Atg8-family members.14
INTRODUCTION
■
Macroautophagy, hereafter referred as autophagy, is a highly
conserved catabolic process that together with proteasomal
degradation serves in the removal and reuse of unwanted
cellular components, such as damaged organelles, protein
aggregates, intracellular pathogens, and other macromolecular
structures (reviewed in refs 1−4). Autophagy is characterized
by the formation of double-membrane vesicles, autophago-
somes, which are fused with the vacuole (in fungi and plants)
or lysosomes (in higher organisms) and which serve to expose
engulfed cargo to the resident degradative enzymes. Among
∼40 autophagy related (Atg) proteins (reviewed in refs 2 and
5), ubiquitin-like (UBL) proteins of the Atg8 family
(autophagy modifiers) play a key role. Atg8 is expressed as a
precursor protein which undergoes processing by the cysteine
protease Atg4 exposing a C-terminal glycine residue.
Subsequently, the Atg8 C-terminus is covalently linked to
phosphatidyl-ethanolamine (PE) by a ubiquitin-like conjuga-
tion cascade, resulting in incorporation into the inner and
outer membrane of the double-membrane autophagosome.2,6
The PE conjugates tether Atg8−PE to lipid bilayers;7 and they
recruit additional components of the autophagic machi-
nery8−10 and, via interaction with selective autophagy
receptors, sequester specific cargo to autophagosomes.11,12
Humans encode six Atg8 homologues: LC3A, LC3B, LC3C
(LC3 subfamily) and GABARAP, GABARAPL1, GABAR-
APL2/GATE-16 (GABARAP subfamily). LC3/GABARAPs
display a similar topology as observed in ubiquitin; however,
Received: September 23, 2020
Published: March 26, 2021
https://doi.org/10.1021/acs.jmedchem.0c01564
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One of the prototypical and most studied mammalian
selective autophagy receptors is p62/SQSTM1 (p62), a
modular protein involved in autophagy-related degradation of
misfolded proteins and aggregates.15 Despite the fact that a
defective autophagy pathway has been linked to the develop-
ment of many human diseases,5 the efforts to generate selective
autophagy small molecule modulators are in the very beginning
stages. Selective targeting of the LIR interaction with LC3/
GABARAP subfamily members would allow studies to
understand the individual role of each family member in
recruiting cargo to the autophagosome, and it would facilitate
the development of small molecule degraders that would
selectively recruit target proteins to the autophagosome
(AUTACs).16 In this study, we present data based on a
screening approach for small molecules identifying ligands for
LC3/GABARAPs that inhibit interaction with p62. We
demonstrate that LC3/GABARAPs represents a druggable
interface, and we characterized the aminocoumarin antibiotic
novobiocin as a lead structure inhibiting the interaction of
LC3A and LC3B with p62.
Figure 1. (A) Structure of novobiocin (1) (B) 1 causes a shift of the
melting temperature of recombinant LC3B in the DSF assay. (C)
1
Representative (fingerprint) areas of LC3B H−15N HSQC spectra
upon titration with 1 (overlaid). The rainbow color code represents
increasing molar ratios from free LC3B (red) upon 8-fold molar
excess of 1 (purple). (D) Representative (fingerprint) areas of LC3B
1H−15N HSQC spectra upon interaction with the p62 LIR peptide.
Spectra of the free (red) and p62 bound form (purple) of LC3B are
shown.
RESULTS AND DISCUSSION
■
We first established a robust AlphaScreen interaction assay
between biotin-LIRtide (polypeptide including the LIR
sequence DDDWTHL from p62) and GST-LC3B. The assay
is based on detecting the proximity of donor and acceptor
beads that attach to biotin and GST. Using this homogeneous
assay, we conducted a medium-throughput screening of the
Prestwick Chemicals library of approved drugs which
comprises 1280 compounds (see the Screening of Libraries
of Compounds That Affect the Interaction between LC3b and
LIRtide section). The most promising hit was the natural
product antibiotic novobiocin (1, Figure 1A). The direct
binding of 1 to LC3B was confirmed using differential scanning
fluorimetry (DSF). 1 markedly shifted the melting point of
recombinant LC3B by 4.4 °C (Figure 1B).
To investigate the interaction further, we performed a NMR
titration experiment, stepwise adding 1 to 15N-labeled LC3B
up to 8-fold molar excess. Upon titration with 1, LC3B
resonances displayed significant chemical shift perturbations
(CSPs), exclusively in the fast exchange mode (Figures 1C and
S1A). These CSPs are similar in direction and magnitude to
the CSPs induced by titration of canonical p62 LIR-containing
peptide (Figure 1D), suggesting similar interaction site for
both 1 and p62 LIR with the LC3B surface. However, p62 LIR
induced CSPs were in the slow to intermediate exchange
modes, indicating higher affinity of LC3B to p62 LIR in
comparison to 1. Mapping of the 1-induced CSPs on the
sequence and surface of LC3B (Figure S1) resulted in
characteristic shifts of residues involved in binding of LIR
peptides, with the largest CSP allocated in the LC3B HP2
pocket. In contrast, only small CSPs were observed for the
LC3B resonances in HP1 area, indicating that, in contrast to
typical LIRs, only one of the two hydrophobic pockets was
occupied by 1 (Figure 2).
nM of LC3s with increasing concentration of sGFP-LIR
chimera led to determination of the Kd of the LIR−LC3
interaction (0.258 μM for LC3A and 0.126 μM for LC3B,
Figure 3B). Additionally, using this assay, we were able to show
that 2 was able to sufficiently displace the p62 LIR on LC3A
and LC3B, potentially by competing its interaction with HP2
(Figure 3C).
Using the aforementioned assay, we investigated the
preliminary SARs of novobiocin (1) by subsequent structure
simplification. In the first step, we reduced the double bond of
the 3-hydroxybenzamide residue. Then, the novobiose
carbohydrate moiety was removed. Subsequent removal of
the methyl moiety in the 8-position of the aminocoumarin core
and the hydroxyl moiety of the benzamide substituent led to
the simplified compound 6 (Table 1).
We investigated the SARs of the novobiocin derivatives
using three orthogonal techniques: HTRF displacement assay,
ITC, and DSF. The hydrogenation of the allyl double bond
(compound 2) led to improved Kd values and an increase in
ΔTm. The aglycon of 2, compound 3, exhibited decreased
affinity toward both LC3A and LC3B. A very pronounced
decrease in ΔTm underlined the important role of the
novobiose part for protein stabilization. In contrast, the
removal of neither the methyl group in the 8-position nor
the hydroxy group of the benzamide part had a pronounced
effect on affinity. The removal of both groups resulted in
compound 6 and even led to an improved potency in
displacement of the LIR peptide from LC3A and LC3B in
comparison to the aglycon 3.
In order to rationalize the SARs of novobiocin derivatives,
we crystallized LC3A with the most potent inhibitor 2. In the
X-ray structure, 2 bound to the interface between LC3A and
p62, or more precisely to the LIR docking site (LDS). The 4-
hydroxy-coumarin core was enclosed by a positively charged
clamp formed by two lysine residues K30 and K49 (Figure 4A,
cyan). The 3-hydroxybenzamide occupied a lipophilic binding
In order to elucidate the preliminary structure−activity
relationships (SARs) of novobiocin derivatives, we developed a
robust orthogonal assay system based on homogeneous time-
resolved FRET (HTRF) technology. Therefore, LC3A and
LC3B were expressed as fusion proteins with a SNAP tag,
which was subsequently used for labeling with terbium cryptate
acting as the FRET donor. LIR motif coupled to superfolder
GFP acted as the FRET acceptor (Figure 3A). Titration of 10
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extend this potency to all LIR motifs, recognizing the LDS on
Atg8/LC3/GABARAPs surface. A vast majority of all func-
tionally active selective autophagy receptors and adaptors
possess such LIRs,4 allowing one to target a broad set of
autophagy-related interactions by the derivatives of 1.
Guided by the X-ray structure, which showed that the
benzamide substituent occupied the hydrophobic pocket HP2,
thereby mimicking the Leu residue of the LIR motif, we
explored the SAR of this moiety (Table 2). We varied the
linker between the hydrophobic residue and the lipophilic part,
choosing between methylene, thioether, sulfonamide, and
ether isosters. In most cases, the methylene linker was
preferred, followed by the thioether, sulfonamide, and ether.
The X-ray structure of 2 in complex with LC3A (Figure 4)
clarifies this preference. The dihedral angle at the methylene is
119°. A comparable angle can be accommodated by the
thioether, but not by sulfonamide and ether bioisosters.17
Furthermore, the nonpolar methylene and thioether linkers are
more suitable for the hydrophobic environment enclosed by
valine, leucine, and isoleucine side chains. However, when
using unsaturated or aromatic lipophilic substituents, the ether
linker (9h−9j) outperformed the methylene bearing com-
pound 6i. Considering the size of the lipophilic residue,
potency increases with the size of the chain. The n-butyl series
(6e−9e) delivers the most potent compounds with all linkers.
However, in the case of the optimal methylene linker, propyl is
already sufficient to reach the same Kd values. Branched series
(6a−8a, 6g−9g) did not outperform the n-alkyl counterparts.
Cyclopropyl isoster (compounds 6f, 7f, and 9f) of series a were
well-tolerated.
In the next step, we varied the position of the isopentyl side
chain and the substituent in the para-position of the benzamide
(Table 3). Although the X-ray structure suggests that the 4-
hydroxy group is involved in hydrogen bond interactions to a
water molecule (Figure 4A), it can be replaced by a fluoro
substituent with a marginal loss in potency (compound 10).
Methylation of the hydroxy group resulted in equipotent
compound 11. As expected, the transition of the isopentyl
group to the ortho-position resulted in loss of potency
(compound 12).
As mentioned before, the 4-isopentyl benzamide moiety
occupies the HP2 pocket like the leucine residue of the LIR
motif. Subsequently, we investigated whether amino acids can
be attached to the coumarin scaffold, and prepared the series
13a−g (Table 4). The SAR of the amino acid series appears to
be very restrictive when compared to the benzamide series.
Small substituents (glycine 13a, alanine 13b, and valine 13c)
are not tolerated. Leucine 13e, but not isoleucine 13f, yielded
some potency. A clear preference of the S-configuration
(compound 13e) over the enantiomer 13d could be observed.
The introduction of the non-natural n-butyl substituent
(compound 13g) yielded only a very weak inhibitor.
Inspection of the X-ray structure of 2 in complex with LC3A
suggested, that the novobiose moiety does not fully occupy the
hydrophobic pocket HP1, which recognizes the aromatic
residue Trp or Phe of the LIR motif. Furthermore, the LIR
motif is flanked by acidic residues which exhibit ionic
interactions with LC3s. Therefore, we hypothesized that a
gain in binding affinity can be reached by exploiting one of
these interactions. HP1 is located next to the 7-position of the
coumarin scaffold. First, we introduced several aromatic
residues via Suzuki coupling to the 7-bromo-substituted
coumarin precursor. 1-Naphthyl substituent 14a was well-
Figure 2. ITC analysis of compound 1 binding to all human Atg8-
family proteins. The top panels represent the raw injection heats while
the bottom panels show the integrated and normalized heat per
titration step. Calculated Kd values (solid line representing a nonlinear
least-squares fit) to a single site binding model for LC3A and LC3B
are shown. To elucidate the selectivity of 1, we performed isothermal
titration calorimetry (ITC) experiments, titrating 1 into all six human
Atg8-proteins. While we observed pronounced binding of 1 to LC3A
and LC3B, no significant interactions were detected for LC3C as well
as to the three GABARAP subfamily proteins. LC3A displayed an
affinity that was in the micromolar range (Kd = 1.3 μM), typical for
Atg8:LIR interactions. The determined Kd value for LC3B (32 μM)
was higher which was in agreement with the NMR titration data.
pocket formed by residues F52, I35, I67, I66, and L63 (Figure
4A, green). The two methyl groups of the novobiose moiety
displayed superficial interactions with the lipophilic pocket
formed by residues I23 and L53 (yellow), while the urethane
group directly interacted with H27 via a directed hydrogen
bond. Superposition with the crystal structure of LC3B in
complex with the LIR peptide revealed that 2 mimics the
peptide interaction (Figure 4B). The branched alkyl chain of
the 3-hydroxybenzamide part occupies the hydrophobic pocket
HP2, which is responsible for recognition of Γ (Figure 4C and
D). The hydrophobic pocket HP1, which is occupied by an
aromatic residue in the peptide complex, is not completely
filled with the two methyl groups of the novobiose moiety.
Finally, the β sheet interactions with the backbone of V54 and
F52 are patterned by the amide NH in the 3-position and the
hydroxy group in the 4-position of the coumarin, respectively
(Figure 4E and F).
We have showed experimentally that novobiocin derivatives
are potent to disrupt interaction between the p62 LIR motif
and LC3A/LC3B proteins. However, our results allowed us to
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Figure 3. (A) Principle of the HTRF displacement assay. The p62-LIR peptide is expressed as sGFP fusion, while LC3s are coupled to Tb-cryptate.
(B) Association curves of the p62-LIR-sGFP to LC3s. (C) Displacement of 1 μM p62-LIR-sGFP from 10 nM LC3s by 2.
Table 1. Investigation of Preliminary Structure−Activity Relationships of Novobiocin Derivatives
LC3A
LC3B
a
b
c
d
a
b
c
d
compd
IC50 [μM]
Kicalc [μM]
Kd [μM]
ΔTm
IC50 [μM]
Kicalc [μM]
Kd [μM]
ΔTm
1
2
3
4
5
6a
42.9 7.1
16.0 1.0
118.6 16.7
159.8 10.6
127.7 4.0
64.8 3.7
8.8
3.3
24.3
32.7
26.2
13.3
7.5
2.8
14.2
15.7
27.5
17.2
4.4
5.6
0.7
0.0
0.8
1.4
172.0 17.4
72.3 7.4
250.8 10.9
260.9 5.8
227.6 11.1
139.6 10.4
19.3
8.1
28.1
29.3
25.5
15.7
17.4
11.4
21.6
23.5
20.5
25.8
4.4
5.6
1.9
1.8
2.5
2.0
a
b
c
d
IC50 SD value is determined by the HRTF displacement assay. Ki calculated from HTRF displacement. Kd determined by ITC. ΔTm
determined by DSF.
tolerated, while 2-benzofurane isoster 14e led to full inactivity
(Table 5). Introduction of the 2-biphenyl moiety resulted in
almost inactive compound 14b. The introduction of meta- and
para-benzoic acids (compounds 14c and 14d) was also
tolerated by LC3s. A series of 5-substituted N-alkyl pyrazoles
14f−14h also did not lead to more potent inhibitory values.
We checked the tolerability of amide and sulfonamide
isosters of the phenolic −OH group in the 7-position which
exhibit potential H-bond donor and acceptor interactions and
would allow further extension. Sulfonamide substituents in the
7-position of the coumarin scaffold could be introduced and
were intended to combine the narrow angle to fit the HP1 with
a polar group pointing toward the solvent. Almost all
compounds of the series 14i−14o were well tolerated,
especially compound 14o, which incorporates a sulfonamide,
and a well-tolerated 2-naphthyl moiety from 14a showed best
inhibitory values in this study. Notably, selectivity of 14o
toward the other members of the Atg8 family remained
comparable to 1 and 2 (Supporting Information, Table S3).
Finally, amide derivatives 14p and 14q, which should reach out
for ionic interactions, were prepared, without observation of a
potency increase. The acyl amide 14r was slightly more active
than the extended derivatives 14p and 14q, which could
probably be an indication of the importance of the potential H-
bond donor and acceptor interactions mentioned above.
Chemistry. Derivatives 2 and 3 could be prepared directly
from 1 (Scheme 1). Thereby, Pd-catalyzed hydrogenation was
used to reduce the double bond of 1, yielding 2
(dihydronovobiocin). The aglycon 3 was prepared by cleaving
the glycoside bond using acetyl chloride in EtOH.
The synthesis of all other derivatives was not possible from
1, and it is therefore split into the preparation of different
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The different isosteric benzoic acids were accessible through
different routes (Scheme 3). Alkoxybenzoic acids 24a−j could
be synthesized through classical alkylation of 25, followed by
alkaline ester hydrolysis of the methyl 3-alkoxybenzoates 26a−
j. The 3-thioalkyl benzoic acids 28a−g could be synthesized
through a procedure published by Migliore et al. by reacting 3-
thiobenzoic acid (27) with the corresponding alkyl halides in
EtOH and potassium hydroxide.21 The 3-alkylsulfonamido-
benzoic acids 32a−g were synthesized by an one-pot reaction
of methyl 3-aminobenzoate 29 with alkylsulfonyl chloride,
followed by ester hydrolysis and cleavage of disulfonamides.
The starting point for the 3-alkylbenzoic acids 36a−i was
methyl 3-formylbenzoate (33), which was converted into the
corresponding methyl 3-alkenylbenzoates (34a−i) by Wittig
reaction with alkyltriphenyl bromides and LiHMDS as the
base. These alkenes could be converted into methyl 3-
alkylbenzoates 35a−i by reduction with hydrogen over
palladium on charcoal. The following ester hydrolysis was
performed as before and yielded the 3-alkylbenzoic acids 36a−
i.
The syntheses of 2-isopentylbenzoic acid (40) and 4-fluoro-
3-isopentylbenzoic acid (44) started with protection of the
corresponding formylbenzoic acids 37 and 41 by benzylation
of the carboxyl group, followed by Wittig reaction with isobutyl
triphenyl phosphonium bromide under the same conditions as
previously described (Scheme 4). The hydrogenation over
palladium on charcoal resulted in simultaneous reduction of
the double bond and cleavage of the benzyl group. Starting
point for the benzoic acids 51 and 53 was methyl 4-
hydroxybenzoate 45 which was ortho-formylated to 46 using
paraformaldehyde in the presence of triethylamine and
magnesium dichloride. Then the 4-hydroxy group was
benzylated, and the protected methyl 4-(benzyloxy)-3-
formylbenzoate 47 was converted to methyl 4-(benzyloxy)-3-
(3-methylbut-1-en-1-yl)benzoate 48 by Wittig reaction as
described before. The following hydrogenation over palladium
on charcoal lead to methyl 4-hydroxy-3-isopentylbenzoate
(49). While alkaline ester hydrolysis followed by acetylation
with acetic anhydride and pyridine yielded 51, methylation
with dimethyl sulfate, followed by ester hydrolysis leads to 4-
methoxy-3-isopentylbenzoic acid 53.
Figure 4. Structural analysis of 2 in complex with LC3A. (A) Directed
interactions of 2 to LC3A. (B) Superposition of the LC3A in complex
with 2 and with the p62 LIR motif. (C, D) The p62 LIR motif
occupies two hydrophobic binding pockets, while only the leucine
binding pocket is occupied by 2. (E, F) 2 mimics the β-strand-like
backbone interactions of p62-LIR peptide.
coumarin cores and various benzoic acids. The synthesis of the
two 7-hydroxy coumarin cores 18a,b could be accomplished in
an adapted procedure published by Gammon et al.18 (Scheme
2). In the first step, 16a,b was chemoselectively benzylated at
the 4-OH group with potassium carbonate as the base and
potassium iodide as the catalyst to get 17a,b. The cyclization
was performed through a Claisen condensation between 17a,b
and diethyl carbonate with the use of NaH as the base. The
following nitration was accomplished in a similar way as
published by Manjappa et al. with the use of sodium nitrite and
nitric acid in acetic acid.19 The reduction and debenzylation of
19a,b over palladium over charcoal and in hydrogen
atmosphere provided 15a,b in a quantitative yield.
The amide coupling between the amino coumarin cores
15a−d and the benzoic acids 24a−j, 28a−g, 32a−g, 36b−i,
40, 44, 51, and 53 or the commercially available N-Boc amino
acids 56a−g was accomplished under standard conditions by
using HOBt and EDC × HCl (Scheme 5).
The reduction of 54 over palladium on charcoal in a
hydrogen atmosphere delivered 57 in quantitative yield. The
sulfonamido derivatives 14i−o were synthesized through the
reaction of 57 and the corresponding aryl- and alky sulfonyl
chlorides, while the reaction between 57 and the respective
acid anhydrides led to the N-acyl derivatives 14p−r (Scheme
6). The aryl and pyrazole derivatives 14a−h could be
synthesized in moderate yields through a Suzuki coupling
between 55 and the corresponding boronic acids or pinacol
boronic esters.
For the 7-NO2 and 7-Br coumarin core 15c,d, the synthesis
procedure according to Stecher et al. was adapted.20 Therefore,
the 2-hydroxy group of 7-nitro and 7-bromo salicylic acid
(20a,b) was protected by acetylation using acetic anhydride,
triethylamine as the base and DMAP as the catalyst. The
resulting intermediates 21a,b were converted in the corre-
sponding acyl chloride by refluxing with thionyl chloride. The
acyl chloride was coupled under decarboxylative conditions
with the malonate derivative 22b, which was previously
synthesized by N-Boc protection and selective monoester
hydrolysis of diethyl 2-aminomalonate 22a. The cyclization of
the resulting crude product was accomplished by an intra-
molecular transesterification under alkaline conditions and
afforded 23a,b in adequate yields. The cleavage of the N-Boc
group with hydrogen chloride in dioxane leads to the 3-amino-
4-hydroxy-coumarins 15c,d as the hydrochloride-dioxane
adducts.
CONCLUSION
■
This study describes the discovery of the first nonpeptidic
druglike inhibitor of the LC3A and LC3B adapter proteins
demonstrating druggability with druglike small molecule
inhibitors. Furthermore, selectivity toward other members of
the Atg8 family was achieved. We provide first insights into the
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a
Table 2. SAR Exploration of the Phenyl Substituent in the 3-Position of the Benzamide Group
a
Ki values in μM are calculated from HTRF displacement. i.a.: inactive.
interaction of compound 2 with LC3A, thereby paving the way
to structure-based design of more potent inhibitors of this key
protein−protein interaction of the autophagy pathway. We
further explored the structure−activity relationships of the
benzamide part which mimics leucine of the LIR motif and
points into the hydrophobic pocket HP2. Although the
interaction with HP2 can be considered as essential, the SAR
investigated in this study seems rather flat and diverse
substituents were identified which were able to occupy this
lipophilic area and displace the p62 LIR from LC3A and
LC3B. Previous studies suggest that interactions with HP1 are
more important; however, novobiocin derivatives insufficiently
occupy this interaction area. Therefore, future efforts should
aim at occupying HP1 either by extending a different position
of the coumarin core or by scaffold hopping to a central moiety
which allows for efficient extension of hydrophobic inter-
actions in HP1. Our synthetic efforts to extend the SAR to the
hydrophobic pocket HP1 led to the identification of
substitution patterns which were tolerated by LC3A and
LC3B, which, however, were not sufficient to get a substantial
increase in affinity. Still, the insights in the SAR provide a basis
for further development of LC3 inhibitors, by targeting
conserved surfaces that are involved in the binding of all
typical LIR motifs, including p62, and have potential
applications in context of AUTACs.
EXPERIMENTAL SECTION
■
General. All starting materials, solvents, and reagents were
purchased from commercial suppliers, (e.g., Sigma-Aldrich, Apollo
Scientific, Acros Organics) and used without further purification.
NMR spectra were recorded on a Bruker AMX 250, Bruker AV 300,
Bruker AV 400, or Bruker AV500 spectrometer (Bruker Corporation,
Billerica, MA, USA). Chemical shifts (δ) are reported in ppm relative
to tetramethylsilane (TMS) as reference. Multiplicity is reported as
follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m,
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purity was analyzed on a LC2020 system (Shimadzu, Kyoto, Japan)
using UV detection at 254 and 280 nm equipped with a Luna column
(10μ C18(2) 100 Å; 250 × 21.2 mm; Phenomenex, Torrance, CA,
USA) using different gradients (solvent A, acetonitrile; solvent B,
H2O + 0.1% formic acid) at a flow rate of 1 mL/min and was if not
otherwise considered >95%. Method A: solvent mixture A/B 5:95
isocratic for 2 min to 90:10 after additional 12 min and holding at
90:10 for additional 6 min, back to 5:95 in 1 min and holding for
additional 4 min. Method B: solvent mixture A/B 50:50 isocratic for 2
min to 90:10 after additional 12 min and holding at 90:10 for
additional 6 min, back to 50:50 in 1 min and holding for additional 4
min. Method C: solvent A/B 60:40 to 95:5 in 7 min, holding for
additional 8 min at 95:5 and back to 60:40 in 3 min and holding for
additional 2 min. Thin layer chromatography for reaction control was
performed on silica gel 60 F254 aluminum sheets (Merck, Germany).
Preparative column chromatography was performed on silica gel 63−
200 μM (Merck, Germany).
Table 3. Further Variations of the Benzamide Moiety
(3R,4S,5R,6R)-5-Hydroxy-6-((4-hydroxy-3-(4-hydroxy-3-isopen-
tylbenzamido)-8-methyl-2-oxo-2H-chromen-7-yl)oxy)-3-methoxy-
2,2-dimethyltetrahydro-2H-pyran-4-yl Carbamate (2). Novobiocin
sodium salt (1) (1.0 g, 1.6 mmol) of was dissolved in MeOH (40
mL), and palladium on charcoal (w = 10%, 100 mg) was added. The
suspension was stirred in an autoclave for 4 h at 50 °C under
hydrogen atmosphere at 4 bar. The suspension was filtered through a
Celite pad, which was rinsed with MeOH (40 mL). The solvent was
removed in vacuo, and product (1.0 g, 1.6 mmol, 99%) was obtained
as a colorless foam. 1H NMR (400 MHz, DMSO-d6) δ = 9.73 (s, 1H),
8.47 (s, 1H), 7.72 (s, 1H), 7.64−7.60 (m, 2H), 6.91 (d, J = 8.9 Hz,
1H), 6.77 (d, J = 8.3 Hz, 1H), 6.66 (bs, 2H), 5.52 (d, J = 4.9 Hz, 1H),
5.44 (d, J = 2.0 Hz, 1H), 5.16 (dd, J = 3.0/9.8 Hz, 1H), 4.05 (m, 1H),
3.48 (s, 4H), 2.54 (m, 2H), 2.17 (s, 3H), 1.58−1.41 (m, 3H), 1.28 (s,
3H), 1.10 (s, 3H), 0.92 (d, J = 6.5 Hz, 6H) ppm; 13C NMR (101
MHz, DMSO-d6) δ = 170.2, 165.6, 162.9, 157.2, 156.3, 155.2, 152.0,
129.3, 127.7, 126.4, 122.9, 122.0, 118.1, 114.0, 111.5, 107.5, 98.5,
96.6, 80.9, 77.7, 70.4, 68.9, 60.9, 28.4, 27.4, 22.8, 22.5, 8.5 ppm; MS
(ESI+) m/z: 615.30 [M + H]+; HRMS (MALDI+) m/z: calcd for
[C31H38N2O11 + H]+: 615.25484, found: 615.25398, Δ = 1.4 ppm;
HPLC: tR (Method A) = 16.43 min.
a
IC50 SD value is determined by the HRTF displacement assay, and
Ki is calculated.
Table 4. Replacement of the Benzamide Moiety by Boc-
Substituted Amino Acids
a
N-(4,7-Dihydroxy-8-methyl-2-oxo-2H-chromen-3-yl)-4-hydroxy-
3-isopentylbenzamide (3). 2 (0.30 g, 0.49 mmol, 1.0 equiv) was
dissolved in EtOH (5 mL) and heated up to 80 °C. Acetyl chloride
(0.10, 0.98 mmol, 2.0 equiv) was added, and the reaction mixture was
stirred for additional 3 h at 80 °C. The mixture was poured on ice
water, and the formed precipitate was separated through filtration to
obtain the product (0.15 mg, 0.38 mmol, 78%) as a colorless solid. 1H
NMR (400 MHz, DMSO-d6) δ = 11.78 (bs, 1H), 10.33 (s, 1H), 9.88
(s, 1H), 9.10 (s, 1H), 7.75 (m, 1H), 7.63 (m, 1H), 7.51 (m, 1H), 6.83
(m, 2H), 2.65 (m, 2H), 2.10 (s, 3H), 1.51−1.39 (m, 3H,), 0.85 (s,
6H) ppm; 13C NMR (101 MHz, DMSO-d6) δ = 166.6, 160.9, 159.7,
159.0, 158.4, 151.3, 130.0, 128.3, 127.4, 124.9, 121.5, 114.3, 111.8,
108.1, 100.5, 100.4, 27.5, 27.4, 22.5, 8.1 ppm; MS (ESI−) m/z:
396.80 [M − H]+ ; HRMS (MALDI+) m/z: calcd for [C22H23NO6 +
H]+: 398.15981, found: 398.15960, Δ = 0.5 ppm; HPLC tR (Method
A) = 16.23 min.
General Procedure for Amide Coupling (Procedure A). The
respective benzoic acid or Boc-protected amino acid (0.9−1.1 equiv)
was dissolved in dry DMF, and HOBt (0.9−1.1 equiv) and EDC ×
HCl (0.9−1.1 equiv) were added. After the solution clarified, the
corresponding 3-aminocoumarin (1.0 equiv) was added together with
triethylamine (2.5 equiv) and the mixture was stirred at room
temperature overnight. The solution was treated with 1 M aqueous
NaOH (2.0 equiv) and stirred for additional 5 h to cleave formed
esters. The reaction mixtures were acidified with aqueous citric acid
(w = 10%), and the aqueous phase was extracted with EtOAc (3×).
The combined organic phases were washed with brine, dried over
MgSO4, and filtered, and the solvent was removed in vacuo. Further
purification of the products was realized by preparative HPLC.
N-(4,7-Dihydroxy-8-methyl-2-oxo-2H-chromen-3-yl)-3-isopen-
tylbenzamide (4). According to general procedure A: 311 mg (1.50
mmol) of 15b, 317 mg (1.65 mmol) of 36a, 260 mg (1.65 mmol) of
a
IC50 SD value is determined by the HRTF displacement assay, and
Ki is calculated. i.a.: inactive.
multiplet. Approximate coupling constants (J) are given in hertz (Hz).
Mass spectra were obtained on a VG Platform II system (Thermo
Fischer Scientific, Inc., Waltham, MA, USA) using electrospray
ionization (ESI). High-resolution mass spectra were recorded on a
MALDI LTQ ORBITRAP XL instrument (Thermo Fisher Scientific).
Compounds were purified by preparative HPLC using a Shimadzu
Preparative Prominence Modular HPLC (Shimadzu, Kyoto, Japan)
with the following conditions: column, Luna (10μ C18(2) 100 Å; 250
× 21.2 mm; Phenomenex, Torrance, CA, USA); mobile phase,
different gradients of acetonitrile/H2O + 0.1% formic acid at a flow
rate of 21 mL/min and UV-detection at 254 and 280 nm. Compound
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Table 5. Exploration of the Hydrophobic Pocket HP1 by Variation of the 7-Position of the Coumarin Core
a
IC50 SD value is determined by the HRTF displacement assay, and Ki is calculated. i.a.: inactive.
a
Scheme 1. Direct Derivatization of 1
a
(a) H2, Pd/C, MeOH, 50 °C, 4 h; (b) AcCl, EtOH, reflux, 2 h.
HOBt, 316 mg (1.65 mmol) of EDC × HCl, 15 mL of DMF and 0.4
mL of triethylamine. The desired product (185 mg, 0.49 mmol, 32%)
was obtained as a colorless solid. 1H NMR (300 MHz, DMSO-d6) δ =
11.69 (bs, 1H), 10.43 (s, 1H), 9.38 (s, 1H), 7.85 (s, 1H), 7.82−7.80
(m, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 5.1 Hz, 2H), 6.89 (d, J
= 8.7 Hz, 1H), 2.66 (t, J = 7.7 Hz, 2H), 2.18 (s, 3H), 1.64−1.48 (m,
3H), 0.93 (d, J = 6.2 Hz, 6H) ppm; 13C NMR (126 MHz, DMSO-d6)
δ = 166.7, 160.9, 160.4, 159.1, 151.5, 142.5, 133.9, 131.5, 128.1,
127.8, 125.4, 121.6, 111.8, 110.4, 108.0, 99.9, 40.3, 33.0, 27.1, 22.4 (2
× C), 8.1 ppm; MS (ESI−) m/z: 379.95 [M − H]−; HRMS (MALDI
+) m/z: calcd for [C22H23NO5 + H]+: 382.16490, found: 382.16459,
Δ = 0.8 ppm; HPLC: tR (Method A) = 18.61 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-4-hydroxy-3-isopen-
tylbenzamide (5). According to general procedure A: 483 mg (2.5
mmol) of 15a, 751 mg (3.0 mmol) of 51, 474 mg (3.0 mmol) of
HOBt, 575 mg (3.0 mmol) of EDC × HCl, 15 mL of DMF, and 0.9
mL of triethylamine. The desired product (84 mg, 0.22 mmol, 9%)
was obtained as a colorless solid. 1H NMR (500 MHz, DMSO-d6) δ =
11.91 (bs, 1H), 10.54 (s, 1H), 9.96 (s, 1H), 9.18 (s, 1H), 7.77 (d, J =
2.0 Hz, 1H), 7.72−7.67 (m, 2H), 6.85 (d, J = 8.4 Hz, 1H), 6.82 (dd, J
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Scheme 2. Preparation of the 7-Substituted Coumarin Cores
a
(a) BnBr, K2CO3, KI, acetone, reflux, 5 h; (b) NaH, EtO2(CO), toluene, 140 °C, 16 h; (c) NaNO2, HNO3 (w = 65%), AcOH, 60 °C, 15 min; (d)
H2, Pd/C, MeOH, RT, 16 h; (e) Ac2O, NEt3, DMAP, EtOAc, 80 °C, 16 h; (f) (i) Boc2O, NaHCO3, DMAP, H2O/dioxane, RT, 5 h; (ii) KOH,
EtOH, RT, 3 h; (g) (i) SOCl2, DCM, reflux, 5 h; (ii) 2-((tert-butoxycarbonyl)amino)-3-ethoxy-3-oxopropanoic acid, NEt3, MgCl2, THF, 0 °C →
RT, 16 h; (iii) NaOHaq, MeOH, RT, 3 h; (h) 4M HCl in dioxane, MeOH, 0 °C → RT, 5 h.
a
Scheme 3. Preparation of the Benzoic Acid Derivatives
a
(a) (MeO)2SO2 or R−X (X = Cl, Br, I), K2CO3, KI, acetone or ACN, 6 h, reflux; (b) KOH, THF/MeOH/H2O (2:1:1), 6 h, reflux; (c) (a) R−X
(X = Cl, Br, I), NaOH, EtOH, 4 h, reflux; (d) R-SO2Cl, NEt3, DCM, 3 h, 0 °C; (e) alkyl triphenyl phosphonium bromide, LiHMDS, THF, 0 °C →
RT, 3 h; (f) H2, Pd/C (w = 10%), MeOH, RT, 5 h.
1
= 2.3/8.7 Hz, 1H), 6.72 (d, J = 2.2 Hz, 1H), 2.59−2.55 (m, 2H), 1.57
(non, J = 6.5 Hz, 1H), 1.48−1.43 (m, 2H), 0.93 (d, J = 6.5 Hz, 6H)
ppm; 13C NMR (126 MHz, DMSO-d6) δ = 166.6, 161.3, 160.9, 159.8,
158.4, 153.4, 130.0, 128.4, 127.4, 125.0, 124.2, 114.3, 113.0, 108.2,
101.9, 100.7, 38.7, 27.5, 27.4, 22.5 (2 × C) ppm; MS (ESI−) m/z:
obtained as a pale-yellow solid. H NMR (300 MHz, DMSO-d6) δ =
11.80 (bs, 1H), 10.55 (s, 1H), 9.37 (s, 1H), 7.86−7.77 (m, 2H), 7.72
(d, J = 8.7 Hz, 1H), 7.43−7.38 (m, 2H), 6.83 (dd, J = 2.2/8.7 Hz,
1H), 6.73 (d, J = 2.2 Hz, 1H), 2.66 (t, J = 7.8 Hz, 2H), 1.64−1.47 (m,
3H), 0.93 (d, J = 6.3 Hz, 6H) ppm; 13C NMR (75 MHz, DMSO-d6) δ
= 166.7, 161.4, 160.8, 160.2, 153.5, 142.5, 133.9, 131.5, 128.1, 127.8,
125.4, 125.0, 113.0, 108.0, 101.9, 100.2, 40.3, 32.9, 27.1, 22.4 (2 × C)
ppm; MS (ESI−) m/z: 366.30 [M − H]−; HRMS (MALDI+) m/z:
calcd for [C21H21NO5 + H]+: 368.14925, found: 368.14926, Δ = 0.03
ppm; HPLC: tR (Method A) = 17.78 min.
382.00 [M
−
H]−; HRMS (MALDI+) m/z: calcd for
[C21H21NO6+Na]+: 406.12611, found: 406.12603, Δ = 0.2 ppm;
HPLC: tR (Method A) = 15.92 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-isopentylbenza-
mide (6a). According to general procedure A: 236 mg (1.22 mmol) of
15a, 235 mg (1.22 mmol) of 36a, 212 mg (1.34 mmol) of HOBt, 258
mg (1.34 mmol) of EDC × HCl, 15 mL of DMF, and 0.3 mL of
triethylamine. The desired product (96 mg, 0.26 mmol, 21%) was
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-propylbenzamide
(6c). According to general procedure A: 209 mg (1.08 mmol) of 15a,
178 mg (1.08 mmol) of 36c, 188 mg (1.19 mmol) of HOBt, 229 mg
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Scheme 4. Synthesis of Benzoic Acids
a
(a) BnBr, K2CO3, acetone, reflux, 1 h; (b) isobutyl triphenyl phosphonium bromide, LiHMDS, THF, 0 °C → RT, 3 h; (c) H2, Pd/C (w = 10%),
MeOH, RT, 5 h; (d) (a) BnBr (1.2 equiv), NaHCO3, DMF, RT, 4 h; (e) paraformaldehyde, MgCl2, (Et)3N, DCE, 70°C, 12 h; (f) benzyl bromide,
K2CO3, DMF, 5.5 h, 85 °C; (g) KOH, THF/MeOH/H2O (2:1:1), 6 h, reflux; (h) Ac2O, pyridine, DCM, 0 °C → RT, 12 h; (i) (MeO)2SO2,
K2CO3, acetone, 80 °C, 2.5 h.
(ESI−) m/z: 338.25 [M − H]−; HRMS (MALDI+) m/z: calcd for
a
Scheme 5. Amide Coupling Procedure
[C19H17NO5 + H]+: 340.11795, found: 340.11811, Δ = 0.5 ppm;
HPLC: tR (Method A) = 16.33 min.
3-Butyl-N-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)benzamide
(6d). According to general procedure A: 365 mg (1.89 mmol) of 15a,
337 mg (1.89 mmol) of 36d, 328 mg (2.08 mmol) of HOBt, 399 mg
(2.08 mmol) of EDC × HCl, 16 mL DMF, and 3 mL of triethylamine.
The desired product (75 mg, 0.21 mmol, 11%) was obtained as a
1
colorless solid. H NMR (300 MHz, DMSO-d6) δ = 11.82 (bs, 1H),
10.55 (s, 1H), 9.37 (s, 1H), 7.85−7.79 (m, 2H), 7.72 (d, J = 8.7 Hz,
1H), 7.42−7.39 (m, 2H), 6.83 (dd, J = 2.2/8.7 Hz, 1H), 6.73 (d, J =
2.2 Hz, 1H), 2.66 (t, J = 7.6 Hz, 2H), 1.66−1.56 (m, 2H), 1.41−1.27
(m, 2H), 0.92 (t, J = 7.4 Hz, 3H) ppm; 13C NMR (75 MHz, DMSO-
d6) δ = 166.7, 161.4, 160.8, 160.2, 153.5, 142.3, 133.9, 131.5, 128.1,
a
(a) HOBt, EDC × HCl, NEt3, DMF, 16 h, RT; (b) HOBt, EDC ×
127.9, 125.4, 125.1, 113.0, 108.0, 101.9, 100.2, 34.7, 33.1, 21.7, 13.7
ppm; MS (ESI−) m/z: 352.25 [M − H]−; HRMS (MALDI+) m/z:
calcd for [C20H19NO5 + H]+: 354.13360, found: 354.13379, Δ = 0.5
ppm; HPLC: tR (Method A) = 17.19 min.
HCl, DMF, 16 h, RT.
(1.19 mmol) of EDC × HCl, 15 mL of DMF, and 3 mL of
triethylamine. The desired product (45 mg, 0.13 mmol, 12%) was
obtained as a colorless solid. H NMR (300 MHz, DMSO-d6) δ =
11.80 (1H, bs), 10.54 (s, 1H), 9.37 (s, 1H), 7.86−7.77 (m, 2H), 7.72
(d, J = 8.6 Hz, 1H), 7.45−7.38 (m, 2H), 6.83 (dd, J = 2.3/8.6 Hz,
1H), 6.73 (d, J = 2.3 Hz, 1H), 2.64 (t, J = 7.5 Hz, 2H), 1.64 (m, 2H),
0.92 (t, J = 7.26, 3H) ppm; 13C NMR (75 MHz, DMSO-d6) δ =
166.7, 161.4, 160.8, 160.3, 153.5, 142.1, 133.9, 131.6, 128.0, 127.9,
125.5, 125.0, 112.9, 108.0, 101.9, 100.2, 37.1, 24.0, 13.6 ppm; MS
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-pentylbenzamide
(6e). According to general procedure A: 297 mg (1.54 mmol) of 15a,
296 mg (1.54 mmol) of 36e, 267 mg (1.69 mmol) of HOBt, 325 mg
(1.69 mmol) of EDC × HCl, 15 mL of DMF and 3 mL of
triethylamine. The desired product (85 mg, 0.23 mmol, 15%) was
1
1
obtained as a colorless solid. H NMR (300 MHz, DMSO-d6) δ =
11.79 (bs, 1H), 10.55 (s, 1H, OH), 9.37 (s, 1H), 7.85−7.78 (m, 2H),
7.72 (d, J = 8.7 Hz, 1H), 7.44−7.38 (m, 2H), 6.83 (dd, J = 2.3/8.7
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Scheme 6. Introduction of Substituents in the 7-Position of the Coumarin Core
a
(a) H2, Pd/C, EtOH, RT, 12 h; (b) R″-SO2Cl or R″-C(O)OC(O)-R″, pyridine, DCM, 0 °C → RT, 16 h; (c) R″-B(OR)2, NaHCO3,
Pd(PPh3)4, 80 °C, 24 h.
Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H), 2.65 (t, J = 7.6 Hz, 2H), 1.66−1.58
(m, 2H), 1.37−1.24 (m, 4H), 0.88 (t, J = 7.0 Hz, 3H) ppm; 13C
NMR (75 MHz, DMSO-d6) δ = 166.7, 161.4, 160.8, 160.2, 153.5,
142.4, 133.9, 131.5, 128.1, 127.9, 125.4, 125.0, 113.0, 108.0, 101.9,
100.2, 35.0, 30.9, 30.6, 21.9, 13.9 ppm; MS (ESI−) m/z: 366.30 [M −
H]−; HRMS (MALDI+) m/z: calcd for [C21H21NO5 + H]+:
368.14925, found: 368.14942, Δ = 0.5 ppm; HPLC: tR (Method A)
= 17.98 min.
3-(2-Cyclopropylethyl)-N-(4,7-dihydroxy-2-oxo-2H-chromen-3-
yl)benzamide (6f). According to general procedure A: 349 mg (1.81
mmol) of 15a, 378 mg (1.99 mmol) of 36f, 314 mg (1.99 mmol) of
HOBt, 381 mg (1.99 mmol) of EDC × HCl, 15 mL of DMF, and 3
mL of triethylamine. The desired product (130 mg, 0.36 mmol, 20%)
was obtained as a colorless solid. 1H NMR (300 MHz, DMSO-d6) δ =
11.80 (bs, 1H), 10.55 (s, 1H), 9.36 (s, 1H), 7.86−7.79 (m, 2H), 7.72
(d, J = 8.7 Hz, 1H), 7.44−7.38 (m, 2H), 6.83 (dd, J = 2.3/8.7 Hz,
1H), 6.73 (d, J = 2.3 Hz, 1H), 2.78−2.72 (m, 2H), 1.57−1.49 (m,
2H), 0.74−0.65 (m, 1H), 0.44−0.38 (m, 2H), 0.09−0.06 (m, 2H)
ppm; 13C NMR (75 MHz, DMSO-d6) δ = 166.7, 161.4, 160.8, 160.2,
153.5, 142.2, 133.9, 131.6, 128.0, 127.9, 125.4, 125.0, 113.0, 108.0,
101.9, 100.2, 39.5, 36.1, 35.3, 10.6, 4.4 (2 × C) ppm; MS (ESI−) m/
z: 364.25 [M − H]−; HRMS (MALDI+) m/z: calcd for [C21H19NO5
+ H]+: 366.13360, found: 366.13370, Δ = 0.3 ppm; HPLC: tR
(Method A) = 17.01 min.
128.3 (2 × C), 128.1, 128.0, 125.9, 125.6, 125.0, 112.9, 108.0, 101.9,
100.1, 37.0, 37.0 ppm; MS (ESI−) m/z: 399.90 [M − H]−; HRMS
(MALDI+) m/z: calcd for [C24H19NO5 + H]+: 402.13360, found:
402.13363, Δ = 0.01 ppm; HPLC: tR (Method A) = 16.94 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-(isobutylthio)-
benzamide (7a). According to general procedure A: 384 mg (1.99
mmol) of 15a, 376 mg (1.79 mmol) of 28a, 343 mg (1.79 mmol) of
HOBt, 274 mg (1.79 mmol) of EDC × HCl, and 10 mL of DMF. The
desired product (112 mg, 0.29 mmol, 15%) was obtained as a
1
colorless solid. H NMR (400 MHz, DMSO-d6) δ = 11.81 (bs, 1H),
10.55 (s, 1H), 9.45 (s, 1H), 7.93 (s, 1H), 7.76 (d, J = 7.8 Hz, 1H),
7.72 (d, J = 8.7, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.44 (t, J = 7.8 Hz,
1H), 6.83 (dd, J = 2.2/8.7 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 2.94 (d,
J = 6.6 Hz, 2H), 1.83 (sept., J = 6.6 Hz, 1H), 1.01 (d, J = 6.6 Hz, 6H)
ppm; 13C NMR (101 MHz, DMSO-d6) δ = 166.0, 161.4, 160.8, 160.6,
153.5, 137.1, 134.7, 130.7, 128.8, 127.3, 125.1, 125.0, 112.9, 108.0,
101.9, 99.8, 40.8, 27.7, 21.7 ppm; MS (ESI−) m/z: 383.85 [M − H]−;
HRMS (MALDI+) m/z: calcd for [C20H19NO5S + H]+: 386.10567,
found:386.10580, Δ = 0.3 ppm; HPLC tR (Method B) = 10.97 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-(ethylthio)-
benzamide (7c). According to general procedure A: 148 mg (0.77
mmol) of 15a, 126 mg (0.69 mmol) of 28c, 106 mg (0.69 mmol) of
HOBt, 133 mg (0.69 mmol) of EDC × HCl, and 5 mL of DMF. The
desired product (82 mg, 0.23 mmol, 30%) was obtained as a colorless
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-isobutylbenzamide
(6g). According to general procedure A: 107 mg (0.56 mmol) of 15a,
109 mg (0.61 mmol) of 36g, 97 mg (0.61 mmol) of HOBt, 117 mg
(0.61 mmol) of EDC × HCl, 8 mL of DMF, and 3 mL of
triethylamine. The desired product (46 mg, 0.13 mmol, 23%) was
1
solid. H NMR (400 MHz, DMSO-d6) δ = 11.80 (bs, 1H), 10.55 (s,
1H), 9.46 (s, 1H), 7.93 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.72 (d, J =
8.7 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 6.83
(dd, J = 2.2/8.7 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 3.06 (q, J = 7.3 Hz,
2H), 1.27 (t, J = 7.3 Hz, 3H) ppm; 13C NMR (101 MHz, DMSO-d6)
δ = 166.0, 161.5, 160.8, 160.5, 153.5, 136.5, 134.7, 130.7, 128.9,
127.3, 125.2, 125.0, 113.0, 108.0, 101.9, 99.9, 26.1, 14.2 ppm; MS
(ESI−) m/z: 355.80 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C18H15NO5S + H]+: 358.07366, found: 358.07437, Δ = 2.0 ppm;
HPLC tR (Method B) = 8.57 min.
1
obtained as a colorless solid. H NMR (300 MHz, DMSO-d6) δ =
11.82 (bs, 1H), 10.54 (s, 1H), 9.37 (s, 1H), 7.85−7.79 (m, 2H), 7.72
(d, J = 8.7 Hz, 1H), 7.44−7.35 (m, 2H), 6.83 (dd, J = 2.3/8.7 Hz,
1H), 6.73 (d, J = 2.3 Hz, 1H), 2.53 (d, J = 7.1 Hz, 2H), 1.91 (sept., J
= 6.7 Hz, 1H), 0.89 (d, J = 6.7 Hz, 6H) ppm; 13C NMR (75 MHz,
DMSO-d6) δ = 166.7, 161.4, 160.8, 160.2, 153.5, 141.2, 133.8, 132.2,
128.5, 127.9, 125.5, 125.0, 113.0, 108.0, 101.9, 100.2, 44.5, 29.6, 22.1
(2 × C) ppm; MS (ESI−) m/z: 352.30 [M − H]−; HRMS (MALDI
+) m/z: calcd for [C20H19NO5 + H]+: 354.13360, found: 354.13358,
Δ = 0.1 ppm; HPLC: tR (Method A) = 16.98 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-(propylthio)-
benzamide (7d). According to general procedure A: 179 mg (0.93
mmol) of 15a, 164 mg (0.84 mmol) of 28d, 128 mg (0.84 mmol) of
HOBt, 160 mg (0.84 mmol) of EDC × HCl, and 5 mL of DMF. The
desired product (87 mg, 0.23 mmol, 25%) was obtained as a colorless
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-phenethylbenza-
mide (6i). According to general procedure A: 138 mg (0.72 mmol) of
15a, 162 mg (0.72 mmol) of 36i, 124 mg (0.79 mmol) of HOBt, 151
mg (0.79 mmol) of EDC × HCl, 10 mL of DMF, and 3 mL of
triethylamine. The desired product (40 mg, 0.10 mmol, 14%) was
1
solid. H NMR (400 MHz, DMSO-d6) δ = 11.81 (bs, 1H), 10.55 (s,
1H), 9.45 (s, 1H), 7.93 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.72 (d, J =
8.7 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 6.82
(d, J = 2.2/8.7 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 3.03 (t, J = 7.2 Hz,
2H), 1.62 (sext, J = 7.2 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H) ppm; 13C
NMR (101 MHz, DMSO-d6) δ = 166.0, 161.4, 160.8, 160.6, 153.5,
136.7, 134.7, 130.8, 128.8, 127.3, 125.2, 125.1, 112.9, 108.0, 101.9,
99.8, 33.9, 21.9, 13.2 ppm; MS (ESI−) m/z: 369.85 [M − H]−;
HRMS (MALDI+) m/z: calcd for [C19H17NO5S + H]+: 372.09002,
found: 372.09042, Δ = 1.1 ppm; HPLC tR (Method B) = 9.87 min.
1
obtained as a colorless solid. H NMR (300 MHz, DMSO-d6) δ =
11.82 (bs, 1H), 10.55 (s, 1H), 9.38 (s,1H), 7.93 (s, 1H), 7.84−7.81
(m, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.42−7.40 (m, 2H), 7.29−7.25 (m,
4H), 7.21−7.18 (m, 1H), 6.83 (dd, J = 2.3/8.7 Hz, 1H), 6.73 (d, J =
2.3 Hz, 1H), 2.99−2.90 (m, 4H) ppm; 13C NMR (75 MHz, DMSO-
d6) δ = 166.6, 161.4, 160.8, 153.5, 141.5, 133.9, 131.6, 128.4 (2 × C),
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Journal of Medicinal Chemistry
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Article
3-(Butylthio)-N-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)-
benzamide (7e). According to general procedure A: 169 mg (0.88
mmol) of 15a, 166 mg (0.79 mmol) of 28e, 121 mg (0.79 mmol) of
HOBt, 151 mg (0.79 mmol) of EDC × HCl, and 5 mL of DMF. The
desired product (94 mg, 0.24 mmol, 28%) was obtained as a colorless
9.42 (s, 1H), 7.79−7.76 (m, 2H), 7.72 (d, J = 8.7 Hz, 1H), 7.48 (t, J =
7.8 Hz, 1H), 7.41 (dd, J = 1.1/8.2 Hz, 1H), 6.83 (dd, J = 2.2/8.7 Hz,
1H), 6.73 (d, J = 2.2 Hz, 1H), 3.04 (s, 3H) ppm; 13C NMR (151
MHz, DMSO-d6) δ = 166.2, 161.4, 160.7, 160.4, 153.4, 138.4, 135.3,
129.0, 125.0, 123.2, 122.9, 119.6, 112.9, 108.0, 101.9, 100.0, 39.1
ppm; MS (ESI−) m/z: 389.15 [M − H]−; HRMS (MALDI+) m/z:
calcd for [C17H14N2O7S + H]+: 391.05945, found: 391.05921, Δ =
0.6 ppm; HPLC tR (Method A) = 12.03 min.
1
solid. H NMR (400 MHz, DMSO-d6) δ = 11.81 (bs, 1H), 10.55 (s,
1H), 9.45 (s, 1H), 7.93 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.72 (d, J =
8.7 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 6.83
(dd, J = 2.2/8.7 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 3.05 (t, J = 7.2 Hz,
1H), 1.59 (quint., J = 7.1 Hz, 2H), 1.42 (sext, J = 7.4 Hz, 2H), 0.90 (t,
J = 7.4 Hz, 3H) ppm; 13C NMR (101 MHz, DMSO-d6): 166.0, 161.4,
160.8, 160.6, 153.5, 136.8, 134.7, 130.7, 128.8, 127.2, 125.1, 125.0,
112.9, 108.0, 101.9, 99.9, 31.6, 30.6, 21.3, 13.5 ppm; MS (ESI−) m/z:
383.85 [M − H]−; HRMS (MALDI+) m/z: calcd for [C20H19NO5S +
H]+: 386.10567, found: 386.10614, Δ = 1.2 ppm; HPLC tR (Method
B) = 11.08 min.
N - ( 4 , 7 - D i h y d r o x y - 2 - o x o - 2 H - c h r o m e n - 3 - y l ) - 3 -
(ethylsulfonamido)benzamide (8c). According to general procedure
A: 174 mg (0.90 mmol) of 15a, 227 mg (0.99 mmol) of 32c, 156 mg
(0.99 mmol) of HOBt, 190 mg (0.99 mmol) of EDC × HCl, 15 mL
of DMF, and 3 mL of triethylamine. The desired product (65 mg,
1
0.16 mmol, 18%) was obtained as a colorless solid. H NMR (600
MHz, DMSO-d6) δ = 11.84 (bs, 1H), 10.56 (s, 1H), 9.97 (s, 1H),
9.42 (s, 1H), 7.79−7.77 (m, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.72 (d, J
= 8.7 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.42 (dd, J = 1.3/8.1 Hz, 1H),
6.83 (dd, J = 2.2/8.7 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 3.14 (q, J =
7.3 Hz, 2H), 1.21 (t, J = 7.3 Hz, 3H) ppm; 13C NMR (151 MHz,
DMSO-d6) δ = 166.2, 161.4, 160.7, 160.3, 153.5, 138.5, 135.3, 129.1,
125.0, 123.0, 122.5, 119.2, 112.9, 107.9, 101.9, 100.0, 45.3, 8.0 ppm;
MS (ESI−) m/z: 403.20 [M − H]−; HRMS (MALDI+) m/z: calcd
for [C18H16N2O7S + H]+: 405.07454, found: 405.07510, Δ = 1.4
ppm; HPLC tR (Method A) = 12.47 min.
3-((Cyclopropylmethyl)thio)-N-(4,7-dihydroxy-2-oxo-2H-chro-
men-3-yl)benzamide (7f). According to general procedure A: 102 mg
(0.53 mmol) of 15a, 99 mg (0.48 mmol) of 28f, 73 mg (0.48 mmol)
of HOBt, 91 mg (0.48 mmol) of EDC × HCl, and 5 mL of DMF. The
desired product (28 mg, 0.07 mmol, 14%) was obtained as a colorless
1
solid. H NMR (250 MHz, DMSO-d6) δ = 11.88 (bs, 1H), 10.52 (s,
1H), 9.41 (s, 1H), 7.94 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.70 (d, J =
8.7 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 6.81
(dd, J = 2.2/8.7, 1H), 6.71 (d, J = 2.2 Hz, 1H), 3.00 (d, J = 7.1 Hz,
2H), 1.09−0.99 (m, 1H), 0.57−0.51 (m, 2H), 0.30−0.25 (m, 2H)
ppm; 13C NMR (101 MHz, DMSO-d6) δ = 166.0, 161.3, 161.0, 160.9,
153.6, 137.1, 134.8, 130.8, 128.8, 127.3, 125.2, 125.1, 112.8, 108.4,
101.9, 99.7, 37.7, 10.4, 5.5 (2 × C) ppm; MS (ESI−) m/z: 381.85 [M
− H]−; HRMS (MALDI+) m/z: calcd for [C20H17NO5S + H]+:
384.09002, found: 384.08981, Δ = 0.5 ppm; HPLC tR (Method B) =
9.64 min.
N - ( 4 , 7 - D i h y d r o x y - 2 - o x o - 2 H - c h r o m e n - 3 - y l ) - 3 -
(propylsulfonamido)benzamide (8d). According to general proce-
dure A: 92 mg (0.48 mmol) of 15a, 127 mg (0.52 mmol) of 32d, 82
mg (0.52 mmol) of HOBt, 100 mg (0.52 mmol) of EDC × HCl, 10
mL of DMF, and 3 mL of triethylamine. The desired product (20 mg,
1
0.05 mmol, 10%) was obtained as a colorless solid. H NMR (600
MHz, DMSO-d6) δ = 11.84 (bs, 1H), 10.55 (s, 1H), 9.96 (s, 1H),
9.41 (s, 1H), 7.79−7.77 (m, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.72 (d, J
= 8.7 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.41 (dd, J = 1.2/8.1, 1H),
6.84 (dd, J = 2.1/8.7 Hz, 1H), 6.73 (d, J = 2.1 Hz, 1H), 3.11 (t, J =
7.7 Hz, 2H), 1.70 (sext, J = 7.6 Hz, 2H), 0.94 (t, J = 7.6 Hz, 3H)
ppm; 13C NMR (151 MHz, DMSO-d6) δ = 166.2, 161.4, 160.7, 160.3,
153.5, 138.5, 135.3, 129.0, 125.0, 123.0, 122.4, 119.2, 112.9, 107.9,
101.9, 100.0, 52.5, 16.8, 12.5 ppm; MS (ESI−) m/z: 416.90 [M −
H]−; HRMS (MALDI+) m/z: calcd for [C19H18N2O7S + H]+:
419.09075, found: 419.09005, Δ = 1.7 ppm; HPLC tR (Method A) =
13.18 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-(isopropylthio)-
benzamide (7g). According to general procedure A: 171 mg (0.88
mmol) of 15a, 156 mg (0.80 mmol) of 28g, 122 mg (0.80 mmol) of
HOBt, 152 mg (0.80 mmol) of EDC × HCl, and 5 mL of DMF. The
desired product (73 mg, 0.20 mmol, 23%) was obtained as a colorless
1
solid. H NMR (400 MHz, DMSO-d6) δ = 11.82 (bs, 1H), 10.55 (s,
1H), 9.47 (s, 1H), 8.00 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.72 (d, J =
8.7 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 6.83
(dd, J = 2.2/8.7 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 3.60 (sept, J = 6.6
Hz, 1H), 1.27 (d, J = 6.6 Hz, 6H) ppm; 13C NMR (101 MHz,
DMSO-d6) δ = 166.0, 161.4, 160.8, 160.6, 153.5, 135.6, 134.8, 133.3,
129.9, 128.9, 126.1, 125.0, 112.9, 108.0, 101.9, 99.8, 37.1, 22.8 ppm;
MS (ESI−) m/z: 369.85 [M − H]−; HRMS (MALDI+) m/z: calcd
for [C19H17NO5S + H]+: 372.09002, found: 372.09008, Δ = 0.2 ppm;
HPLC tR (Method B) = 9.60 min.
N - ( 4 , 7 - Di h y d r o x y - 2 - o x o - 2 H -c h r o m e n- 3 - y l ) - 3 - ( 2 -
methylpropylsulfonamido)benzamide (8a). According to general
procedure A: 280 mg (1.45 mmol) of 15a, 410 mg (1.59 mmol) of
32a, 252 mg (1.59 mmol) of HOBt, 305 mg (1.59 mmol) of EDC ×
HCl, 20 mL of DMF, and 3 mL of triethylamine. The desired product
(65 mg, 0.15 mmol, 10%) was obtained as a colorless solid. 1H NMR
(600 MHz, DMSO-d6) δ = 11.84 (bs, 1H), 10.56 (s, 1H), 9.97 (s,
1H), 9.42 (s, 1H), 7.77−7.74 (m, 2H), 7.72 (d, J = 8.6 Hz, 1H), 7.47
(t, J = 7.9 Hz, 1H), 7.42−7.39 (m, 1H), 6.83 (dd, J = 2.1/8.6 Hz, 1H)
6.73 (d, J = 2.1 Hz, 1H), 3.02 (d, J = 6.6 Hz, 2H), 2.15 (non., J = 6.6
Hz, 1H), 0.99 (d, J = 6.6 Hz, 6H) ppm; 13C NMR (151 MHz,
DMSO-d6) δ = 166.2, 161.4, 160.7, 160.3, 153.5, 138.5, 135.3, 129.1,
125.0, 123.0, 122.4, 119.2, 112.9, 107.9, 101.9, 100.0, 58.4, 48.6, 24.3,
22.1 ppm; MS (ESI−) m/z: 431.30 [M − H]−; HRMS (MALDI+)
m/z: calcd for [C20H20N2O7S + H]+: 433.10640, found: 433.10563, Δ
= 1.8 ppm; HPLC tR (Method A) = 13.79 min.
3-(Butylsulfonamido)-N-(4,7-dihydroxy-2-oxo-2H-chromen-3-
yl)benzamide (8e). According to general procedure A: 246 mg (1.28
mmol) of 15a, 361 mg (1.40 mmol) of 32e, 221 mg (1.40 mmol) of
HOBt, 269 mg (1.40 mmol) of EDC × HCl, 20 mL of DMF, and 3
mL of triethylamine. The desired product (60 mg, 0.14 mmol, 11%)
was obtained as a colorless solid. 1H NMR (600 MHz, DMSO-d6) δ =
11.84 (bs, 1H), 10.55 (s, 1H), 9.96 (s, 1H), 9.41 (s, 1H), 7.79−7.76
(m, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.47 (t, J
= 8.0 Hz, 1H), 7.41 (dd, J = 1.2/8.0 Hz, 1H), 6.83 (dd, J = 2.2/8.8
Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 3.14−3.11 (m, 2H), 1.69−1.63 (m,
2H), 1.39−1.33 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H) ppm; 13C NMR
(151 MHz, DMSO-d6) δ = 166.2, 161.4, 160.7, 160.3, 153.5, 138.5,
135.3, 129.0, 125.0, 122.4, 119.2, 112.9, 107.9, 101.9, 100.0, 50.5,
25.1, 20.6, 13.4 ppm; MS (ESI−) m/z: 431.30 [M − H]−; HRMS
(MALDI+) m/z: calcd for [C20H20N2O7S + H]+: 433.10640, found:
433.10559, Δ = 1.9 ppm; HPLC tR (Method A) = 13.79 min.
N -( 4 , 7 -D i hy dr o x y - 2 - o x o -2 H - c h r o m e n - 3 - y l ) -3 - ( 1 -
methylethylsulfonamido)benzamide (8g). According to general
procedure A: 130 mg (0.67 mmol) of 15a, 180 mg (0.74 mmol) of
32g, 117 mg (0.74 mmol) of HOBt, 142 mg (0.74 mmol) of EDC ×
HCl, 10 mL of DMF, and 3 mL of triethylamine. The desired product
(72 mg, 0.17 mmol, 26%) was obtained as a colorless solid. 1H NMR
(300 MHz, DMSO-d6) δ = 11.92 (bs, 1H), 10.55 (s, 1H), 9.93 (s,
1H), 9.40 (s, 1H), 7.84−7.71 (m, 3H), 7.48−7.42 (m, 2H), 6.83 (dd,
J = 2.2/8.8 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 1.26 (d, J = 6.9 Hz, 6H)
ppm; 13C NMR (75 MHz, DMSO-d6) δ = 166.2, 161.5, 160.8, 160.4,
153.5, 138.8, 135.3, 129.1, 125.0, 122.9, 122.3, 119.1, 113.0, 107.9,
101.9, 100.0, 51.5, 16.1 ppm; MS (ESI−) m/z: 417.25 [M − H]−;
N - ( 4 , 7 - D i h y d r o x y - 2 - o x o - 2 H - c h r o m e n - 3 - y l ) - 3 -
(methylsulfonamido)benzamide (8b). According to general proce-
dure A: 117 mg (0.60 mmol) of 15a, 143 mg (0.66 mmol) of 32b,
105 mg (0.66 mmol) of HOBt, 127 mg (0.66 mmol) of EDC × HCl,
12 mL of DMF, and 3 mL of triethylamine. The desired product (25
mg, 0.06 mmol, 11%) was obtained as a colorless solid. 1H NMR (600
MHz, DMSO-d6) δ = 11.86 (bs, 1H), 10.55 (s, 1H), 9.91 (s, 1H),
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Article
HRMS (MALDI+) m/z: calcd for [C19H18N2O7S + H]+: 419.09074,
found: 419.09041, Δ = 0.8 ppm; HPLC tR (Method A) = 12.91 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-methoxybenza-
mide (9b). According to general procedure A: 193 mg (1.0 mmol) of
15a, 152 mg (1.0 mmol) of 24b, 168 mg (1.1 mmol) of HOBt, 211
mg (1.1 mmol) of EDC × HCl, and 5 mL of DMF. The desired
product (96 mg, 0.29 mmol, 29%) was obtained as a colorless solid.
1H NMR (400 MHz, DMSO-d6) δ = 11.81 (bs, 1H), 10.54 (s, 1H),
9.39 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.59−7.56 (m, 2H), 7.42 (t, J =
8.1 Hz, 1H), 7.14 (dd, J = 2.0/8.1 Hz, 1H), 6.83 (dd, J = 2.2/8.7 Hz,
1H), 6.73 (d, J = 2.2 Hz, 1H), 3.83 (s, 3H) ppm; 13C NMR (101
MHz, DMSO-d6) δ = 166.3, 161.4, 160.7, 160.4, 159.0, 153.5, 135.4,
129.3, 125.0, 120.3, 117.4, 113.1, 112.9, 108.0, 101.9, 100.1, 55.3
ppm; MS (ESI+) m/z: 327.80 [M + H]+; HRMS (MALDI+) m/z:
calcd for [C17H13NO6 + H]+: 328.08156, found: 328.08159, Δ = 0.1
ppm; HPLC tR (Method A) = 13.93 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-ethoxybenzamide
(9c). According to general procedure A: 193 mg (1.0 mmol) of 15a,
166 mg (1.0 mmol) of 24c, 168 mg (1.1 mmol) of HOBt, 211 mg
(1.1 mmol) of EDC × HCl, and 5 mL of DMF. The desired product
(50 mg, 0.15 mmol, 15%) was obtained as a colorless solid. 1H NMR
(400 MHz, DMSO-d6) δ = 11.87 (bs, 1H), 10.51 (s, 1H), 9.34 (s,
1H), 7.70 (d, J = 8.7 Hz, 1H), 7.57−7.54 (m, 2H), 7.40 (t, J = 8.1 Hz,
1H), 7.11 (dd, J = 2.0/8.1 Hz, 1H), 6.81 (dd, J = 2.2/8.7 Hz, 1H),
6.71 (d, J = 2.2 Hz, 1H), 4.10 (q, J = 7.0 Hz, 2H), 1.36 (t, J = 7.0 Hz,
3H) ppm; 13C NMR (101 MHz, DMSO-d6) δ = 166.2, 161.3, 160.8,
158.3, 153.5, 135.4, 129.3, 125.0, 120.1, 117.9, 113.6, 112.8, 101.9,
99.9, 63.3, 14.6 ppm; MS (ESI+) m/z: 341.85 [M + H]+; HRMS
(MALDI+) m/z: calcd for [C18H15NO6 + H]+: 342.09721, found:
342.09715, Δ = 0.2 ppm; HPLC tR (Method A) = 14.91 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-propoxybenzamide
(9d). According to general procedure A: 193 mg (1.0 mmol) of 15a,
180 mg (1.0 mmol) of 24d, 168 mg (1.1 mmol) of HOBt, 211 mg
(1.1 mmol) of EDC × HCl, and 5 mL of DMF. The desired product
(90 mg, 0.25 mmol, 25%) was obtained as a colorless solid. 1H NMR
(400 MHz, DMSO-d6) δ = 11.80 (bs, 1H), 10.54 (s, 1H), 9.38 (s,
1H), 7.72 (d, J = 8.7 Hz, 1H), 7.58−7.54 (m, 2H), 7.41 (t, J = 8.2 Hz,
1H), 7.13 (dd, J = 1.8/8.1 Hz, 1H), 6.83 (dd, J = 2.2/8.7 Hz, 1H),
6.73 (d, J = 2.2 Hz, 1H), 4.01 (t, J = 6.5 Hz, 2H), 1.76 (sext, J = 7.0
Hz, 2H), 1.00 (t, J = 7.5 Hz, 3H) ppm; 13C NMR (101 MHz, DMSO-
d6) δ = 166.3, 161.4, 160.7, 160.3, 158.5, 153.5, 135.3, 129.3, 125.0,
120.2, 117.9, 113.7, 112.9, 108.0, 101.9, 100.1, 69.2, 22.0, 10.4 ppm;
MS (ESI+) m/z: 355.85 [M + H]+; HRMS (MALDI+) m/z: calcd for
[C19H17NO6 + H]+: 356.11286, found: 356.11262, Δ = 0.7 ppm;
HPLC tR (Method A) = 15.91 min.
NMR (101 MHz, DMSO-d6) δ = 166.3, 161.4, 160.8, 160.5, 158.4,
153.5, 135.3, 129.2, 125.0, 120.1, 118.0, 113.6, 112.9, 108.1, 101.9,
100.0, 72.2, 10.1, 3.1 (2 × C) ppm; MS (ESI+) m/z: 367.80 [M +
H]+; HRMS (MALDI+) m/z: calcd for [C20H17NO6 + H]+
368.11286, found: 368.11301, Δ = 0.4 ppm; HPLC tR (Method A)
= 15.54 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-isopropoxybenza-
mide (9g). According to general procedure A: 193 mg (1.0 mmol) of
15a, 180 mg (1.0 mmol) of 24g, 168 mg (1.1 mmol) of HOBt, 211
mg (1.1 mmol) of EDC × HCl, and 5 mL of DMF. The desired
product (67 mg, 0.19 mmol, 19%) was obtained as a colorless solid.
1H NMR (400 MHz, DMSO-d6) δ = 11.84 (bs, 1H), 10.52 (s, 1H),
9.34 (s, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.54−7.52 (m, 2H), 7.39 (t, J =
8.1 Hz, 1H), 7.10 (dd, J = 1.8/8.1 Hz, 1H), 6.81 (dd, J = 2.2/8.7 Hz,
1H), 6.72 (d, J = 2.2 Hz, 1H), 4.69 (sept, J = 6.1 Hz, 1H), 1.30 (d, J =
6.1 Hz, 6H) ppm; 13C NMR (101 MHz, DMSO-d6) δ = 166.3, 161.3,
160.8, 160.7, 157.2, 153.5, 135.4, 129.3, 125.0, 120.1, 119.0, 115.0,
112.8, 108.2, 101.9, 99.9, 69.5, 21.8 (2 × C) ppm; MS (ESI+) m/z:
355.85 [M + H]+; HRMS (MALDI+) m/z: calcd for [C19H17NO6 +
H]+: 356.11286, found: 356.11282, Δ = 0.1 ppm; HPLC tR (Method
A) = 15.56 min.
3-(Allyloxy)-N-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)-
benzamide (9h). According to general procedure A: 193 mg (1.0
mmol) of 15a, 178 mg (1.0 mmol) of 24h, 168 mg (1.1 mmol) of
HOBt, 211 mg (1.1 mmol) of EDC × HCl, and 5 mL of DMF. The
desired product (49 mg, 0.14 mmol, 14%) was obtained as a colorless
1
solid. H NMR (400 MHz, DMSO-d6) δ = 11.90 (bs, 1H), 10.51 (s,
1H), 9.34 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.59−7.55 (m, 2H), 7.41
(t, J = 8.1 Hz, 1H), 7.19−7.14 (m, 1H), 6.81 (dd, J = 2.2/8.7 Hz,
1H), 6.71 (d, J = 2.2 Hz, 1H), 6.13−6.03 (m, 1H), 5.42 (dd, J = 1.5/
17.3 Hz, 1H), 5.28 (dd, J = 1.5/10.6 Hz, 1H), 4.64 (d, J = 5.2 Hz,
2H) ppm; 13C NMR (101 MHz, DMSO-d6) δ = 166.2, 161.3, 160.8,
158.0, 153.5, 135.4, 133.6, 129.3, 125.1, 120.4, 118.0, 117.5, 114.0,
112.8, 101.9, 68.3 ppm; MS (ESI+) m/z: 353.85 [M + H]+; HRMS
(MALDI+) m/z: calcd for [C19H15NO6 + H]+: 354.09721, found:
354.09699, Δ = 0.6 ppm; HPLC tR (Method A) = 15.18 min.
3-(Benzyloxy)-N-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)-
benzamide (9i). According to general procedure A: 193 mg (1.0
mmol) of 15a, 228 mg (1.0 mmol) of 24i, 168 mg (1.1 mmol) of
HOBt, 211 mg (1.1 mmol) of EDC × HCl, and 5 mL of DMF. The
desired product (87 mg, 0.22 mmol, 22%) was obtained as a colorless
1
solid. H NMR (400 MHz, DMSO-d6) δ = 11.87 (bs, 1H), 10.53 (s,
1H), 9.37 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.66 (s, 1H), 7.59 (d, J =
7.7 Hz, 1H), 7.49−7.47 (m, 2H), 7.43−7.38 (m, 3H), 7.36−7.32 (m,
1H), 7.21 (dd, J = 2.1/8.1 Hz, 1H), 6.82 (dd, J = 2.2 Hz, 8.7 Hz, 1H),
6.72 (d, J = 2.2 Hz, 1H), 5.19 (s, 2H) ppm; 13C NMR (101 MHz,
DMSO-d6) δ = 166.2, 161.3, 160.8, 160.7, 158.1, 153.5, 136.9, 135.4,
129.3, 128.5, 127.9, 127.6, 125.0, 120.5, 118.1, 114.2, 112.8, 108.2,
101.9, 99.9, 69.4 ppm; MS (ESI+) m/z: 403.90 [M + H]+; HRMS
(MALDI+) m/z: calcd for [C23H17NO6 + H]+: 404.11286, found:
404.11198, Δ = 2.2 ppm; HPLC tR (Method A) = 16.11 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-phenoxybenza-
mide (9j). According to general procedure A: 232 mg (1.2 mmol) of
15a, 257 mg (1.2 mmol) of 3-phenoxybenzoic acid (24j), 235 mg
(1.3 mmol) of HOBt, 253 mg (1.3 mmol) of EDC × HCl, and 5 mL
of DMF. The desired product (300 mg, 0.77 mmol, 64%) was
3-Butoxy-N-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)benzamide
(9e). According to general procedure A: 193 mg (1.0 mmol) of 15a,
194 mg (1.0 mmol) of 24e, 168 mg (1.1 mmol) of HOBt, 211 mg
(1.1 mmol) of EDC × HCl, and 5 mL of DMF. The desired product
(82 mg, 0.22 mmol, 22%) was obtained as a colorless solid. 1H NMR
(400 MHz, DMSO-d6) δ = 11.82 (bs, 1H), 10.54 (s, 1H), 9.37 (s,
1H), 7.72 (d, J = 8.7 Hz, 1H), 7.57−7.54 (m, 2H), 7.40 (t, J = 8.1 Hz,
1H), 7.13 (dd, J = 1.6/8.1 Hz, 1H), 6.83 (dd, J = 2.2/8.7 Hz, 1H),
6.73 (d, J = 2.2 Hz, 1H), 4.04 (t, J = 6.5 Hz, 2H), 1.77−169 (m, 2H),
1.51−1.42 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H) ppm; 13C NMR (101
MHz, DMSO-d6) δ = 166.3, 161.4, 160.8, 160.4, 158.5, 153.5, 135.3,
129.3, 125.0, 120.7, 120.2, 118.1, 117.9, 113.7, 112.9, 108.0, 101.9,
100.0, 67.4, 30.7, 18.7, 13.7 ppm; MS (ESI+) m/z: 369.90 [M + H]+;
HRMS (MALDI+) m/z: calcd for [C20H19NO6 + H]+: 370.12851,
found: 370.12870, Δ = 0.5 ppm; HPLC tR (Method A) = 16.71 min.
3-(Cyclopropylmethoxy)-N-(4,7-dihydroxy-2-oxo-2H-chromen-3-
yl)benzamide (9f). According to general procedure A: 193 mg (1.0
mmol) of 15a, 192 mg (1.0 mmol) of 24f, 168 mg (1.1 mmol) of
HOBt, 211 mg (1.1 mmol) of EDC × HCl, and 5 mL of DMF. The
desired product (94 mg, 0.26 mmol, 26%) was obtained as a colorless
1
obtained as a colorless solid. H NMR (400 MHz, DMSO-d6) δ =
11.82 (bs, 1H), 10.53 (s, 1H), 9.42 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H),
7.70 (d, J = 8.7 Hz, 1H), 7.62 (s, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.43
(t, J = 7.8 Hz, 2H), 7.24 (dd, J = 2.0/8.1 Hz, 1H), 7.18 (t, J = 7.4 Hz,
1H), 7.05 (d, J = 8.1 Hz, 2H), 6.83 (dd, J = 1.9/8.7 Hz, 1H), 6.71 (d,
J = 1.9 Hz, 1H) ppm; 13C NMR (126 MHz, DMSO-d6) δ = 166.1,
161.6, 161.0, 160.7, 156.7, 156.6, 153.7, 136.1, 130.4, 130.2, 125.2,
124.0, 123.2, 119.0, 118.2, 113.2, 108.0, 102.1, 100.0 ppm; MS (ESI
+) m/z: 390.11 [M + H]+; HRMS (MALDI+) m/z: calcd for
[C22H15NO6 + H]+: 390.09721, found: 390.09650, Δ = 1.8 ppm;
HPLC tR (Method A) = 9.24 min.
1
solid. H NMR (400 MHz, DMSO-d6) δ = 11.86 (bs, 1H), 10.53 (s,
1H), 9.35 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.57−7.53 (m, 2H), 7.39
(t, J = 8.1 Hz, 1H), 7.12 (dd, J = 1.9/8.1 Hz, 1H), 6.82 (dd, J = 2.2/
8.7 Hz, 1H), 6.72 (d, J = 2.2 Hz, 1H), 3.89 (d, J = 7.0 Hz, 2H), 1.31−
1.21 (m, 1H), 0.61−0.55 (m, 2H), 0.36−0.32 (m, 2H) ppm; 13C
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-4-fluoro-3-isopentyl-
benzamide (10). According to general procedure A: 386 mg (2.0
mmol) of 15a, 463 mg (2.2 mmol) of 44, 422 mg (2.2 mmol) of
3732
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J. Med. Chem. 2021, 64, 3720−3746

Journal of Medicinal Chemistry
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Article
HOBt, 347 mg (2.2 mmol) of EDC × HCl, 10 mL of DMF, and 0.6
mL of triethylamine. The desired product (146 mg, 0.38 mmol, 19%)
was obtained as a colorless solid. 1H NMR (400 MHz, DMSO-d6) δ =
11.79 (bs, 1H), 10.57 (s, 1H), 9.39 (s, 1H), 7.95 (dd, J = 2.1/7.4 Hz,
1H), 7.89−7.85 (m, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.30−7.25 (m,
1H), 6.83 (dd, J = 2.3/8.7 Hz, 1H), 6.73 (d, J = 2.3 Hz), 2.69−2.65
(m, 2H), 1.62 (m, 3H), 0.94 (d, J = 6.4 Hz, 6H) ppm; 13C NMR (101
MHz, DMSO-d6) δ = 165.7, 161.5, 160.8, 160.4, 153.5, 130.9, 130.3,
129.0, 128.0, 125.1, 115.1, 114.9, 113.0, 107.9, 101.9, 100.0, 40.2,
27.3, 26.2, 22.3 (2 × C) ppm; MS (ESI−) m/z: 384.16 [M − H]−;
HRMS (MALDI+) m/z: calcd for [C21H20FNO5 + H]+: 386.13983,
found: 386.13985, Δ = 0.1 ppm; HPLC: tR (Method A) = 18.08 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-3-isopentyl-4-me-
thoxybenzamide (11). According to general procedure A: 155 mg
(0.80 mmol) of 15a, 213 mg (0.96 mmol) of 53, 152 mg (0.96 mmol)
of HOBt, 184 mg (0.96 mmol) of EDC × HCl, 5 mL of DMF, and
0.3 mL of triethylamine. The desired product (25 mg, 0.06 mmol,
101.9, 100.7, 78.5, 49.7, 28.2 (3 × C), 18.1 ppm; MS (ESI−) m/z:
363.25 [M
−
H]−; HRMS (MALDI+) m/z: calcd for
[C17H20N2O7+Na]+: 387.11627, found: 387.11592, Δ = 0.9 ppm;
HPLC: tR (Method A) = 13.38 min.
(S)-tert-Butyl (1-((4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-
amino)-1-oxopropan-2-yl)carbamate (13c). According to general
procedure A: 193 mg (1.0 mmol) of 15a, 196 mg (0.9 mmol) of Boc-
L-Val (56c), 138 mg (0.9 mmol) of HOBt, 173 mg (0.9 mmol) of
EDC × HCl, and 5 mL of DMF. The desired product (128 mg, 0.33
1
mmol, 36%) was obtained as a colorless solid. H NMR (300 MHz,
DMSO-d6) δ = 12.03 (bs, 1H), 10.57 (s, 1H), 9.35 (s, 1H), 7.70 (d, J
= 8.7 Hz, 1H), 6.87−6.80 (m, 2H), 6.71 (d, J = 2.2 Hz, 1H), 4.20−
4.15 (m, 1H), 2.18−2.03 (m, 1H), 1.40 (s, 9H), 0.92 (d, J = 6.7 Hz,
3H), 0.88 (d, J = 6.7 Hz, 3H) ppm; 13C NMR (75 MHz, DMSO-d6) δ
= 172.8, 161.4, 160.2, 157.9, 155.7, 153.1, 125.1, 113.2, 108.0, 101.9,
100.6, 78.4, 59.3, 30.6, 28.2 (3 × C), 19.2, 17.8 ppm; MS (ESI−) m/
z: 391.95 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C19H24N2O7+Na]+: 415.14757, found: 415.14737, Δ = 0.5 ppm;
HPLC: tR (Method A) = 14.66 min.
(R)-tert-Butyl (1-((4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-
amino)-4-methyl-1-oxopentan-2-yl)-carbamate (13d). According
to general procedure A: 193 mg (1.0 mmol) of 15a, 208 mg (0.9
mmol) of Boc-D-Leu (56d), 138 mg (0.9 mmol) of HOBt, 173 mg
(0.9 mmol) of EDC × HCl, and 5 mL of DMF. The desired product
(87 mg, 0.21 mmol, 24%) was obtained as a colorless solid. 1H NMR
(250 MHz, DMSO-d6) δ = 12.13 (bs, 1H), 10.57 (s, 1H), 9.35 (s,
1H), 7.70 (d, J = 8.7 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 6.83 (dd, J =
2.2/8.7 Hz, 1H), 6.72 (d, J = 2.2 Hz, 1H), 4.29 (q, J = 7.6 Hz, 1H),
1.68 (sept, J = 6.4 Hz, 1H), 1.54 (t, J = 7.0 Hz), 1.40 (s, 9H), 0.90
(dd, J = 1.3/6.4 Hz, 6H) ppm; 13C NMR (101 MHz, DMSO-d6) δ =
174.1, 161.4, 160.2, 157.1, 155.6, 152.9, 125.0, 113.2, 108.0, 101.9,
100.9, 78.5, 52.7, 40.6, 28.2 (3 × C), 24.3, 23.2, 21.4 ppm; MS
(ESI−) m/z: 405.30 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C20H26N2O7+Na]+: 429.16322, found: 429.16269, Δ = 1.2 ppm;
HPLC: tR (Method A) = 15.49 min.
1
8%) was obtained as a colorless solid. H NMR (500 MHz, DMSO-
d6) δ = 11.85 (bs, 1H), 10.55 (s, 1H), 9.27 (s, 1H), 7.86 (dd, J = 2.2/
8.6 Hz, 1H), 7.81 (d, J = 2.2 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.05
(d, J = 8.6 Hz, 1H), 6.83 (dd, J = 2.3/8.7 Hz, 1H), 6.73 (d, J = 2.3 Hz,
1H), 3.86 (s, 3H), 2.62−2.58 (m, 2H), 1.57 (non, J = 6.7 Hz, 1H),
1.47−1.42 (m, 2H), 0.93 (d, J = 6.7 Hz, 6H) ppm; 13C NMR (126
MHz, DMSO-d6) δ = 166.4, 161.4, 160.9, 160.0, 159.8, 153.4, 130.0,
129.4, 127.7, 125.6, 125.0, 113.0, 110.0, 108.1, 101.9, 100.5, 55.7,
38.7, 27.5, 27.4, 22.5 (2 × C) ppm; MS (ESI−) m/z: 395.95 [M −
H]−; HRMS (MALDI+) m/z: calcd for [C22H23NO6 + H]+:
398.15981, found: 398.15979, Δ = 0.05 ppm; HPLC: tR (Method
A) = 18.40 min.
N-(4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-2-isopentylbenza-
mide (12). According to general procedure A: 115 mg (0.59 mmol) of
15a, 137 mg (0.71 mmol) of 40, 113 mg (0.71 mmol) of HOBt, 137
mg (0.71 mmol) of EDC × HCl, 8 mL of DMF, and 3 mL of
triethylamine. The desired product (33 mg, 0.09 mmol, 15%) was
1
obtained as a colorless solid. H NMR (500 MHz, DMSO-d6) δ =
(S)-tert-Butyl (1-((4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-
amino)-4-methyl-1-oxopentan-2-yl)-carbamate (13e). According
to general procedure A: 193 mg (1.0 mmol) of 15a, 224 mg (0.9
mmol) of Boc-L-Leu × H2O (56e), 138 mg (0.9 mmol) of HOBt, 173
mg (0.9 mmol) of EDC × HCl, of 5 mL of DMF. The desired
product (97 mg, 0.24 mmol, 27%) was obtained as a colorless solid.
1H NMR (300 MHz, DMSO-d6) δ = 12.13 (bs, 1H), 10.57 (s, 1H),
9.35 (s, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 7.4 Hz, 1H), 6.82
(dd, J = 2.2/8.7 Hz, 1H), 6.72 (d, J = 2.2 Hz, 1H), 4.29 (q, J = 7.4 Hz,
1H)), 1.74−1.51 (m, 3H), 1.40 (s, 9H), 0.91−0.89 (m, 6H) ppm; 13C
NMR (75 MHz, DMSO-d6) δ = 174.1, 161.4, 160.2, 157.1, 155.6,
152.9, 125.0, 113.2, 108.0, 101.9, 100.8, 78.5, 52.7, 40.6, 28.2 (3 × C),
24.3, 23.2, 21.4 ppm; MS (ESI−) m/z: 405.30 [M − H]−; HRMS
(MALDI+) m/z: calcd for [C20H26N2O7+Na]+: 429.16322, found:
429.16256, Δ = 1.5 ppm; HPLC: tR (Method A) = 15.49 min.
tert-Butyl ((2S,3S)-1-((4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-
amino)-3-methyl-1-oxopentan-2-yl)-carbamate (13f). According
to general procedure A: 193 mg (1.0 mmol) of 15a, 212 mg (0.9
mmol) of Boc-L-Ile × 1/2H2O (56f), 138 mg (0.9 mmol) of HOBt,
173 mg (0.9 mmol) of EDC × HCl, and 5 mL of DMF. The desired
product (62 mg, 0.15 mmol, 17%) was obtained as a colorless solid.
1H NMR (300 MHz, DMSO-d6) δ = 12.03 (bs, 1H), 10.57 (s, 1H),
9.41 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.82
(dd, J = 2.2/8.7 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 4.19 (t, J = 7.6 Hz,
1H), 1.89−1.74 (m, 1H), 1.54−1.33 (m, 10H), 1.25−1.09 (m, 1H),
0.90 (d, J = 6.8 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H) ppm; 13C NMR (75
MHz, DMSO-d6) δ = 173.0, 161.4, 160.2, 157.6, 155.6, 153.0, 125.0,
113.2, 108.0, 101.9, 100.8, 78.4, 58.6, 36.7, 28.2 (3 × C), 24.3, 15.3,
11.1 ppm; MS (ESI−) m/z: 405.35 [M − H]−; HRMS (MALDI+)
m/z: calcd for [C20H26N2O7+Na]+: 429.16322, found: 429.16255, Δ
= 1.6 ppm; HPLC: tR (Method A) = 15.35 min.
(S)-tert-Butyl (1-((4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-
amino)-1-oxohexan-2-yl)carbamate (13g). According to general
procedure A: 193 mg (1.0 mmol) of 15a, 208 mg (0.9 mmol) of Boc-
L-norleucine (56g), 138 mg (0.9 mmol) of HOBt, 173 mg (0.9
11.86 (bs, 1H), 10.56 (s, 1H), 9.28 (s, 1H), 7.75 (d, J = 8.7 Hz, 1H),
7.73−7.71 (m, 1H), 7.40−7.37 (m, 1H), 7.29−7.26 (m, 2H), 6.83
(dd, J = 2.3/8.7 Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H), 2.81−2.77 (m,
2H), 1.56−1.51 (m, 1H), 1.50−1.44 (m, 2H), 0.88 (d, J = 6.5 Hz,
6H) ppm; 13C NMR (126 MHz, DMSO-d6) δ = 170.2, 161.9, 161.0,
160.3, 153.9, 141.9, 136.2, 130.2, 130.0, 128.8, 125.6, 125.5, 113.4,
108.5, 102.4, 100.9, 41.0, 31.1, 28.2, 22.9 (2 × C) ppm; MS (ESI−)
m/z: 366.00 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C21H21NO5 + H]+: 368.14925, found: 368.14951, Δ = 0.7 ppm;
HPLC: tR (Method A) = 17.19 min.
tert-Butyl (2-((4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-2-
oxoethyl)carbamate (13a). According to general procedure A: 193
mg (1.0 mmol) of 15a, 161 mg (0.9 mmol) of Boc-Gly (56a), 138 mg
(0.9 mmol) of HOBt, 173 mg (0.9 mmol) of EDC × HCl, and 5 mL
of DMF. The desired product (118 mg, 0.34 mmol, 37%) was
1
obtained as a colorless solid. H NMR (300 MHz, DMSO-d6) δ =
12.02 (bs, 1H), 10.56 (s, 1H), 9.17 (s, 1H), 7.69 (d, J = 8.7 Hz, 1H),
7.02 (t, J = 5.8 Hz, 1H), 6.82 (dd, J = 2.2/8.7 Hz, 1H). 6.70 (d, J =
2.2 Hz. 1H), 3.84 (d, J = 5.8 Hz, 2H), 1.39 (s, 9H) ppm; 13C NMR
(75 MHz, DMSO-d6) δ = 170.6, 161.4, 160.5, 158.3, 155.8, 153.1,
125.1, 113.1, 108.0, 101.9, 100.1, 78.2, 43.2, 28.2 (3 × C) ppm; MS
(ESI−) m/z: 349,20 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C16H18N2O7+Na]+: 373.10062, found: 373.10061, Δ = 0.03 ppm;
HPLC: tR (Method A) = 12.67 min.
(S)-tert-Butyl (1-((4,7-Dihydroxy-2-oxo-2H-chromen-3-yl)-
amino)-1-oxopropan-2-yl)carbamate (13b). According to general
procedure A: 193 mg (1.0 mmol) of 15a, 170 mg (0.9 mmol) of Boc-
L-Ala (56b), 138 mg (0.9 mmol) of HOBt, 173 mg (0.9 mmol) of
EDC × HCl, and 5 mL of DMF. The desired product (115 mg, 0.32
1
mmol, 35%) was obtained as a colorless solid. H NMR (300 MHz,
DMSO-d6) δ = 12.14 (bs, 1H), 10.57 (s, 1H), 9.31 (s, 1H), 7.70 (d, J
= 8.7 Hz, 1H), 7.14 (d, J = 7.1 Hz, 1H), 6.82 (dd, J = 2.2/8.7 Hz,
1H), 6.72 (d, J = 2.2 Hz, 1H), 4.30 (quint, J = 7.1 Hz, 1H), 1.40 (s,
9H), 1.28 (d, J = 7.1 Hz, 3H) ppm; 13C NMR (101 MHz, DMSO-d6)
δ = 174.1, 161.4, 160.2, 157.3, 155.2, 152.9, 125.1, 113.2, 108.0,
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Article
mmol) of EDC × HCl, and 5 mL of DMF. The desired product (116
mg, 0.29 mmol, 32%) was obtained as a colorless solid. 1H NMR (300
MHz, DMSO-d6) δ = 12.12 (bs, 1H), 10.57 (s, 1H), 9.36 (s, 1H),
7.70 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H), 6.82 (dd, J = 2.2/
8.7 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 4.29−4.19 (m, 1H), 1.78−1.51
(m, 2H), 1.46−1.23 (m, 13H), 0.87 (t, J = 6.5 Hz, 3H) ppm; 13C
NMR (75 MHz, DMSO-d6) δ = 173.8, 161.4, 160.2, 157.2, 155.5,
152.9, 125.0, 113.2, 108.0, 101.9, 100.8, 78.4, 54.2, 31.6, 28.2 (3 × C),
27.5, 21.9, 13.9 ppm; MS (ESI−) m/z: 405.35 [M − H]−; HRMS
(MALDI+) m/z: calcd for [C20H26N2O7+Na]+: 429.16322, found:
429.16268, Δ = 1.3 ppm; HPLC: tR (Method A) = 15.54 min.
N-(4-Hydroxy-7-nitro-2-oxo-2H-chromen-3-yl)-3-isopentylben-
zamide (54). According to general procedure A: 1.20 g (4.65 mmol)
of 15c × HCl, 0.98 g (5.12 mmol) of 36a, 0.98 (5.12 mmol) of EDC
× HCl, 0.78 g (5.12 mmol) of HOBt × H2O, 2.0 mL of triethylamine,
and 15 mL of DMF. The desired product (1.60 g, 4.04 mmol, 87%)
33.8, 27.9, 22.6 (2 × C) ppm; MS (ESI−) m/z: 502.05 [M − H]−;
HRMS (MALDI+) m/z: calcd for [C33H29NO4+Na]+: 526.19888,
found: 526.19886, Δ = 0.04 ppm; HPLC: tR (Method C) = 16.60
min.
4-(4-Hydroxy-3-(3-isopentylbenzamido)-2-oxo-2H-chromen-7-
yl)benzoic Acid (14c). According to general procedure B: 198 mg
(0.46 mmol) of 55, 95 mg (0.55 mmol) of 4-boronobenzoic acid, 293
mg (2.76 mmol) of NaHCO3, 54 mg (0.05 mmol) of Pd(PPh3)4, 5
mL of DMF, and 1.4 mL of H2O. The desired product (100 mg, 0.21
1
mmol, 46%) was obtained as a colorless solid. H NMR (500 MHz,
DMSO-d6) δ = 13.10 (bs, 1H), 12.20 (bs, 1H), 9.55 (s, 1H), 8.07−
8.06 (m, 2H), 8.01 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.87
(s, 1H), 7.85−7.79 (m, 3H), 7.43 (d, J = 4.7 Hz, 2H), 2.69−2.65 (m,
2H), 1.60−1.49 (m, 3H), 0.94 (d, J = 6.4 Hz, 6H) ppm; 13C NMR
(126 MHz, DMSO-d6) δ = 167.0, 166.3, 160.4, 159.2, 152.1, 142.7,
142.6, 142.4, 133.8, 131.7, 130.6, 130.1 (2 × C), 128.2, 127.9, 127.3
(2 × C), 125.5, 124.4, 123.0, 115.9, 114.4, 103.3, 40.6, 33.0, 27.1, 22.4
(2 × C) ppm; MS (ESI−) m/z: 470.05 [M − H]−; HRMS (MALDI
+) m/z: calcd for [C28H25NO6 + H]+: 494.15741, found: 494.15890,
Δ = 3.0 ppm; HPLC: tR (Method C) = 12.29 min.
1
was obtained as an orange solid. H NMR (500 MHz, CDCl3) δ =
14.11 (s, 1H), 8.90 (s, 1H), 8.22 (s, 3H), 7.78−7.74 (m, 2H), 7.50−
7.45 (m, 2H), 2.72 (t, J = 8.1 Hz, 2H), 1.67−1.53 (m, 3H), 0.97 (d, J
= 6.5 Hz, 6H) ppm; MS (ESI−) m/z: 395.25 [M − H]−.
N-(7-Bromo-4-hydroxy-2-oxo-2H-chromen-3-yl)-3-isopentylben-
zamide (55). According to general procedure A: 2.08 g (8.14 mmol)
of 15d × HCl, 1.30 g (6.78 mmol) of 36a, 1.56 g (8.14 mmol) of
EDC × HCl, 1.29 g (8.14 mmol) of HOBt, 2.9 mL of triethylamine,
and 20 mL of DMF. The desired product (1.81 g, 4.20 mmol, 62%)
was obtained as a light brown solid. 1H NMR (300 MHz, DMSO-d6)
δ = 14.00 (s, 1H), 8.84 (s, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.76−7.73
(m, 2H), 7.53 (d, J = 1.6 Hz, 1H), 7.49 (dd, J = 1.8/8.5 Hz, 1H),
7.46−7.41 (m, 2H), 2.74−2.68 (m, 2H), 1.69−1.51 (m, 3H), 0.96 (d,
J = 6.3 Hz, 6H) ppm; MS (ESI−) m/z: 428.24 [M − H]−.
General Procedure for Suzuki Coupling (Procedure B). 55
(1.0 equiv), the corresponding boronic acid or boronic ester (1.2
equiv), and Pd(PPh3)4 (0.1 equiv) were combined under argon
atmosphere. Then DMF and a 2 M aqueous Na2CO3 solution (6.0
equiv) were added. The reaction mixture was stirred for 12h at 80 °C.
The reaction was quenched by the addition of 2 M aqueous HCl, and
the aqueous phase was extracted three times with EtOAc. The
combined organic phases were washed with brine, dried over
anhydrous MgSO4, filtered, and evaporated under reduced pressure.
The crude product was purified by preparative HPLC.
N-(4-Hydroxy-7-(naphthalen-1-yl)-2-oxo-2H-chromen-3-yl)-3-
isopentylbenzamide (14a). According to general procedure B: 198
mg (0.46 mmol) of 55, 95 mg (0.55 mmol) of naphthalen-1-ylboronic
acid, 293 mg (2.76 mmol) of NaHCO3, 54 mg (0.05 mmol) of
Pd(PPh3)4, 5 mL of DMF, and 1.4 mL of H2O. The desired product
(167 mg, 0.35 mmol, 76%) was obtained as a colorless solid. 1H NMR
(500 MHz, CDCl3) δ = 13.90 (s, 1H), 8.84 (s, 1H), 8.05 (d, J = 8.1
Hz, 1H), 7.83 (t, J = 8.2 Hz, 2H), 7.79 (d, J = 8.6 Hz, 1H), 7.70−7.67
(m, 2H), 7.48−7.34 (m, 8H), 2.64−2.61 (m, 2H), 1.54 (non, J = 6.6
Hz, 1H), 1.49−1.44 (m, 2H), 0.87 (d, J = 6.5 Hz, 6H) ppm; 13C
NMR (126 MHz, CDCl3) δ = 167.9, 161.5, 153.0, 150.6, 145.0,
144.6, 138.3, 133.9, 133.6, 131.6, 131.2, 129.2, 128.8, 128.6, 127.7,
127.3, 127.0, 126.7, 126.3, 125.5, 124.9, 124.5, 117.7, 116.2, 104.9,
40.8, 33.8, 27.9, 22.6 (2 × C) ppm; MS (ESI−) m/z: 476.05 [M −
H]−; HRMS (MALDI+) m/z: calcd for [C31H27NO4+Na]+:
500.18323, found: 500.18327, Δ = 0.08 ppm; HPLC: tR (Method
C) = 17.19 min.
N-(7-([1,1′-Biphenyl]-2-yl)-4-hydroxy-2-oxo-2H-chromen-3-yl)-3-
isopentylbenzamide (14b). According to general procedure B: 198
mg (0.46 mmol) of 55, 109 mg (0.55 mmol) of [1,1′-biphenyl]-2-
ylboronic acid, 293 mg (2.76 mmol) of NaHCO3, 54 mg (0.05 mmol)
of Pd(PPh3)4, 5 mL of DMF, and 1.4 mL of H2O. The desired
product (175 mg, 0.35 mmol, 76%) was obtained as a colorless solid.
1H NMR (500 MHz, CDCl3) δ = 13.85 (s, 1H), 8.87 (s, 1H), 7.85 (d,
3-(4-Hydroxy-3-(3-isopentylbenzamido)-2-oxo-2H-chromen-7-
yl)benzoic Acid (14d). According to general procedure B: 198 mg
(0.46 mmol) of 55, 92 mg (0.55 mmol) of 3-boronobenzoic acid, 293
mg (2.76 mmol) of NaHCO3, 54 mg (0.05 mmol) of Pd(PPh3)4, 5
mL of DMF, and 1.4 mL of H2O. The desired product (105 mg, 0.22
1
mmol, 48%) was obtained as a colorless solid. H NMR (500 MHz,
DMSO-d6) δ = 13.19 (bs, 1H), 12.19 (bs, 1H), 9.55 (s, 1H), 8.30 (s,
1H), 8.08 (d, J = 8.3 Hz, 1H), 8.02−8.00 (m, 2H), 7.87 (s, 1H),
7.84−7.81 (m, 2H), 7.78 (dd, J = 1.6/8.3 Hz, 1H), 7.67 (t, J = 7.8 Hz,
1H), 7.64−7.60 (m, 1H), 7.50−7.46 (m, 1H), 7.42 (d, J = 4.9 Hz,
2H), 2.68−2.65 (m, 2H), 1.62−1.50 (m, 3H), 0.94 (d, J = 6.3 Hz,
6H) ppm; 13C NMR (126 MHz, DMSO-d6) δ = 167.1, 166.6, 160.5,
159.3, 152.1, 143.0, 142.6, 138.7, 134.7, 134.6, 133.8, 131.7, 131.6,
130.7, 129.6, 129.3, 128.2, 127.9, 127.7, 125.5, 124.5, 122.9, 115.6,
114.2, 103.2, 40.4, 33.0, 27.1, 22.4 (2 × C) ppm; MS (ESI−) m/z:
470.00 [M − H]−; HRMS (MALDI+) m/z: calcd for [C28H25NO6 +
H]+: 494.15741, found: 494.15812, Δ = 1.4 ppm; HPLC: tR (Method
C) = 12.11 and 12.23 min.
N-(7-(Benzofuran-3-yl)-4-hydroxy-2-oxo-2H-chromen-3-yl)-3-
isopentylbenzamide (14e). According to general procedure B: 198
mg (0.46 mmol) of 55, 89 mg (0.55 mmol) of benzofuran-3-ylboronic
acid, 293 mg (2.76 mmol) of NaHCO3, 54 mg (0.05 mmol) of
Pd(PPh3)4, 5 mL of DMF, and 1.4 mL of H2O. The desired product
1
(140 mg, 0.30 mmol, 65%) was obtained as a pale-yellow solid. H
NMR (500 MHz, CDCl3) δ = 13.98 (s, 1H), 8.89 (s, 1H), 8.10 (d, J =
8.6 Hz, 1H), 7.92 (s, 1H), 7.89−7.86 (m, 1H), 7.77−7.74 (m, 2H),
7.65−7.63 (m, 2H), 7.58−7.56 (m, 1H), 7.46−7.36 (m, 4H), 2.73−
2.70 (m, 2H), 1.63 (non, J = 6.6 Hz, 1H), 1.58−1.53 (m, 2H), 0.97
(d, J = 6.5 Hz, 6H) ppm; 13C NMR (126 MHz, CDCl3) δ = 167.8,
161.3, 156.1, 152.8, 151.1, 144.6, 142.6, 136.2, 133.6, 131.5, 129.2,
127.7, 125.7, 125.2, 125.2, 124.9, 123.9, 123.7, 121.0, 120.3, 116.2,
114.5, 112.2, 104.8, 40.8, 33.8, 27.9, 22.6 (2 × C) ppm; MS (ESI−)
m/z: 466.00 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C29H25NO5+Na]+: 490.16249, found: 490.16242, Δ = 0.1 ppm;
HPLC: tR (Method C) = 15.40 min.
N-(7-(1-Benzyl-1H-pyrazol-5-yl)-4-hydroxy-2-oxo-2H-chromen-
3-yl)-3-isopentylbenzamide (14f). According to general procedure B:
198 mg (0.46 mmol) of 55, 157 mg (0.55 mmol) of 1-benzyl-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, 293 mg
(2.76 mmol) of NaHCO3, 54 mg (0.05 mmol) of Pd(PPh3)4, 5 mL
of DMF, and 1.4 mL of H2O. The desired product (130 mg, 0.26
mmol, 57%) was obtained as a pale-yellow solid. 1H NMR (500 MHz,
CDCl3) δ = 13.90 (s, 1H), 8.81 (s, 1H), 7.96 (d, J = 8.8 Hz, 1H),
7.70−7.67 (m, 2H), 7.59 (d, J = 2.0 Hz, 1H), 7.40−7.36 (m, 2H),
7.27−7.18 (m, 5H), 6.98 (d, J = 6.9 Hz, 2H), 6.39 (d, J = 1.9 Hz,
1H), 5.35 (s, 2H), 2.66−2.62 (m, 2H), 1.55 (non, J = 6.6 Hz, 1H),
1.50−1.45 (m, 2H), 0.89 (d, J = 6.4 Hz, 6H) ppm; 13C NMR (126
MHz, CDCl3) δ = 168.0, 161.1, 152.4, 150.4, 144.6, 142.4, 139.6,
137.1, 134.1, 133.7, 131.4, 129.2, 129.0 (2 × C), 127.9, 127.7, 126.8
(2 × C), 125.3, 125.1, 124.9, 117.2, 116.3, 107.5, 105.3, 53.7, 40.8,
J = 8.2 Hz, 1H), 7.76−7.73 (m, 2H), 7.48−7.41 (m, 6H), 7.24−7.21
(m, 3H), 7.17−7.11 (m, 4H), 2.72−2.69 (m, 2H), 1.66−1.52 (m,
3H), 0.96 (d, J = 6.5 Hz, 6H) ppm; 13C NMR (126 MHz, CDCl3) δ =
167.8, 161.5, 153.0, 150.3, 145.9, 144.6, 140.9, 140.9, 138.8, 133.5,
131.6, 131.0, 130.6, 129.9 (2 × C), 129.1, 128.6, 128.3 (2 × C),
127.9, 127.7, 127.1, 126.8, 124.9, 124.0, 117.5, 115.5, 104.8, 40.8,
3734
https://doi.org/10.1021/acs.jmedchem.0c01564
J. Med. Chem. 2021, 64, 3720−3746

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
33.8, 27.9, 22.6 (2 × C) ppm; MS (ESI−) m/z: 506.20 [M − H]−;
HRMS (MALDI+) m/z: calcd for [C31H29N3O4 + H]+: 508.22308,
found: 508.22289, Δ = 0.4 ppm; HPLC: tR (Method C) = 12.77 min.
N-(7-(1-Ethyl-1H-pyrazol-5-yl)-4-hydroxy-2-oxo-2H-chromen-3-
yl)-3-isopentylbenzamide (14g). According to general procedure B:
198 mg (0.46 mmol) of 55, 123 mg (0.55 mmol) of 1-ethyl-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, 293 mg
(2.76 mmol) of NaHCO3, 54 mg (0.05 mmol) of Pd(PPh3)4, 5 mL
of DMF, and 1.4 mL of H2O. The desired product (63 mg, 0.14
mmol, 30%) was obtained as a pale-yellow solid. 1H NMR (500 MHz,
CDCl3) δ = 13.91 (s, 1H), 8.80 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H),
7.68−7.65 (m, 2H), 7.49 (d, J = 1.9 Hz, 1H), 7.38−7.33 (m, 2H),
7.31 (dd, J = 1.6/8.1 Hz, 1H), 7.29 (d, J = 1.6 Hz, 1H), 6.28 (d, J =
1.9 Hz, 1H), 4.14 (q, J = 7.3 Hz, 2H), 2.63−2.59 (m, 2H), 1.52 (non,
J = 6.6 Hz, 1H), 1.47−1.42 (m, 2H), 1.37 (t, J = 7.3 Hz, 3H), 0.86 (d,
J = 6.5 Hz, 6H) ppm; 13C NMR (126 MHz, CDCl3) δ = 168.0, 161.2,
152.5, 150.5, 144.6, 141.4, 139.1, 134.4, 133.7, 129.2, 127.7, 125.3,
125.1, 124.9, 117.1, 116.2, 107.0, 105.3, 45.0, 40.8, 33.8, 27.9, 22.6 (2
× C), 15.9 ppm; MS (ESI−) m/z: 444.00 [M − H]−; HRMS
(MALDI+) m/z: calcd for [C26H27N3O4 + H]+: 446.20743, found:
446.20745, Δ = 0.04 ppm; HPLC: tR (Method C) = 12.31 min.
N-(4-Hydroxy-7-(1-methyl-1H-pyrazol-5-yl)-2-oxo-2H-chromen-
3-yl)-3-isopentylbenzamide (14h). According to general procedure
B: 198 mg (0.46 mmol) of 55, 157 mg (0.55 mmol) of 1-methyl-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol, 293 mg
(2.76 mmol) of NaHCO3, 54 mg (0.05 mmol) of Pd(PPh3)4, 5 mL
of DMF, and 1.4 mL of H2O. The desired product (110 mg, 0.25
12.00 (bs, 1H), 10.45 (s, 1H), 9.45 (s, 1H), 7.86−7.80 (m, 3H),
7.43−7.40 (m, 2H), 7.37−7.32 (m, 3H), 7.30−7.26 (m, 2H), 7.17−
7.13 (m, 2H), 2.70−2.64 (m, 2H), 1.62−1.47 (m, 3H), 0.93 (d, J =
6.2 Hz, 6H) ppm; 13C NMR (75 MHz, DMSO-d6) δ = 166.7, 160.5,
159.7, 152.5, 142.6, 142.4, 133.8, 131.6, 131.0 (2 × C), 129.2, 128.4
(2 × C), 128.4, 128.1, 127.8, 125.4, 124.9, 114.2, 111.0, 104.2, 101.5,
52.3, 40.4, 32.9, 27.1, 22.4 (2 × C) ppm; MS (ESI−) m/z: 519.00 [M
− H]−; HRMS (MALDI+) m/z: calcd for [C28H28N2O6S + H]+:
521.17408, found: 521.17214, Δ = 3.7 ppm; HPLC: tR (Method A) =
19.32 min.
N-(4-Hydroxy-7-(2-methylpropylsulfonamido)-2-oxo-2H-chro-
men-3-yl)-3-isopentylbenzamide (14k). According to general
procedure C: 150 mg (0.41 mmol) of 57, 66 μL (0.49 mmol) of 2-
methylpropane-1-sulfonyl chloride, and 66 μL (0.81 mmol) of
pyridine in 5 mL of dry DCM. The desired product (56 mg, 0.12
1
mmol, 28%) was obtained as a colorless solid. H NMR (300 MHz,
DMSO-d6) δ = 12.06 (bs, 1H), 10.45 (s, 1H), 9.44 (s, 1H), 7.86−
7.80 (m, 3H), 7.46−7.38 (m, 2H), 7.21−7.17 (m, 2H), 3.15 (d, J =
6.3 Hz, 2H), 2.69−2.63 (m, 2H), 2.15 (non, J = 6.8 Hz, 1H), 1.64−
1.47 (m, 3H), 1.02 (d, J = 6.8 Hz, 6H), 0.94 (d, J = 6.3 Hz, 6H) ppm;
13C NMR (75 MHz, DMSO-d6) δ = 166.6, 160.5, 159.7, 152.6, 142.6,
142.3, 133.8, 131.6, 128.1, 127.8, 125.4, 125.0, 114.4, 111.2, 104.3,
101.6, 58.6, 40.4, 32.9, 27.1, 24.4, 22.4 (2 × C), 22.0 (2 × C) ppm;
MS (ESI−) m/z: 485.05 [M − H]−; HRMS (MALDI+) m/z: calcd
for [C25H30N2O6S + H]+: 487.18973, found: 487.18796, Δ = 3.6
ppm; HPLC: tR (Method A) = 19.66 min.
N-(4-Hydroxy-2-oxo-7-(phenylsulfonamido)-2H-chromen-3-yl)-
3-isopentylbenzamide (14l). According to general procedure C: 300
mg (0.82 mmol) of 57, 126 μL (0,98 mmol) of benzolsulfonyl
chloride, and 132 μL (1,64 mmol) of pyridine in 10 mL of dry DCM.
The desired product (293 mg, 0.58 mmol, 71%) was obtained as a
1
mmol, 54%) was obtained as a colorless solid. H NMR (500 MHz,
CDCl3) δ = 13.91 (s, 1H), 8.80 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H),
7.68−7.65 (m, 2H), 7.46 (d, J = 1.9 Hz, 1H), 7.37−7.33 (m, 3H),
7.32 (d, J = 1.3 Hz, 1H), 6.33 (d, J = 1.9 Hz, 1H), 3.88 (s, 3H), 2.63−
2.60 (m, 2H), 1.52 (non, J = 6.7 Hz, 1H), 1.47−1.43 (m, 2H), 0.86
(d, J = 6.5 Hz, 6H) ppm; 13C NMR (126 MHz, CDCl3) δ = 168.0,
161.1, 152.4, 150.5, 144.7, 142.0, 138.9, 134.0, 133.7, 131.4, 129.2,
127.7, 125.1, 124.9, 117.1, 116.1, 107.1, 105.3, 40.8, 38.0, 33.8, 27.9,
22.6 (2 × C) ppm; MS (ESI−) m/z: 430.00 [M − H]−; HRMS
(MALDI+) m/z: calcd for [C25H25N3O4 + H]+: 432.19178, found:
432.19154, Δ = 0.6 ppm; HPLC: tR (Method C) = 11.60 min.
General Procedure for 7-N-Sulfonation and Acylation
(Procedure C). 57 (1.0 equiv) and pyridine (1.2−2.0 equiv) were
dissolved in dry DCM and cooled to 0 °C. Then the corresponding
alkylsulfonyl chloride or acid anhydride (1.2 equiv) was added, and
the reaction mixture was stirred for 12 h, while it was allowed to warm
up to room temperature. The reaction was quenched by addition of 2
M aqueous HCl, and the aqueous phase was extracted three times
with EtOAc. The combined organic phase was dried over anhydrous
MgSO4, filtered, and evaporated under reduced pressure. The crude
product was purified by preparative HPLC.
N-(4-Hydroxy-2-oxo-7-(2-phenylethylsulfonamido)-2H-chro-
men-3-yl)-3-isopentylbenzamide (14i). According to general proce-
dure C: 150 mg (0.41 mmol) of 57, 100 mg (0.49 mmol) of 2-
phenylethanesulfonyl chloride, and 66 μL (0.81 mmol) of pyridine in
5 mL of dry DCM. The desired product (74 mg, 0.14 mmol, 34%)
was obtained as a colorless solid. 1H NMR (300 MHz, DMSO-d6) δ =
12.01 (bs, 1H), 10.53 (s, 1H), 9.45 (s, 1H), 7.85−7.79 (m, 3H),
7.42−7.40 (m, 2H), 7.29−7.16 (m, 7H), 3.59−3.53 (m, 2H), 3.05−
2.99 (m, 2H), 2.72−2.62 (m, 2H), 1.64−1.47 (m, 3H), 0.93 (d, J =
6.2 Hz, 6H) ppm; 13C NMR (75 MHz, DMSO-d6) δ = 166.6, 160.4,
159.6, 152.5, 142.6, 142.1, 137.8, 133.8, 131.6, 128.5 (2 × C), 128.4
(2 × C), 128.1, 127.8, 126.5, 125.4, 125.0, 114.6, 111.4, 104.8, 101.7,
52.1, 40.4, 32.9, 29.1, 27.1, 22.4 (2 × C) ppm; MS (ESI−) m/z:
533.00 [M − H]−; HRMS (MALDI+) m/z: calcd for [C29H30N2O6S
+ H]+: 535.18973, found: 535.18792, Δ = 3.4 ppm; HPLC: tR
(Method A) = 19.98 min.
1
colorless solid. H NMR (500 MHz, DMSO-d6) δ = 12.02 (bs, 1H),
10.99 (s, 1H), 9.42 (s, 1H), 7.87−7.85 (m, 2H), 7.83 (s, 1H), 7.80−
7.77 (m, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.67−7.63 (m, 1H), 7.61−
7.58 (m, 2H), 7.40−7.39 (m, 2H), 7.15 (dd, J = 2.0/8.6 Hz, 1H),
7.08 (d, J = 2.0 Hz, 1H), 2.65 (t, J = 7.8 Hz, 2H) 1.60−1.47 (m, 3H),
0.93 (d, J = 6.4 Hz, 6H) ppm; 13C NMR (126 MHz, DMSO-d6) δ =
166.6, 160.3, 159.5, 152.2, 142.5, 141.5, 139.1, 133.8, 133.4, 131.5,
129.6 (2 × C), 128.1, 127.8, 126.7 (2 × C), 125.4, 125.0, 115.0,
111.7, 105.0, 101.8, 40.3, 32.9, 27.1, 22.4 (2 × C) ppm; MS (ESI−)
m/z: 504.95 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C27H26N2O6S + H]+: 507.15843, found: 507.15781, Δ = 1.2 ppm;
HPLC: tR (Method C) = 10.35 min.
N-(4-Hydroxy-7-(4-methylphenylsulfonamido)-2-oxo-2H-chro-
men-3-yl)-3-isopentylbenzamide (14m). According to general
procedure C: 302 mg (0.82 mmol) of 57, 191 mg (0.98 mmol) of
para-toluenesulfonyl chloride, and 132 μL (1.64 mmol) of pyridine in
10 mL of dry DCM. The desired product (269 mg, 0.52 mmol, 63%)
was obtained as a colorless solid. 1H NMR (500 MHz, DMSO-d6) δ =
12.09 (s, 1H), 10.92 (s, 1H), 9.42 (s, 1H), 7.84−7.73 (m, 4H), 7.40−
7.37 (m, 3H), 7.13 (dd, J = 1.6/8.6 Hz, 1H), 7.08 (d, J = 1.6 Hz, 1H),
2.64 (t, J = 7.8 Hz, 2H), 2.33 (s, 3H), 1.60−1.47 (m, 3H), 0.92 (d, J =
6.4 Hz, 6H) ppm; 13C NMR (126 MHz, DMSO-d6) δ = 166.6, 160.3,
159.6, 152.3, 143.9, 142.5, 141.7, 136.2, 133.8, 131.5, 130.0 (2 × C),
128.1, 127.8, 126.8 (2 × C), 125.4, 124.9, 114.9, 111.7, 104.9, 101.7,
40.3, 32.9, 27.1, 22.4 (2 × C), 21.0 ppm; MS (ESI−) m/z: 518.95 [M
− H]−; HRMS (MALDI+) m/z: calcd for [C28H28N2O6S + H]+:
521.17408, found = 521.17342, Δ = 1.3 ppm; HPLC: tR (Method C)
= 10.84 min.
N-(4-Hydroxy-7-(2-methylphenylsulfonamido)-2-oxo-2H-chro-
men-3-yl)-3-isopentylbenzamide (14n). According to general
procedure C: 293 mg (0.8 mmol) of 57, 191 mg (0.96 mmol) of
ortho-toluenesulfonyl chloride, and 128 μL (1.6 mmol) of pyridine in
10 mL of dry DCM. The desired product (234 mg, 0.45 mmol, 56%)
was obtained as a colorless solid. 1H NMR (500 MHz, DMSO-d6) δ =
12.00 (s, 1H), 11.26 (s, 1H), 9.40 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H),
7.82 (s, 1H), 7.79−7.76 (m, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.54 (td, J
= 0.9/7.5 Hz, 1H), 7.44−7.37 (m, 4H), 7.10 (dd, J = 1.9/8.7 Hz,
1H), 7.00 (d, J = 1.9 Hz, 1H), 2.65−2.62 (m, 5H), 1.59−1.47 (m,
3H), 0.92 (d, J = 6.4 Hz, 6H) ppm; 13C NMR (126 MHz, DMSO-d6)
N-(4-Hydroxy-2-oxo-7-(phenylmethylsulfonamido)-2H-chro-
men-3-yl)-3-isopentylbenzamide (14j). According to general proce-
dure C: 150 mg (0.41 mmol) of 57, 95 mg (0.49 mmol) of
phenylmethanesulfonyl chloride, and 66 μL (0.81 mmol) of pyridine
in 5 mL of dry DCM. The desired product (92 mg, 0.18 mmol, 43%)
was obtained as a colorless solid. 1H NMR (300 MHz, DMSO-d6) δ =
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δ = 166.6, 160.3, 159.6, 152.3, 142.5, 141.4, 137.0, 136.9, 133.8,
133.5, 132.8, 131.5, 129.5, 128.1, 127.8, 126.6, 125.4, 125.0, 114.0,
111.2, 103.9, 101.6, 40.3, 32.9, 27.1, 22.4 (2 × C), 19.6 ppm; MS
(ESI−) m/z: 519.00 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C28H28N2O6S + H]+: 521.17408, found: 521.17351, Δ = 1.1 ppm;
HPLC: tR (Method C) = 10.84 min.
General Procedure for Debenzylation and Reduction of the
Nitro Group (Procedure D). The corresponding 7-benzyloxy-4-
hydroxy-3-nitro-2H-chromen-2-one (1.0 equiv) was suspended in
MeOH or EtOH, and palladium on charcoal (w = 10%) was added.
The reaction mixture was stirred under hydrogen atmosphere at room
temperature for 6 h before it was filtered through a Celite pad, which
was rinsed with MeOH or EtOH (3 × 10 mL). The filtrate was dried
in vacuo to obtain the desired product.
N-(4-Hydroxy-7-(naphthalene-1-sulfonamido)-2-oxo-2H-chro-
men-3-yl)-3-isopentylbenzamide (14o). According to general
procedure C: 300 mg (0.82 mmol) of 57, 223 mg (0.98 mmol) of
naphthalene-1-sulfonyl chloride, and 132 μL (1.64 mmol) of pyridine
in 10 mL of dry DCM. The desired product (213 mg, 0.38 mmol,
46%) was obtained as a colorless solid. 1H NMR (500 MHz, DMSO-
d6) δ = 11.98 (bs, 1H), 11.42 (s, 1H), 9.41 (s, 1H), 8.75 (d, J = 8.6
Hz, 1H), 8.38 (d, J = 7.0 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 8.08 (d, J
= 8.1 Hz, 1H), 7.82−7.77 (m, 3H), 7.70−7.66 (m, 3H), 7.39−7.35
(m, 2H), 7.09 (dd, J = 1.9/8.7 Hz, 1H), 7.03 (d, J = 1.9 Hz, 1H), 2.62
(t, J = 7.8 Hz, 2H), 1.55−1.45 (m, 3H), 0.89 (d, J = 6.3 Hz, 6H)
ppm; 13C NMR (126 MHz, DMSO-d6) δ = 166.6, 160.3, 159.4, 152.3,
142.6, 141.4, 135.0, 133.8, 133.8, 133.7, 131.6, 130.4, 129.3, 128.5,
128.1, 127.8, 127.3, 127.2, 125.4, 125.0, 124.6, 124.0, 114.1, 111.3,
104.0, 101.7, 40.3, 32.9, 27.1, 22.4 (2 × C) ppm; MS (ESI−) m/z:
555.00 [M − H]−; HRMS (MALDI+) m/z: calcd for [C31H28N2O6S
+ H]+: 557.17408, found: 557.17344, Δ = 1.1 ppm; HPLC: tR
(Method C) = 11.24 min.
3-Amino-4,7-dihydroxy-2H-chromen-2-one (15a). According to
general procedure D: 3.0 g (9.6 mmol) of 19a and 0.3 g of palladium
on charcoal (w = 10%) in 180 mL of EtOH. The desired product (1.8
1
g, 9.2 mmol, 96%) was obtained as a brown solid. H NMR (400
MHz, DMSO-d6) δ = 10.06 (bs, 1H), 7.64 (d, J = 8.5 Hz, 1H), 6.65
(dd, J = 2.2/8.5 Hz, 1H), 6.53 (d, J = 2.2 Hz, 1H) ppm; MS (ESI+)
m/z: 234.85 [M + H+ACN]+.
3-Amino-4,7-dihydroxy-8-methyl-2H-chromen-2-one (15b). Ac-
cording to general procedure D: 1.60 g (4.89 mmol) of 19b, 0.16 g
palladium on charcoal (w = 10%) in 100 mL of EtOH. The desired
product (943 mg, 4.55 mmol, 93%) was obtained as a brown solid. 1H
NMR (400 MHz, DMSO-d6) δ = 9.90 (bs, 1H), 7.50 (d, J = 8.4 Hz,
1H), 6.72 (d, J = 8.4 Hz, 1H), 3.65 (bs, 2H), 2.11 (s, 3H) ppm; MS
(ESI+) m/z: 208.06 [M + H]+.
Methyl 4-Hydroxy-3-isopentylbenzoate (49). According to
general procedure D: 1.27 g (4.09 mmol) of 48 and 127 mg of
palladium on charcoal w = 10% in 35 mL of MeOH. The desired
product (894 mg, 4.02 mmol, 98%) was obtained as a colorless oil. 1H
NMR (250 MHz, CDCl3) δ = 7.84 (d, J = 2.0 Hz, 1H), 7.78 (dd, J =
2.0/8.3 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 3.89 (s, 3H), 2.66−2.59 (t,
J = 8.1 Hz, 2H), 1.69−1.46 (m, 3H), 0.95 (d, J = 6.4 Hz, 6H) ppm;
MS (ESI+) m/z: 222.90 [M + H]+.
4-((4-Hydroxy-3-(3-isopentylbenzamido)-2-oxo-2H-chromen-7-
yl)amino)-4-oxobutanoic Acid (14p). According to general proce-
dure C: 149 mg (0.41 mmol) of 57, 50 mg (0.49 mmol) of succinic
anhydride, and 66 μL (0.81 mmol) of pyridine in 5 mL of dry DCM.
The desired product (51 mg, 0.11 mmol, 27%) was obtained as a
1
colorless solid. H NMR (300 MHz, DMSO-d6) δ = 12.16 (bs, 1H),
N-(7-Amino-4-hydroxy-2-oxo-2H-chromen-3-yl)-3-isopentylben-
zamide (57). According to general procedure D: 1.39 g (3.50 mmol)
of 54 and 100 mg of palladium on charcoal (w = 10%) in 100 mL of
EtOH, H2 atmosphere. The desired product (1.25 g, 3.41 mmol,
97%) was obtained as a light brown solid. 1H NMR (500 MHz,
DMSO-d6) δ = 11.48 (bs, 1H), 9.30 (s, 1H), 7.85 (s, 1H), 7.82−7.79
(m, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.43−7.38 (m, 2H), 6.59 (dd, J =
1.9/8.6 Hz, 1H), 6.43 (d, J = 1.9 Hz, 1H), 6.12 (bs, 2H), 2.67 (t, J =
7.8 Hz, 2H), 1.60−1.49 (m, 3H), 0.94 (d, J = 6.4 Hz, 6H) ppm; MS
(ESI−) m/z: 365.30 [M − H]−.
2-Isopentylbenzoic Acid (40). According to general procedure D:
299 mg (1.0 mmol) of 39 and 45 mg of palladium on charcoal (w =
10%) in 15 mL of MeOH. The desired product (137 mg, 0.7 mmol,
66%) was obtained as a colorless solid. 1H NMR (250 MHz, CDCl3)
δ = 8.02 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.30−7.24 (m,
2H), 3.02 (t, J = 7.8 Hz, 2H), 1.74−1.46 (m, 3H), 0.96 (d, J = 6.2 Hz,
6H) ppm; MS (ESI+) m/z: 215.12 [M + H]+.
4-Fluoro-3-isopentylbenzoic Acid (44). According to general
procedure D: 1.54 g (5.2 mmol) of 43 and 150 mg of palladium
on charcoal (w = 10%) in 20 mL of MeOH. The desired product
(1.05 g, 5.0 mmol, 96%) was obtained as a colorless solid and directly
used for amide coupling.
General Procedure for Boc Cleavage (Procedure E). The
corresponding Boc protected 3-aminocoumarin was dissolved in
MeOH and cooled to 0 °C. Then 4 M HCl in 1,4-dioxane (10 equiv)
was added dropwise, and after addition was completed the reaction
mixture was stirred for 5 h at room temperature. The resulting
precipitate was filtrated and washed three times with diethyl ether and
dried in vacuo.
11.97 (bs, 1H), 10.44 (s, 1H), 9.43 (s, 1H), 7.85−7.77 (m, 4H), 7.51
(dd, J = 1.9/8.7 Hz, 1H), 7.42−7.40 (m, 2H), 2.69−2.61 (m, 4H),
2.58−2.54 (m, 2H), 1.64−1.47 (m, 3H), 0.94 (d, J = 6.2 Hz, 6H)
ppm; 13C NMR (75 MHz, DMSO-d6) δ = 173.7, 171.0, 166.7, 160.6,
159.6, 152.3, 142.8, 142.6, 133.8, 131.6, 128.1, 127.8, 125.4, 124.3,
115.0, 111.0, 105.1, 101.5, 40.4, 33.0, 31.3, 28.6, 27.1, 22.4 ppm; MS
(ESI−) m/z: 465.30 [M − H]−; HRMS (MALDI+) m/z: calcd for
[C25H26N2O7 + H]+: 467.18128, found: 467.17978, Δ = 3.2 ppm;
HPLC: tR (Method A) = 17.09 min.
5-((4-Hydroxy-3-(3-isopentylbenzamido)-2-oxo-2H-chromen-7-
yl)amino)-5-oxopentanoic Acid (14q). According to general
procedure C: 149 mg (0.41 mmol) of 57, 56 mg (0.49 mmol) of
glutaric anhydride, and 66 μL (0.81 mmol) of pyridine in 5 mL of dry
DCM. The desired product (97 mg, 0.20 mmol, 50%) was obtained as
a colorless solid. 1H NMR (300 MHz, DMSO-d6) δ = 12.09 (bs, 1H),
11.97 (bs, 1H), 10.36 (s, 1H), 9.43 (s, 1H), 7.85−7.78 (m, 4H), 7.52
(dd, J = 1.9/8.7 Hz, 1H), 7.42−7.40 (m, 2H), 2.66 (t, J = 7.7 Hz,
2H), 2.43 (d, J = 7.3 Hz, 2H), 2.30 (d, J = 7.3 Hz, 2H), 1.83 (quint, J
= 7.3 Hz, 2H), 1.64−1.47 (m, 3H), 0.94 (d, J = 6.2 Hz, 6H) ppm; 13C
NMR (75 MHz, DMSO-d6) δ = 174.1, 171.6, 166.7, 160.6, 159.7,
152.3, 142.8, 142.6, 133.8, 131.6, 128.1, 127.8, 125.4, 124.3, 115.1,
111.0, 105.2, 101.5, 40.3, 35.5, 32.9, 32.9, 27.1, 22.4 (2 × C), 20.2
ppm; MS (ESI−) m/z: 479.15 [M − H]−; HRMS (MALDI+) m/z:
calcd for [C26H28N2O7+Na]+: 503.17887, found: 503.17773, Δ = 2.3
ppm; HPLC: tR (Method A) = 17.83 min.
N-(7-Acetamido-4-hydroxy-2-oxo-2H-chromen-3-yl)-3-isopen-
tylbenzamide (14r). According to general procedure C: 149 mg (0.41
mmol) of 57, 46 μL (0.49 mmol) of acetic anhydride, and 66 μL
(0.81 mmol) of pyridine in 5 mL of dry DCM. The desired product
(60 mg, 0.15 mmol, 36%) was obtained as a colorless solid. 1H NMR
(300 MHz, DMSO-d6) δ = 11.94 (bs, 1H), 10.40 (s, 1H), 9.44 (s,
1H), 7.85−7.78 (m, 4H), 7.50 (dd, J = 1.9/8.7 Hz, 1H), 7.43−7.39
(m, 2H), 2.67 (t, J = 7.7 Hz, 2H), 2.11 (s, 3H), 1.62−1.48 (m, 3H),
0.94 (d, J = 6.3 Hz, 6H) ppm; 13C NMR (75 MHz, DMSO-d6) δ =
169.1, 166.7, 160.6, 159.6, 152.3, 142.9, 142.6, 133.8, 131.5, 128.1,
127.8, 125.4, 124.3, 115.1, 111.0, 105.1, 101.5, 40.4, 32.9, 27.1, 24.2,
22.4 ppm; MS (ESI−) m/z: 407.10 [M − H]−; HRMS (MALDI+)
m/z: calcd for [C23H24N2O5+Na]+: 431.15774, found: 431.15700, Δ
= 1.7 ppm; HPLC: tR (Method A) = 18.33 min.
3-Amino-4-hydroxy-7-nitro-2H-chromen-2-one (15c). According
to general procedure E: 2.35 g (7.28 mmol) of 23a and 18 mL (72
mmol) of 4 M HCl in 1,4-dioxane in 15 mL of MeOH. The desired
product (2.20 g, 6.35 mmol, 99%) was obtained in the form of its
1
hydrochloride 1,4-dioxane adduct as a yellow solid. H NMR (250
MHz, DMSO-d6) δ = 8.51 (bs, 4H), 8.12−8.07 (m, 2H), 8.03−7.99
(m, 1H) ppm; MS (ESI−) m/z: 221.10 [M − H]−.
3-Amino-7-bromo-4-hydroxy-2H-chromen-2-one (15d). Accord-
ing to general procedure E: 4.70 g (13.2 mmol) of 23b and 33 mL
(132 mmol) of 4 M HCl in 1,4-dioxane in 27 mL of MeOH. The
desired product (4.67, 12.3 mmol, 93%) was obtained in the form of
3736
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its hydrochloride 1,4-dioxane adduct as a yellow solid. 1H NMR (250
MHz, DMSO-d6) δ = 7.86 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 1.8 Hz,
1H), 7.48 (dd, J = 1.8/8.4 Hz, 1H), 6.90 (bs, 4H) ppm; MS (ESI−)
m/z: 255.75 [M − H]−.
General Procedure for Benzylation of 2′,4′-Dihydroxyace-
tophenones (Procedure F). The respective 2′,4′-dihydroxyaceto-
phenone (1.0 equiv) was dissolved in acetone, and potassium
carbonate (2.0 equiv) and potassium iodide (0.1 equiv) were added.
The suspension was stirred at 50 °C for 30 min before benzyl
bromide (1.1 equiv) was added slowly. After the addition was
completed, the reaction mixture was heated to reflux for 5 h. The
suspension was cooled to room temperature and filtered, and the
solvent was removed in vacuo. The crude product was purified by
column chromatography (hexane/EtOAc 4:1).
1-(4-(Benzyloxy)-2-hydroxyphenyl)ethanone (17a). According to
general procedure F: 2.0 g (13.2 mmol) of 2′,4′-dihydroxyacetophe-
none (16a), 1.7 mL (14.5 mmol) of benzyl bromide, 3.6 (26.3 mmol)
of K2CO3, and 0.2 g (1.3 mmol) of potassium iodide in 66 mL of
acetone. The desired product (3.1 g, 12.8 mmol, 97%) was obtained
as an orange-colored solid. 1H NMR (250 MHz, DMSO-d6) δ = 12.61
(s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.47−7.34 (m, 5H), 6.60 (dd, J =
2.4/8.9 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 5.19 (s, 2H), 2.56 (s, 3H)
ppm; MS (ESI+) m/z: 242.90 [M + H]+.
1-(4-(Benzyloxy)-2-hydroxy-3-methylphenyl)ethanone (17b). Ac-
cording to general procedure F: 5.0 g (30.0 mmol) of 2′,4′-dihydroxy-
3′methylacetophenone (16b), 4.0 mL (33.0 mmol) of benzyl
bromide, 8.3 g (60.0 mmol) of potassium carbonate, and 0.5 g (3.0
mmol) of potassium iodide in 150 mL of acetone. The desired
product (7.3 g, 28.5 mmol, 95%) was obtained as an orange-colored
solid. 1H NMR (250 MHz, DMSO-d6) δ = 12.86 (s, 1H), 7.81 (d, J =
9.0 Hz, 1H), 7.51−7.29 (m, 5H), 6.74 (d, J = 9.0 Hz, 1H), 5.27 (s,
2H), 2.58 (s, 3H), 2.06 (s, 3H) ppm; MS (ESI−) m/z: 255.00 [M −
H]−.
General Procedure for Cyclization of Acetophenones
(Procedure G). 4-Benzyloxy-2-hydroxyacetophenone (1.0 equiv)
was added slowly to a solution of sodium hydride (w = 60% in mineral
oil, 4.0 equiv) in dry toluene, and the mixture was heated up to 120
°C. Diethyl carbonate (4.0 equiv) was added dropwise, and after
completion the reaction mixture was stirred at 140 °C overnight.
Most of the solvent was removed under reduced pressure, and the
residue was treated with ice cold water. The two phases were
separated, and the organic phase was extracted with water (2 × 100
mL). The toluene phase was discarded, while all aqueous phases were
combined. The combined aqueous phases were acidified with 2 M
aqueous HCl and extracted with EtOAc (3 × 100 mL). The
combined organic phases were dried over MgSO4 and filtered, and the
solvent was removed in vacuo. The products were pure enough
without any further purification.
heated up and stirred for 15 min at 60 °C. The suspension was cooled
to room temperature, and the precipitate was separated by filtration
and washed with ice cold water and dried in vacuo.
7-(Benzyloxy)-4-hydroxy-3-nitro-2H-chromen-2-one (19a). Ac-
cording to general procedure H: 2.15 g (8.0 mmol) of 18a, 0.03 g
(0.38 mmol) of NaNO2, 1.1 mL of HNO3 (w = 65%, 16.0 mmol), and
15 mL of AcOH. The desired product (1.72 g, 5.49 mmol, 69%) was
obtained as a light brown solid. 1H NMR (400 MHz, DMSO-d6) δ =
7.80 (d, J = 8.8 Hz, 1H), 7.41 (m, 5H), 6.88 (m, 2H), 5.18 (s, 2H)
ppm; MS (ESI−) m/z: 311.80 [M − H]−.
7-(Benzyloxy)-4-hydroxy-8-methyl-3-nitro-2H-chromen-2-one
(19b). According to general procedure H: 2.09 g (7.4 mmol) of 18b,
0.03 g (0.35 mmol) of NaNO2, 1,0 mL of HNO3 (w = 65%, 14.8
mmol), and 15 mL of AcOH. The desired product (1.62 g, 4.95
mmol, 67%) was obtained as a light brown solid. 1H NMR (300 MHz,
DMSO-d6) δ = 7.73 (d, J = 8.8 Hz, 1H), 7.49−7.33 (m, 5H), 7.02 (d,
J = 8.8 Hz, 1H), 5.22 (s, 2H), 2.17 (s, 3H) ppm; MS (ESI−) m/z:
325.80 [M − H]−.
General Procedure for Acetylation of 2-Hydroxy Benzoic
Acid Derivatives (Procedure I). The reactions were realized under
exclusion of light. The corresponding 2-hydroxybenzoic acid (1.0
equiv) was dissolved in EtOAc. Then triethylamine (2.0 equiv), acetic
anhydride (2.0 equiv) and DMAP (0.1 equiv) were added. The
reaction mixture was stirred for 12 h at 80 °C. After the reaction was
completed, the reaction mixture was filtered over Celite. The filtrate
was acidified with 2 N aqueous HCl and extracted with EtOAc (3 ×
50 mL). The combined organic phase was washed with brine (50
mL), dried over MgSO4, filtered, and evaporated under reduced
pressure. The product could be isolated after purification through
column chromatography (DCM/MeOH 95:5 + 1% acetic acid).
2-Acetoxy-4-nitrobenzoic Acid (21a). According to general
procedure I: 3.96 (21 mmol) of 2-hydroxy-4-nitrobenzoic acid
(20a), 5.9 mL (42 mmol) of triethylamine, 3.97 mL (42 mmol) of
Ac2O, 0.26 g (2.1 mmol) of DMAP, and 21 mL of EtOAc. The
desired product (4.10 g, 18.2 mmol, 87%) was obtained as a fawn
solid. 1H NMR (250 MHz, DMSO-d6) δ = 8.19 (d, J = 2.6 Hz, 1H),
8.15 (s, 1H), 8.12 (d, J = 1.7 Hz, 1H), 2.29 (s, 3H) ppm; MS (ESI+)
m/z: 225.95 [M + H]+.
2-Acetoxy-4-bromobenzoic Acid (21b). According to general
procedure I: 4.99 g (23.0 mmol) of 4-bromo-2-hydroxybenzoic acid
(20b), 4.39 mL (46.0 mmol) of Ac2O, 6.46 mL (46.0 mmol) of
triethylamine, 0.28 g (2.30 mmol) of DMAP, and 23 mL of EtOAc.
The desired product (5.23 g, 20.2 mmol, 88%) was obtained as a fawn
solid. 1H NMR (250 MHz, DMSO-d6) δ = 12.88 (bs, 1H), 7.86 (d, J
= 8.4 Hz, 1H), 7.59 (dd, J = 2.0/8.4 Hz, 1H), 7.52 (d, J = 2.0 Hz,
1H), 2.24 (s, 3H) ppm; MS (ESI−) m/z: 216.70 [M − Ac]−.
2-((tert-Butoxycarbonyl)amino)-3-ethoxy-3-oxopropanoic Acid
(22b). NaHCO3 (4.15 g, 49.3 mmol, 1.05 equiv) was added slowly
to a suspension of diethyl aminomalonate hydrochloride (22a) (10.2
g, 47.0 mmol, 1.0 equiv) in a mixture of water (60 mL) and 1,4-
dioxane (88 mL) and stirred at room temperature. After the solution
turned clear, a catalyst amount of DMAP (0.06 g, 0.47 mmol, 1.0
equiv) was added. A solution of Boc2O (10.8 g, 49.3 mmol, 1.05
equiv) in 1,4-dioxane (32 mL) was added dropwise, and the reaction
mixture was stirred for an additional 5 h at room temperature. The
solvents were evaporated in vacuo, and the residue was dissolved in
EtOAc. The organic phase was washed with aqueous solutions of
KHSO4 (w = 5%), sat. NaHCO3, and brine. The organic phase was
dried over anhydrous MgSO4, filtered, and evaporated in vacuo. The
desired product (12.3 g, 44.7 mmol, 95%) was obtained as a colorless
viscous oil and was pure enough for the next step without further
purification needed.
7-(Benzyloxy)-4-hydroxy-2H-chromen-2-one (18a). According to
general procedure G: 4.90 g (20.3 mmol) of 17a, 9.6 mL (78.0 mmol)
of diethyl carbonate, 2.00 g (78 mmol) of sodium hydride (w = 60%
in mineral oil), and 200 mL of toluene. The desired product (4.90 g,
1
18.3 mmol, 90%) was obtained as a pale orange-colored solid. H
NMR (400 MHz, DMSO-d6) δ = 12.38 (s, 1H), 7.71 (d, J = 8.7, 1H),
7.50−7.30 (m, 5H), 7.05−6.97 (m, 2H), 5.47 (s, 1H), 5.21 (s, 2H)
ppm; MS (ESI−) m/z: 267.09 [M − H]−.
7-(Benzyloxy)-4-hydroxy-8-methyl-2H-chromen-2-one (18b). Ac-
cording to general procedure G: 4.0 g (15.6 mmol) of 17b, 7.6 mL
(62.4 mmol) of diethyl carbonate, 2.5 g (62,4 mmol) of sodium
hydride (w = 60% in mineral oil), and 150 mL of toluene. The desired
product (3.95 g, 13.99 mmol, 90%) was obtained as a pale orange-
1
colored solid. H NMR (250 MHz, DMSO-d6) δ = 12.27 (s, 1H),
The crude product was dissolved in abs. EtOH (30 mL) ,and a
solution of KOH (2.51 g, w = 85%, 38 mmol, 1.0 equiv) in abs. EtOH
(50 mL) was added dropwise. The reaction mixture was stirred at
room temperature for 3 h. After the reaction was completed, the
solvent was removed in vacuo. The residue was taken up in aqueous
1N NaHCO3 solution (80 mL) and washed two times with EtOAc (2
× 25 mL). The aqueous phase was cooled to 0 °C and acidified by
solid KHSO4 and extracted three times with EtOAc (3 × 50 mL). The
7.64 (d, J = 8.8, 1H), 7.52−7.28 (m, 5H), 7.12 (d, J = 8.8 Hz, 1H),
5.46 (s, 1H), 5.25 (s, 2H), 2.22 (s, 3H) ppm; MS (ESI−) m/z:
281.30 [M − H]−.
General Procedure for Nitration of 4-Hydroxycoumarins
(Procedure H). The corresponding 4-hydroxycoumarin (1.0 equiv)
and sodium nitrite (0.05 equiv) were suspended in acetic acid. A
solution of nitric acid (w = 65%, 2.0 equiv) and acetic acid was added
slowly. After the addition was completed, the reaction mixture was
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organic phase was washed with brine, dried over anhydrous MgSO4,
filtered, and evaporated in vacuo. The desired product (8.2 g, 33.2
mmol, 87%) was obtained as a colorless viscous oil and was pure
enough for the next step without further purification needed. 1H
NMR (400 MHz, DMSO-d6) δ = 13.34 (bs, 1H), 7.47 (d, J = 8.0 Hz,
1H), 4.71 (d, J = 8.0 Hz, 1H), 4.18−4.10 (m, 2H), 1.39 (s, 9H), 1.19
(t, J = 7.1 Hz, 3H) ppm; MS (ESI−) m/z: 245.90 [M − H]−.
General Procedure for Benzoic Acid Chloride Formation
(Procedure J). The corresponding 2-acetoxybenzoic acid (1.0 equiv)
was dissolved in dry DCM under argon atmosphere. After the
dropwise addition of thionyl chloride (20 equiv), the reaction mixture
was refluxed for 5 h. Then the solvent and unreacted thionyl chloride
were removed under reduced pressure. The resulting residue was
dried in vacuo and used in the next synthesis step without further
purification.
General Procedure for C−C Coupling (Procedure K). To an
ice-cooled solution of 22b (1.4 equiv) in dry THF and triethylamine
(6.3 equiv), anhydrous MgCl2 (3.4 equiv) was added. Then a solution
of the corresponding benzoic acid chloride (1.0 equiv) in dry THF
was added, and the resulting suspension was stirred for 12 h, while it
was allowed to warm up to room temperature. The reaction was
quenched by addition of sat. aqueous NH4Cl solution until the
reaction mixture turned clear and then was extracted three times with
EtOAc (3 × 50 mL). The combined organic phase was dried over
MgSO4, filtered, and evaporated under reduced pressure. The
resulting oil was used in the next synthesis step without further
purification.
the aqueous residue was acidified with 2 M aqueous HCl. After
extraction with DCM (3 × 15 mL), the combined organic phases
were dried over MgSO4 and filtered and the solvent was removed in
vacuo. The obtained products could be used in following synthesis
without further purification.
3-Methoxybenzoic Acid (24b). According to general procedure M:
0.98 g (5.9 mmol) of 26b, 1.95 g (29.5 mmol) of KOH in 15 mL of
THF, 15 mL of MeOH, and 30 mL of H2O. The desired product
(0.82 g, 5.39 mmol, 91%) was obtained as a colorless solid. 1H NMR
(400 MHz, DMSO-d6) δ = 12.97 (bs, 1H), 7.53 (d, J = 7.6 Hz, 1H),
7.44−7.39 (m, 2H), 7.20−7.17 (m, 1H), 3.80 (s, 3H) ppm; MS
(ESI−) m/z: 151.90 [M − H]−.
3-Ethoxybenzoic Acid (24c). According to general procedure M:
1.09 g (6.05 mmol) of 26c, 2.00 g (30.3 mmol) of KOH in 15 mL of
THF, 15 mL of MeOH, and 30 mL of H2O. The desired product
(0.88 g, 5.30 mmol, 88%) was obtained as a colorless solid. 1H NMR
(400 MHz, DMSO-d6) δ = 12.95 (bs, 1H), 7.51 (d, J = 7.6 Hz, 1H),
7.42−7.37 (m, 2H), 7.18−7.15 (m, 1H), 4.06 (q, J = 7.0 Hz, 2H),
1.33 (t, J = 7.0 Hz, 3H) ppm; MS (ESI−) m/z: 164.85 [M − H]−.
3-Propoxybenzoic Acid (24d). According to general procedure M:
0.32 g (1.65 mmol) of 26d, 0.55 g (8.25 mmol) of KOH in 10 mL of
THF, 10 mL of MeOH, and 20 mL of H2O. The desired product
(0.27 g, 1.50 mmol, 91%) was obtained as a colorless solid. 1H NMR
(250 MHz, DMSO-d6) δ = 12.94 (bs, 1H), 7.51 (d, J = 7.6 Hz, 1H),
7.42−7.36 (m, 2H), 7.19−7.15 (m, 1H), 3.97 (t, J = 6.5 Hz, 2H),
1.73 (sext, J = 7.0 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H) ppm; MS (ESI−)
m/z: 178.90 [M − H]−.
3-Butoxybenzoic Acid (24e). According to general procedure M:
1.29 g (6.20 mmol) of 26e, 2.05 g (31.0 mmol) of KOH in 20 mL of
THF, 20 mL of MeOH, and 40 mL of H2O. The desired product
(1.14 g, 5.87 mmol, 95%) was obtained as a colorless solid. 1H NMR
(400 MHz, DMSO-d6) δ = 12.96 (bs, 1H), 7.53 (d, J = 7.7 Hz, 1H),
7.44−7.38 (m, 2H), 7.20−7.16 (m, 1H), 4.01 (t, J = 6.3 Hz, 2H),
1.75−1.68 (m, 2H), 1.50−1.40 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H)
ppm; MS (ESI−) m/z: 193.00 [M − H]−.
3-(Cyclopropylmethoxy)benzoic Acid (24f). According to general
procedure M: 1.22 g (5.92 mmol) of 26f, 1.95 g (29.6 mmol) of KOH
in 25 mL of THF, 25 mL of MeOH, and 50 mL of H2O. The desired
product (0.94 g, 4.89 mmol, 83%) was obtained as a pale-yellow solid.
1H NMR (400 MHz, DMSO-d6) δ = 12.94 (bs, 1H), 7.52−7.49 (m,
1H), 7.41−7.37 (m, 2H), 7.19−7.16 (m, 1H), 3.85 (d, J = 7.0 Hz,
2H), 1.27−1.16 (m, 1H), 0.58−0.55 (m, 2H), 0.36−0.32 (m, 2H)
ppm; MS (ESI−) m/z: 190.85 [M − H]−.
General Procedure for Cyclization of Salicyl Derivatives
(Procedure L). The corresponding product of the C−C coupling
(1.0 equiv) was dissolved in MeOH, and 1.5 M aqueous NaOH
solution (6.0 equiv) was added. The reaction mixture was stirred for 3
h at room temperature. Then the solution was adjusted to pH 3 using
1.5 M aqueous HCl. The aqueous phase was extracted three times
with EtOAc (3 × 30 mL). The combined organic phase was washed
with brine, dried over MgSO4, and filtered, and the solvent was
removed under reduced pressure. The crude product was purified by
column chromatography (DCM/MeOH 9:1).
tert-Butyl (4-Hydroxy-7-nitro-2-oxo-2H-chromen-3-yl)-
carbamate (23a). The acyl chloride intermediate was prepared
according to general procedure J and was used without purification:
6.08 g (27.0 mmol) of 21a and 39 mL (540 mmol) of SOCl2 in 54
mL of DCM. Coupling was performed according to procedure K and
used without purification: 9.35 g (37.8 mmol) of 22b, 8.74 g (91.8
mmol) of MgCl2, and 23.9 mL (170 mmol) of triethylamine in 160
mL of THF. Cyclization was performed according to procedure L:
6.48 g (162 mmol) of NaOH in 100 mL of H2O and 81 mL of
MeOH. 4.70 g (14.6 mmol, 54% over three steps) of product 23a was
3-Isopropoxybenzoic Acid (24g). According to general procedure
M: 0.98 g (5.05 mmol) of 26g, 1.67 g (25.3 mmol) of KOH in 15 mL
of THF, 15 mL of MeOH, and 30 mL of H2O. The desired product
(0.83 g, 4.61 mmol, 91%) was obtained as a colorless solid. 1H NMR
(400 MHz, DMSO-d6) δ = 12.95 (bs, 1H), 7.52−7.50 (m, 1H),
7.43−7.37 (m, 2H), 7.18−7.15 (m, 1H), 4.66 (sept, J = 6.0 Hz, 1H),
1.28 (d, J = 6.0 Hz, 6H) ppm; MS (ESI−) m/z: 178.95 [M − H]−.
3-(Allyloxy)benzoic Acid (24h). According to general procedure
M: 1.15 g (5.98 mmol) of 26h, 1.97 g (29.9 mmol) of KOH in 25 mL
of THF, 25 mL of MeOH, and 50 mL of H2O. The desired product
1
obtained. H NMR (250.0 MHz, DMSO-d6) δ = 12.43 (s, 1H), 8.22
(d, J = 1.9 Hz, 1H), 8.19 (d, J = 2.2 Hz, 1H), 8.16 (d, J = 2.2 Hz, 1H),
8.10 (s, 1H), 1.41 (s, 9H) ppm; MS (ESI−) m/z: 321.10 [M − H]−.
tert-Butyl (7-Bromo-4-hydroxy-2-oxo-2H-chromen-3-yl)-
carbamate (23b). The acyl chloride intermediate was prepared
according to general procedure J and was used without purification:
5.96 g (23.0 mmol) of 21b and 33 mL (460 mmol) of SOCl2 in 46
mL of DCM. Coupling was performed according to procedure K and
used without purification: 7.96 g (32.2 mmol) of 22b, 7.45 g (78.2
mmol) of MgCl2, and 20.4 mL (145 mmol) of triethylamine in 130
mL of THF. Cyclization was performed according to procedure L:
5.52 g (138 mmol) of NaOH in 92 mL of H2O and 70 mL of MeOH.
4.70 g (13.2 mmol, 57% over three steps) of product 23b was
1
(1.02 g, 5.72 mmol, 96%) was obtained as a pale-yellow solid. H
NMR (400 MHz, DMSO-d6) δ = 12.98 (bs, 1H), 7.56−7.51 (m, 1H),
7.47−7.44 (m, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.21−7.18 (m, 1H),
6.09−5.99 (m, 1H), 5.42−5.36 (m, 1H), 5.28−5.25 (m, 1H) ppm;
MS (ESI−) m/z: 176.85 [M − H]−.
3-(Benzyloxy)benzoic Acid (24i). According to general procedure
M: 1.58 g (6.52 mmol) of 26i, 2.15 g (32.6 mmol) of KOH in 25 mL
of THF, 25 mL of MeOH, and 50 mL of H2O. The desired product
1
obtained. H NMR (500 MHz, DMSO-d6) δ = 12.24 (bs, 1H), 8.01
1
(1.41 g, 6.18 mmol, 95%) was obtained as a pale-yellow solid. H
(bs, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 1.8 Hz, 1H), 7.56 (dd,
J = 1.8/8.5 Hz, 1H), 1.42 (s, 9H) ppm; MS (ESI−) m/z: 355.75 [M
− H]−.
General Procedure for Alkaline Hydrolysis of Esters
(Procedure M). The respective product of alkene reduction (1.0
equiv) was dissolved in a 1:2 mixture of MeOH and THF. An
aqueous solution of NaOH or KOH (1M, 5.0−6.0 equiv) was added,
and the reaction mixture was heated to reflux for 5 h. After cooling to
room temperature, the organic solvents were removed in vacuo and
NMR (400 MHz, DMSO-d6) δ = 12.97 (bs, 1H), 7.56−7.52 (m, 2H),
7.47−7.38 (m, 5H), 7.35−7.33 (m, 1H), 7.28−7.25 (m, 1H), 5.16 (s,
2H) ppm; MS (ESI−) m/z: 226.95 [M − H]−.
3-Isopentylbenzoic Acid (36a). According to general procedure
M: 339 mg (1.64 mmol) of 35a, 461 mg (8.22 mmol) of NaOH, 20
mL of THF, 10 mL of MeOH, and 10 mL of H2O. The desired
product (235 mg, 1.22 mmol, 75%) was obtained as a colorless solid.
1H NMR (250 MHz, CDCl3) δ = 7.95−7.91 (m, 2H), 7.46−7.35 (m,
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2H), 2.71−2.65 (m, 2H), 1.65−1.49 (m, 3H), 0.95 (d, J = 6.3 Hz,
6H) ppm; MS (ESI+) m/z: 190.95 [M − H]−.
The filtrate was evaporated in vacuo, and the residue was purified by
column chromatography (hexane/EtOAc 9:1).
3-Propylbenzoic Acid (36c). According to general procedure M:
235 mg (1.32 mmol) of 35c, 435 mg (6.59 mmol) of NaOH, 20 mL
of THF, 10 mL of MeOH, and 10 mL of H2O. The desired product
(178 mg, 1.08 mmol, 82%) was obtained as a colorless solid. 1H NMR
(250 MHz, CDCl3): 7.96−7.93 (m, 2H), 7.47−7.36 (m, 2H), 2.69−
2.63 (m, 2H), 1.68 (sext, J = 7.6 Hz, 2H), 0.96 (t, J = 7.3 Hz, 3H)
ppm; MS (ESI+) m/z: 162.85 [M − H]−.
Methyl 3-Methoxybenzoate (26b). According to general proce-
dure N: 1.00 g (6.57 mmol) of methyl 3-hydroxybenzoate (25), 0.69
mL (7.23 mmol) of dimethyl sulfate, and 1.82 g (13.1 mmol) of
K2CO3 in 30 mL of acetone. The desired product (1.0 g, 5.9 mmol,
90%) was obtained as a colorless liquid. 1H NMR (400 MHz, DMSO-
d6) δ = 7.56−7.54 (1H, m), 7.46−7.42 (m, 2H), 7.24−7.21(m, 1H),
3.85 (s, 3H), 3.81 (s,3H) ppm; MS (ESI+) m/z: 167.00 [M + H]+.
Ethyl 3-Methoxybenzoate (26c). According to general procedure
N: 1.00 g (6.57 mmol) of methyl 3-hydroxybenzoate (25), 0.54 mL
(7.23 mmol) of bromoethane, and 1.82 g (13.1 mmol) of K2CO3 in
30 mL of acetone. The desired product (1.09 g, 5.99 mmol, 91%) was
3-Butylbenzoic Acid (36d). According to general procedure M:
403 mg (2.10 mmol) of 35d, 692 mg (10.5 mmol) of NaOH, 20 mL
of THF, 10 mL of MeOH, and 10 mL of H2O. The desired product
(337 mg, 1.89 mmol, 90%) was obtained as a colorless solid. 1H NMR
(250 MHz, CDCl3) δ = 7.96−7.92 (m, 2H), 7.46−7.35 (m, 2H),
2.72−2.65 (m, 2H), 1.70−1.58 (m, 2H), 1.48−1.22 (m, 2H), 0.94 (t,
J = 7.3 Hz, 3H) ppm; MS (ESI+) m/z: 176.85 [M − H]−.
3-Pentylbenzoic Acid (36e). According to general procedure M:
379 mg (1.84 mmol) of 35e, 606 mg (9.19 mmol) of NaOH, 20 mL
of THF, 10 mL of MeOH, and 10 mL of H2O. The desired product
(296 mg, 1.54 mmol, 84%) was obtained as a colorless solid. 1H NMR
(250 MHz, CDCl3) δ = 7.95−7.92 (m, 2H), 7.43−7.38 (m, 2H),
2.71−2.64 (m, 2H), 1.71−1.59 (m, 2H), 1.41−1.25 (m, 4H), 0.91 (t,
J = 6.8 Hz, 3H) ppm; MS (ESI+) m/z: 190.85 [M − H]−.
3-(2-Cyclopropylethyl)benzoic Acid (36f). According to general
procedure M: 469 mg (2.30 mmol) of 35f, 758 mg (11.5 mmol) of
NaOH, 20 mL of THF, 10 mL of MeOH, and 10 mL of H2O. The
desired product (378 mg, 1.99 mmol, 86%) was obtained as a
colorless solid. 1H NMR (250 MHz, CDCl3) δ = 7.96−7.92 (m, 2H),
7.47−7.35 (m, 2H), 2.82−2.65 (m, 2H), 1.68−1.48 (m, 2H), 1.36−
1.28 (m, 1H), 0.47−0.40 (m, 2H), 0.08−0.02 (m, 2H) ppm; MS (ESI
+) m/z: 188.90 [M − H]−.
1
obtained as a colorless liquid. H NMR (400 MHz, DMSO-d6) δ =
7.54−7.52 (m, 1H), 7.44−7.40 (m, 2H), 7.22−7.21 (m, 1H), 4.07 (q,
J = 7.0 Hz, 2H), 3.84 (s, 3H), 1.34 (t, J = 7.0 Hz, 3H) ppm; MS (ESI
+) m/z: 180.90 [M + H]+.
Methyl 3-Propoxybenzoate (26d). According to general procedure
N: 1.00 g (6.57 mmol) of methyl 3-hydroxybenzoate (25), 0.64 mL
(7.23 mmol) of 1-chloropropane, and 1.82 g (13.1 mmol) of K2CO3
in 30 mL of acetone. The desired product (0.32 g, 1.65 mmol, 25%)
was obtained as a colorless liquid. 1H NMR (400 MHz, DMSO-d6) δ
= 7.54−7.52 (m, 1H), 7.44−7.40 (m, 2H), 7.22−7.20 (m, 1H), 3.97
(t, J = 6.5 Hz, 2H), 3.84 (s, 3H), 1.74 (sext, J = 7.0 Hz, 2H), 0.98 (t, J
= 7.4 Hz, 3H) ppm; MS (ESI+) m/z: 194.40 [M + H]+.
Methyl 3-Butoxybenzoate (26e). According to general procedure
N: 1.00 g (6.57 mmol) of methyl 3-hydroxybenzoate (25), 0.78 mL
(7.23 mmol) of 1-bromobutane, 1.82 g (13.1 mmol) of K2CO3, and
0.55 g (3.29 mmol) of potassium iodide in 30 mL of acetonitrile. The
desired product (1.29 g, 6.19 mmol, 94%) was obtained as a colorless
liquid. 1H NMR (400 MHz, DMSO-d6) δ = 7.52 (d, J = 7.6 Hz, 1H),
7.44−7.40 (m, 2H), 7.22−7.19 (m, 1H), 4.02 (t, J = 6.5 Hz, 2H),
3.84 (s, 3H), 1.74−1.66 (m, 2H), 1.49−1.39 (m, 2H), 0.93 (t, J = 7.4
Hz, 3H) ppm; MS (ESI+) m/z: 208.90 [M + H]+.
Methyl 3-(Cyclopropylmethoxy)benzoate (26f). According to
general procedure N: 1.00 g (6.57 mmol) of methyl 3-
hydroxybenzoate (25), 0.70 mL (7.23 mmol) of (bromomethyl)-
cyclopropane, 1.82 g (13.1 mmol) of K2CO3, and 0.55 g (3.29 mmol)
of potassium iodide in 30 mL of acetonitrile. The desired product
(1.22 g, 5.92 mmol, 90%) was obtained as a pale-yellow liquid. H
NMR (400 MHz, DMSO-d6) δ = 7.54−7.51 (m, 1H), 7.44−7.39 (m,
2H), 7.23−7.19 (m, 1H), 3.86 (d, J = 7.2 Hz, 2H), 3.84 (s, 3H),
1.27−1.18 (m, 1H), 0.59−0.55 (m, 2H), 0.36−0.32 (m, 2H) ppm;
MS (ESI+) m/z: 206.85 [M + H]+.
Methyl 3-Isopropoxybenzoate (26g). According to general
procedure N: 1.00 g (6.57 mmol) of methyl 3-hydroxybenzoate
(25), 0.72 mL (7.23 mmol) of 2-iodopropane, and 1.82 g (13.1
mmol) of K2CO3 in 30 mL of acetonitrile. The desired product (0.98
g, 5.05 mmol, 77%) was obtained as a colorless liquid. 1H NMR (400
MHz, DMSO-d6) δ = 7.50 (d, J = 7.7 Hz, 1H), 7.43−7.39 (m, 2H),
7.21−7.18 (m, 1H), 4.66 (sept, J = 6.0 Hz, 1H), 3.84 (s, 3H), 1.27 (d,
J = 6.0 Hz, 6H) ppm; MS (ESI+) m/z: 194.95 [M + H]+.
3-Isobutylbenzoic Acid (36g). According to general procedure M:
163 mg (0.85 mmol) of 35g, 280 mg (4.24 mmol) of NaOH, 20 mL
of THF, 10 mL of MeOH, and 10 mL of H2O. The desired product
(109 mg, 0.61 mmol, 72%) was obtained as a colorless solid. 1H NMR
(250 MHz, CDCl3) δ = 7.97−7.90 (m, 2H), 7.40−7.37 (m, 2H), 2.55
(d, J = 7.2 Hz, 2H), 1.91 (sept., J = 6.8 Hz, 1H), 0.92 (d, J = 6.6 Hz,
6H) ppm; MS (ESI+) m/z: 177.05 [M − H]−.
3-Phenethylbenzoic Acid (36i). According to general procedure
M: 221 mg (0.92 mmol) of 35i, 304 mg (4.60 mmol) of NaOH, 20
mL of THF, 10 mL of MeOH, and 10 mL of H2O. The desired
product (162 mg, 0.72 mmol, 78%) was obtained as a colorless solid.
1H NMR (250 MHz, CDCl3) δ = 7.90−7.86 (m, 2H), 7.33−7.30 (m,
2H), 7.25−7.09 (m, 5H), 2.97−2.84 (m, 4H) ppm; MS (ESI+) m/z:
224.90 [M − H]−.
3-Isopentyl-4-methoxybenzoic Acid (53). According to general
procedure M: 260 mg (1.1 mmol) of 52, 363 mg (5.5 mmol) of
KOH, 12 mL of THF, 6 mL of MeOH, and 6 mL of H2O. The
desired product (232 mg, 1.04 mmol, 95%) was obtained as a
colorless solid. 1H NMR (300 MHz, CDCl3) δ = 7.97 (dd, J = 2.2/8.6
Hz, 1H), 7.89 (d, J = 2.2 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 3.90 (s,
3H), 2.66−2.61 (m, 2H), 1.61 (non, J = 6.5 Hz, 1H), 1.52−1.44 (m,
2H), 0.95 (d, J = 6.5 Hz, 6H) ppm; MS (ESI+) m/z: 220.95 [M −
H]−.
Methyl 3-(Allyloxy)benzoate (26h). According to general
procedure N: 1.00 g (6.57 mmol) of methyl 3-hydroxybenzoate
(25), 0.62 mL (7.23 mmol) of allyl bromide, 1.82 g (13.1 mmol) of
K2CO3, 0.55 g (3.29 mmol) of potassium iodide in 30 mL of acetone.
The desired product (1.15 g, 5.98 mmol, 91%) was obtained as a
4-Hydroxy-3-isopentylbenzoic Acid (50). According to general
procedure M: 534 mg (2.4 mmol) of 49, 793 mg (12.0 mmol) of
KOH, 30 mL of THF, 15 mL of MeOH, and 15 mL of H2O. The
desired product (485 mg, 2.33 mmol, 97%) was obtained as a
colorless solid. 1H NMR (300 MHz, CDCl3) δ = 7.91 (d, J = 2.1 Hz,
1H), 7.86 (dd, J = 2.1/8.3 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 2.67−
2.2.61 (m, 2H), 1.64 (non, J = 6.6 Hz, 1H), 1.56−1.49 (m, 2H), 0.97
(d, J = 6.6 Hz, 6H) ppm; MS (ESI−) m/z: 206.95 [M − H]−.
General Procedure for Alkylation of Methyl 3-Hydroxyben-
zoate (Procedure N). Methyl 3-hydroxy-benzoate (25, 1.0 equiv)
was dissolved in acetone, AcN, or DMF, and K2CO3 (2.0 equiv) was
added. Depending on the alkylating agent, potassium iodide (0.5
equiv) was added. After stirring for 30 min, a solution of the
corresponding alkylating agent (1.1 equiv) in 5 mL of solvent was
added dropwise and the reaction mixture was refluxed for 6 h. Then
the reaction mixture was cooled to room temperature and filtered.
1
colorless liquid. H NMR (400 MHz, DMSO-d6) δ = 7.56−7.53 (m,
1H), 7.46−7.41 (m, 2H), 7.25−7.22 (m, 1H), 6.09−6.00 (m, 1H),
5.43−5.37 (m, 1H), 5.29−5.25 (m, 1H), 4.64−4.62 (m, 2H) ppm;
MS (ESI+) m/z: 193.30 [M + H]+.
Methyl 3-(Benzyloxy)benzoate (26i). According to general
procedure N: 1.00 g (6.57 mmol) of methyl 3-hydroxybenzoate
(25), 0.86 mL (7.23 mmol) of benzyl bromide, 1.82 g (13.1 mmol) of
K2CO3, and 0.55 g (3.29 mmol) of potassium iodide in 30 mL of
acetone. The desired product (1.56 g, 6.44 mmol, 98%) was obtained
1
as a pale-yellow liquid. H NMR (400 MHz, DMSO-d6) δ = 7.57−
7.52 (m, 2H), 7.47−7.38 (m, 5H), 7.35−7.29 (m, 2H), 5.17 (s, 2H),
3.84 (s, 3H) ppm; MS (ESI−) m/z: 241.95 [M − H]−.
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J. Med. Chem. 2021, 64, 3720−3746

Journal of Medicinal Chemistry
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Article
General Procedure for Alkylation of 3-Mercaptobenzoic
Acid (Procedure O). 3-Mercaptobenzoic acid (27, 1.0 equiv) was
dissolved in aqueous ethanol solution (v/v = 30%), and a solution of
NaOH (2.0 equiv) in 5 mL of H2O was added. Then a solution of the
corresponding alkyl halide (2.0 equiv) in 5 mL of EtOH was added
dropwise, and the reaction mixture was stirred at 60 °C for 12 h. Then
the solvent was removed under vacuo, and the aqueous residue was
acidified with 2 M aqueous HCl. The resulting precipitate was filtered
and dried in vacuo to give the desired product.
3-(Isobutylthio)benzoic Acid (28a). According to general
procedure O: 308 mg (2.0 mmol) of 3-mercaptobenzoic acid (27),
431 μL (4.0 mmol) of 1-bromo-2-methylpropane, 160 mg (4.0
mmol) of NaOH in 35 mL of H2O and 15 mL of EtOH. The desired
product (376 mg, 1.79 mmol, 89%) was obtained as a pale-yellow
solid. 1H NMR (250 MHz, DMSO-d6) δ = 13.07 (s, 1H), 7.81 (t, J =
1.6 Hz, 1H), 7.72 (dt, J = 1.3/7.6 Hz, 1H), 7.59−7.52 (m, 1H), 7.43
(t, J = 7.6 Hz, 1H), 2.90 (d, J = 6.7 Hz, 2H), 1.80 (non, J = 6.7 Hz,
1H), 0.99 (d, J = 6.6 Hz, 6H) ppm; MS (ESI−) m/z: 209.05 [M −
H]−.
3-(Ethylthio)benzoic Acid (28c). According to general procedure
O: 154 mg (1.0 mmol) of 3-mercaptobenzoic acid (27), 150 μL (2.0
mmol) of bromoethane, 80 mg (2.0 mmol) of NaOH in 35 mL of
H2O and 15 mL of EtOH. The desired product (126 mg, 0.69 mmol,
69%) was obtained as a colorless solid. 1H NMR (250 MHz, DMSO-
d6) δ = 13.08 (s, 1H), 7.80 (t, J = 1.6 Hz, 1H), 7.73 (dt, J = 1.3/7.6
Hz, 1H), 7.59−7.52 (m, 1H), 7.44 (t, J = 7.6 Hz, 1H), 3.02 (q, J = 7.3
Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H) ppm; MS (ESI−) m/z: 180.80 [M
− H]−.
3-(Propylthio)benzoic Acid (28d). According to general procedure
O: 154 mg (1.0 mmol) of 3-mercaptobenzoic acid (27), 176 μL (2.0
mmol) of 1-chloropropane, 80 mg (2.0 mmol) of NaOH in 35 mL of
H2O and 15 mL of EtOH. The desired product (164 mg, 0.84 mmol,
84%) was obtained as a pale-yellow solid. 1H NMR (250 MHz,
DMSO-d6) δ = 13.08 (s, 1H), 7.80 (t, J = 1.6 Hz, 1H), 7.73 (dt, J =
1.3/7.7 Hz, 1H), 7.58−7.54 (m, 1H), 7.44 (t, J = 7.7 Hz, 1H), 2.99
(q, J = 7.3 Hz, 2H), 1.60 (sext, J = 7.3 Hz, 2H) 0.98 (t, J = 7.3 Hz,
3H) ppm; MS (ESI−) m/z: 194.85 [M − H]−.
3-(Butylthio)benzoic Acid (28e). According to general procedure
O: 154 mg (1.0 mmol) of 3-mercaptobenzoic acid (27), 216 μL (2.0
mmol) of 1-bromobutane, 80 mg (2.0 mmol) of NaOH in 35 mL of
H2O and 15 mL of EtOH. The desired product (166 mg, 0.79 mmol,
79%) was obtained as a pale-yellow solid. 1H NMR (250 MHz,
DMSO-d6) δ = 13.07 (1H, s), 7.80 (t, J = 1.6 Hz, 1H), 7.73 (dt, J =
1.3/7.5 Hz, 1H), 7.58−7.53 (m, 1H), 7.43 (t, J = 7.5 Hz, 1H), 3.00 (t,
J = 7.3 Hz, 2H), 1.62−1.50 (m, 2H), 1.48−1.33 (m, 2H), 0.88 (t, J =
7.3 Hz, 3H) ppm; MS (ESI−) m/z: 208.90 [M − H]−.
3-((Cyclopropylmethyl)thio)benzoic Acid (28f). According to
general procedure O: 154 mg (1.0 mmol) of 3-mercaptobenzoic
acid (27), 194 μL (2.0 mmol) of (bromomethyl)cyclopropane, 80 mg
(2.0 mmol) of NaOH in 35 mL of H2O and 15 mL of EtOH. The
desired product (99 mg, 0.48 mmol, 48%) was obtained as a colorless
solid. 1H NMR (250 MHz, DMSO-d6) δ = 13.07 (s, 1H), 7.83 (t, J =
1.6 Hz, 1H) 7.73 (dt, J = 1.6/7.5 Hz, 1H), 7.60−7.55 (m, 1H), 7.43
(t, J = 7.5 Hz, 1H), 2.97 (d, J = 7.0, 2H), 1.09−0.93 (m, 1H), 0.56−
0.18 (m, 4H) ppm; MS (ESI−) m/z: 206.25 [M − H]−.
residue was taken up in EtOAc. The organic phase was washed
successive with aqueous solutions of 2 M HCl and sat. NaHCO3,
dried over anhydrous MgSO4, and filtered, and the solvent was
removed under reduced pressure. The residue was taken up in a
mixture of THF and MeOH (4:1), and 1 M aqueous NaOH (3.0
equiv) was added. The reaction mixture was stirred overnight at room
temperature before being acidified with 2 M aqueous HCl. The
precipitate was dissolved in EtOAc, and the aqueous phase was
extracted two times with EtOAc. The combined organic phase was
dried over anhydrous MgSO4, filtered, and evaporated under reduced
pressure.
3-(2-Methylpropylsulfonamido)benzoic Acid (MH32a). Accord-
ing to general procedure P: 284 mg (2.0 mmol) of methyl 3-
aminobenzoate (29), 323 μL (2.4 mmol) of 2-methylpropane-1-
sulfonyl chloride, 337 μL (2.4 mmol) of triethylamine, 240 mg (6.0
mmol) of NaOH, 6 mL of DCM, 4 mL of THF, 1 mL of MeOH, and
6 mL of H2O. The desired product (410 mg, 1.60 mmol, 80%) was
obtained as a pale-yellow solid. 1H NMR (300 MHz, acetone-d6) δ =
8.80 (bs, 1H), 8.03−8.01 (m, 1H), 7.81−7.78 (m, 1H), 7.59 (ddd, J =
1.1/2.3/8.1 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 3.05 (d, J = 6.5 Hz,
2H), 2.25 (sept, J = 6.6 Hz, 1H), 1.05 (d, J = 6.6 Hz, 6H) ppm; MS
(ESI−) m/z: 256.15 [M − H]−.
3-(Methylsulfonamido)benzoic Acid (32b). According to general
procedure P: 284 mg (2.0 mmol) of methyl 3-aminobenzoate (29),
186 μL (2.4 mmol) of methanesulfonyl chloride, 337 μL (2.4 mmol)
of triethylamine, 240 mg (6.0 mmol) of NaOH, 6 mL of DCM, 4 mL
of THF, 1 mL of MeOH, and 6 mL of H2O. The desired product
(143 mg, 0.66 mmol, 33%) was obtained as a colorless solid. 1H NMR
(300 MHz, acetone-d6) δ = 8.76 (bs, 1H), 8.03 (t, J = 1.8 Hz, 1H),
7.81 (dt, J = 1.3/7.6 Hz, 1H), 7.60 (ddd, J = 1.2/2.3/8.1 Hz, 1H),
7.50 (t, J = 7.8 Hz, 1H), 3.04 (s, 3H) ppm; MS (ESI−) m/z: 214.00
[M − H]−.
3-(Ethylsulfonamido)benzoic Acid (32c). According to general
procedure P: 284 mg (2.0 mmol) of methyl 3-aminobenzoate (29),
232 μL (2.4 mmol) of ethansulfonyl chloride, 337 μL (2.4 mmol) of
triethylamine, 240 mg (6.0 mmol) of NaOH, 6 mL of DCM, 4 mL of
THF, 1 mL of MeOH, and 6 mL of H2O. The desired product (227
1
mg, 0.99 mmol, 50%) was obtained as a pale-yellow solid. H NMR
(300 MHz, acetone-d6) δ = 8.82 (bs, 1H), 8.05−8.03 (m, 1H), 7.81−
7.76 (m, 1H), 7.63−7.58 (m, 1H), 7.49 (t, J = 7.9 Hz, 1H), 3.16 (q, J
= 7.4 Hz, 2H), 1.30 (t, J = 7.4 Hz, 3H) ppm; MS (ESI−) m/z: 228.05
[M − H]−.
3-(Propylsulfonamido)benzoic Acid (32d). According to general
procedure P: 284 mg (2.0 mmol) of methyl 3-aminobenzoate (29),
278 μL (2.4 mmol) of propane-1-sulfonyl chloride, 337 μL (2.4
mmol) of triethylamine, 240 mg (6.0 mmol) of NaOH, 6 mL of
DCM, 4 mL of THF, 1 mL of MeOH, and 6 mL of H2O. The desired
product (127 mg, 0.52 mmol, 26%) was obtained as a pale-yellow
solid. 1H NMR (300 MHz, acetone-d6) δ = 8.80 (bs, 1H), 8.04−8.02
(m, 1H), 7.81−7.77 (m, 1H), 7.60 (ddd, J = 1.2/2.3/8.1 Hz, 1H),
7.49 (t, J = 7.8 Hz, 1H), 3.16−3.10 (m, 2H), 1.87−1.74 (m, 2H),
1.00 (t, J = 7.5 Hz, 3H) ppm; MS (ESI−) m/z: 242.15 [M − H]−.
3-(Butylsulfonamido)benzoic Acid (32e). According to general
procedure P: 284 mg (2.0 mmol) of methyl 3-aminobenzoate (29),
318 μL (2.4 mmol) of butane-1-sulfonyl chloride, 337 μL (2.4 mmol)
of triethylamine, 240 mg (6.0 mmol) of NaOH, 6 mL of DCM, 4 mL
of THF, 1 mL of MeOH, and 6 mL of H2O. The desired product
3-(Isopropylthio)benzoic Acid (28g). According to general
procedure O: 154 mg (1.0 mmol) of 3-mercaptobenzoic acid (27),
200 μL (2.0 mmol) of 2-iodopropane, 80 mg (2.0 mmol) of NaOH in
35 mL of H2O and 15 mL of EtOH. The desired product (156 mg,
1
(361 mg, 1.40 mmol, 70%) was obtained as a pale-yellow solid. H
NMR (300 MHz, acetone-d6) δ = 8.80 (bs, 1H), 8.04 (t, J = 1.9 Hz,
1H), 7.79 (dt, J = 1.4/7.6 Hz, 1H), 7.60 (ddd, J = 1.1/2.3/8.13 Hz,
1H), 7.49 (t, J = 7.8 Hz, 1H), 3.18−3.13 (m, 2H), 1.81−1.71 (m,
2H), 1.46−1.37 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H) ppm; MS (ESI−)
m/z: 256.10 [M − H]−.
3-(Cyclopropanesulfonamido)benzoic Acid (32f). According to
general procedure P: 2.13 g (15.0 mmol) of methyl 3-aminobenzoate
(29), 1.94 mL (18.0 mmol) of cyclopropanesulfonyl chloride, 2.53
mL (18.0 mmol) of triethylamine, 1.80 g (45.0 mmol) of NaOH, 45
mL of DCM, 30 mL of THF, 7 mL of MeOH, and 45 mL of H2O.
The desired product (1.77 g, 7.34 mmol, 49%) was obtained as a pale-
yellow solid. 1H NMR (300 MHz, DMSO-d6) δ = COOH not
1
0.80 mmol, 80%) was obtained as a pale-yellow solid. H NMR (250
MHz, DMSO-d6) δ = 13.10 (s, 1H), 7.87 (t, J = 1.6 Hz, 1H), 7.79 (dt,
J = 1.3/7.7 Hz, 1H), 7.63−7.59 (m, 1H), 7.46 (t, J = 7.7 Hz, 1H),
3.54 (sept, J = 6.6 Hz, 1H), 0.88 (d, J = 6.6 Hz, 6H) ppm; MS (ESI−)
m/z: 194.90 [M − H]−.
General Procedure for the Synthesis of 3-Alkylsulfonami-
dobenzoic Acids (Procedure P). Methyl 3-aminobenzoate (29, 1.0
equiv) was dissolved in DCM and cooled to 0 °C. The corresponding
alkylsulfonyl chloride (1.2 equiv) and triethylamine (1.2 equiv) were
added. The reaction mixture was stirred for 3 h at room temperature.
Then the solvent was removed under reduced pressure, and the
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observed, 9.93 (s, 1H), 7.85−7.83 (m, 1H), 7.69−7.64 (m, 1H),
7.50−7.41 (m, 2H), 2.63 (quint, J = 6.3 Hz, 1H), 0.93 (d, J = 6.3 Hz,
4H) ppm; MS (ESI−) m/z: 240.02 [M − H]−.
1.02 mmol, 34%) was obtained as a pale-yellow oil. MS (ESI+) m/z:
239.05 [M + H]+.
Benzyl 2-(3-Methylbut-1-en-1-yl)benzoate (39). According to
general procedure Q: 1.0 g (4.2 mmol) of 38, 2.0 g (5.0 mmol) of
isobutyltriphenylphosphonium bromide, and 5.0 mL of LiHMDS (1
M in THF, 5.0 mmol) in 30 mL of THF. The desired product (299
mg, 1.1 mmol, 26%) was obtained as a colorless oil. MS (ESI+) m/z:
303.15 [M + Na]+.
Benzyl 4-Fluoro-3-(3-methylbut-1-en-1-yl)benzoate (43). Ac-
cording to general procedure Q: 1.50 g (5.7 mmol) of 42, 2.78 g
(6.84 mmol) of isobutyltriphenylphosphonium bromide, and 6.8 mL
of LiHMDS (1 M in THF, 6.8 mmol) in 20 mL of THF. The desired
product (1.54 g, 5.17 mmol, 43%) was obtained as a pale-yellow oil.
MS (ESI+) m/z: 298.95 [M + H]+.
Methyl 4-(Benzyloxy)-3-(3-methylbut-1-en-1-yl)benzoate (48).
According to general procedure Q: 1.40 g (5.18 mmol) of 47, 2.53
g (6.22 mmol) of isobutyltriphenylphosphonium bromide, and 6.22
mL of LiHMDS (1 M in THF, 6.22 mmol) in 26 mL of THF. The
desired product (1.27 g, 4.09 mmol, 79%) was obtained as a pale-
yellow oil. MS (ESI+) m/z: 310.95 [M + H]+.
General Procedure for Reduction of Alkenes (Procedure R).
The respective product of the Wittig reaction was dissolved in MeOH,
and palladium on charcoal (w = 10%) was added. The suspension was
stirred for 5 h at room temperature under hydrogen atmosphere. The
suspension was poured and filtered through a Celite pad, which was
rinsed several times with MeOH (3 × 10 mL). The filtrate was dried
in vacuo, and the obtained products were pure enough for further
synthesis.
3-(1-Methylethylsulfonamido)benzoic Acid (32g). According to
general procedure P: 568 mg (4.0 mmol) of methyl 3-aminobenzoate
(29), 538 μL (4.8 mmol) of propane-2-sulfonyl chloride, 675 μL (4.8
mmol) of triethylamine, 480 mg (12 mmol) of NaOH, 12 mL of
DCM, 8 mL of THF, 2 mL of MeOH, and 12 mL of H2O. The
desired product (180 mg, 0.74 mmol, 19%) was obtained as a pale-
yellow solid. 1H NMR (300 MHz, DMSO-d6): 13.04 (s, 1H), 9.98 (s,
1H), 7.84−7.82 (m, 1H), 7.63 (dt, J = 1.7/6.9 Hz, 1H), 7.49−7.40
(m, 2H), 3.23 (sept, J = 6.8 Hz, 1H), 1.24 (d, J = 6.8 Hz, 6H) ppm;
MS (ESI−) m/z: 242.04 [M − H]−.
General Procedure for the Wittig Reaction (Procedure Q).
Alkyltriphenylphosphonium bromide (1.2 equiv) was suspended in
dry THF under argon and cooled with an ice bath. LiHMDS (1 M in
THF, 1.2 equiv) was added slowly, and the reaction mixture was
stirred for 30 min before a solution of the corresponding methyl 3-
formylbenzoate (1.0 equiv) in dry THF was added. The ice bath was
removed after the addition was completed, and the reaction mixture
was stirred for additional 3 h at room temperature. The reaction was
quenched by the addition of a saturated solution of NH4Cl (20 mL),
and the aqueous phase was extracted with EtOAc. The combined
organic phases were dried over MgSO4, filtered, and dried in vacuo.
The crude product was purified by flash chromatography (hexane/
EtOAc 9:1) and was obtained as a colorless liquid, that was not
characterized by NMR but rather hydrogenated in the next synthesis
step.
Methyl 3-Isopentylbenzoate (35a). According to general
procedure R: 383 mg (1.87 mmol) of 34a and 45 mg of palladium
on charcoal (w = 10%) in 15 mL of MeOH. The desired product (339
Methyl 3-(3-Methylbut-1-en-1-yl)benzoate (34a). According to
general procedure Q: 500 mg (2.98 mmol) of methyl 3-
formylbenzoate (33), 1.46 g (3.58 mmol) of isobutyltriphenylphos-
phonium bromide, and 3.6 mL of LiHMDS (1 M in THF, 3.6 mmol)
in 15 mL of THF. The desired product (383 mg, 1.87 mmol, 63%)
was obtained as a colorless oil. MS (ESI+) m/z: 204.90 [M + H]+.
Methyl 3-(Prop-1-en-1-yl)benzoate (34c). According to general
procedure Q: 500 mg (2.98 mmol) of methyl 3-formylbenzoate (33),
1.36 g (3.58 mmol) of ethyltriphenylphosphonium bromide, and 3.6
mL of LiHMDS (1 M in THF, 3.6 mmol) in 15 mL of THF. The
desired product (254 mg, 1.44 mmol, 48%) was obtained as a
colorless oil. MS (ESI+) m/z: 176.85 [M + H]+.
Methyl 3-(But-1-en-1-yl)benzoate (34d). According to general
procedure Q: 500 mg (2.98 mmol) of methyl 3-formylbenzoate (33),
1.39 g (3.58 mmol) of propyltriphenylphosphonium bromide, and 3.6
mL of LiHMDS (1 M in THF, 3.6 mmol) in 15 mL of THF. The
desired product (413 mg, 2.17 mmol, 73%) was obtained as a
colorless oil. MS (ESI+) m/z: 190.85 [M + H]+.
1
mg, 1.64 mmol, 88%) was obtained as a colorless oil. H NMR (250
MHz, CDCl3) δ = 7.87−7.82 (m, 2H), 7.40−7.33 (m, 2H), 3.91 (s,
3H), 2.69−2.62 (m, 2H), 1.68−1.44 (m, 3H), 0.94 (d, J = 6.3 Hz,
6H) ppm; MS (ESI+) m/z: 206.90 [M + H]+.
Methyl 3-Propylbenzoate (35c). According to general procedure
R: 254 mg (1.44 mmol) of 34c and 45 mg of palladium on charcoal
(w = 10%) in 15 mL of MeOH. The desired product (235 mg, 1.32
1
mmol, 92%) was obtained as a colorless oil. H NMR (250 MHz,
CDCl3) δ = 7.88−7.83 (m, 2H), 7.39−7.30 (m, 2H), 3.91 (s, 3H),
2.67−2.60 (m, 2H), 1.74−1.59 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H)
ppm; MS (ESI+) m/z: 178.90 [M + H]+.
Methyl 3-Butylbenzoate (35d). According to general procedure R:
413 mg (2.17 mmol) of 34d and 45 mg of palladium on charcoal (w =
10%) in 15 mL of MeOH. The desired product (403 mg, 2.10 mmol,
1
97%) was obtained as a colorless oil. H NMR (250 MHz, CDCl3) δ
Methyl 3-(Pent-1-en-1-yl)benzoate (34e). According to general
procedure Q: 500 mg (2.98 mmol) of methyl 3-formylbenzoate (33),
1.45 g (3.6 mmol) of butyltriphenylphosphonium bromide, and 3.6
mL of LiHMDS (1 M in THF, 3.6 mmol) in 15 mL of THF. The
desired product (363 mg, 1.78 mmol, 60%) was obtained as a
colorless oil. MS (ESI+) m/z: 204.80 [M + H]+.
Methyl 3-(2-Cyclopropylvinyl)benzoate (34f). According to
general procedure Q: 500 mg (2.98 mmol) of methyl 3-
formylbenzoate (33), 1.45 g (3.58 mmol) of (cyclopropylmethyl)-
triphenylphosphonium bromide, and 3.6 mL of LiHMDS (1 M in
THF, 3.6 mmol) in 15 mL of THF. The desired product (493 mg,
2.44 mmol, 82%) was obtained as a colorless oil. MS (ESI+) m/z:
202.95 [M + H]+.
Methyl 3-(2-Methylprop-1-en-1-yl)benzoate (34g). According to
general procedure Q: 500 mg (2.98 mmol) of methyl 3-
formylbenzoate (33), 1.58 g (3.58 mmol) of isopropyltriphenylphos-
phonium bromide, and 3.6 mL of LiHMDS (1 M in THF, 3.6 mmol)
in 15 mL of THF. The desired product (183 mg, 0.96 mmol, 32%)
was obtained as a colorless oil. MS (ESI+) m/z: 190.90 [M + H]+.
Methyl 3-Styrylbenzoate (34i). According to general procedure Q:
500 mg (2.98 mmol) of methyl 3-formylbenzoate (33), 817 g (3.58
mmol) of dimethyl benzylphosphonate, and 3.6 mL of LiHMDS (1 M
in THF, 3.6 mmol) in 15 mL of THF. The desired product (244 mg,
= 7.87−7.83 (m, 2H), 7.37−7.33 (m, 2H), 3.91 (s, 3H), 2.66 (t, J =
7.7 Hz, 2H), 1.68−1.56 (m, 2H), 1.44−1.29 (m, 2H), 0.93 (t, J = 7.3
Hz, 3H) ppm; MS (ESI+) m/z: 192.90 [M + H]+.
Methyl 3-Pentylbenzoate (35e). According to general procedure
R: 393 mg (1.92 mmol) of 34e and 45 mg of palladium on charcoal
(w = 10%) in 15 mL of MeOH. The desired product (379 mg, 1.84
1
mmol, 96%) was obtained as a colorless oil. H NMR (250 MHz,
CDCl3) δ = 7.87−7.83 (m, 2H), 7.37−7.33 (m, 2H), 3.91 (s, 3H),
2.65 (t, J = 7.7 Hz, 2H), 1.69−1.56 (m, 2H), 1.38−1.28 (m, 4H),
0.89 (t, J = 6.8 Hz, 3H) ppm; MS (ESI+) m/z: 206.85 [M + H]+.
Methyl 3-(2-Cyclopropylethyl)benzoate (35f). According to
general procedure R: 493 mg (2.44 mmol) of 34f and 45 mg of
palladium on charcoal (w = 10%) in 15 mL of MeOH. The desired
product (469 mg, 2.30 mmol, 94%) was obtained as a colorless oil. 1H
NMR (250 MHz, CDCl3) δ = 7.88−7.83 (m, 2H), 7.40−7.30 (m,
2H), 3.91 (s, 3H), 2.79−2.62 (m, 2H), 1.58−1.48 (m, 2H), 1.38−
1.27 (m, 1H), 0.46−0.39 (m, 2H), 0.06−0.01 (m, 2H) ppm; MS (ESI
+) m/z: 204.90 [M + H]+.
Methyl 3-Isobutylbenzoate (35g). According to general procedure
R: 183 mg (0.96 mmol) of 34g and 45 mg of palladium on charcoal
(w = 10%) in 15 mL of MeOH. The desired product (163 mg, 0.85
1
mmol, 89%) was obtained as a colorless oil. H NMR (250 MHz,
CDCl3) δ = 7.89−7.82 (m, 2H), 7.35−7.32 (m, 2H), 3.91 (s, 3H),
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Article
2.52 (d, J = 7.2 Hz, 2H), 1.89 (non, J = 6.9 Hz, 1H), 0.90 (d, J = 6.6
Hz, 6H) ppm; MS (ESI+) m/z: 192.90 [M + H]+.
additional 5 h at 85 °C. The reaction was quenched by the addition of
water (50 mL), and the aqueous phase was extracted with DCM (3 ×
25 mL). The combined organic phases were dried over MgSO4 and
filtered, and the solvent was removed in vacuo. Column chromato-
graphic purification (hexane/EtOAc 4:1) gave the product (1.40 g,
5.18 mmol, 96%) as a colorless liquid. 1H NMR (300 MHz, CDCl3) δ
= 10.53 (s, 1H), 8.53 (d, J = 2.4 Hz, 1H), 8.21 (dd, J = 2.4/8.8 Hz,
1H), 7.46−7.35 (m, 5H), 7.10 (d, J = 8.8 Hz, 1H), 5.27 (s, 2H), 3.91
(s, 3H) ppm; MS (ESI+) m/z: 293.16 [M + Na]+.
Methyl 3-Phenethylbenzoate (35i). According to general
procedure R: 244 mg (1.02 mmol) of 34i and 45 mg of palladium
on charcoal (w = 10%) in 15 mL of MeOH. The desired product (221
1
mg, 0.92 mmol, 90%) was obtained as a colorless oil. H NMR (250
MHz, CDCl3) δ = 7.91−7.84 (m, 2H), 7.35−7.16 (m, 7H), 3.92 (s,
3H), 3.01−2.91 (m, 4H) ppm; MS (ESI+) m/z: 240.85 [M + H]+.
Benzyl 2-Formylbenzoate (38). 2-Formylbenzoic acid (37, 3.0 g,
20.0 mmol, 1.0 equiv) was dissolved in 50 mL of acetone, and K2CO3
(5.5 g, 40.0 mmol, 2.0 equiv) was added. Then benzyl bromide (2.9
mL, 24 mmol, 1.2 equiv) was added, and the reaction mixture was
refluxed for 1 h. After cooling to room temperature, the reaction
mixture was filtered and the solvent was evaporated under reduced
pressure. The crude product was purified by column chromatography
(hexane/EtOAc 9:1) to obtain the desired product (4.50 g, 18.7
mmol, 94%) as a colorless oil. 1H NMR (250 MHz, CDCl3) δ = 10.63
(s, 1H), 8.03−7.98 (m, 1H), 7.96−7.92 (m, 1H), 7.69−7.60 (m, 2H),
7.48−7.34 (m, 5H), 5.42 (s, 2H) ppm; MS (ESI+) m/z: 262.80 [M +
Na]+.
4-Acetoxy-3-isopentylbenzoic Acid (51). 50 (534 mg, 2.56 mmol,
1.0 equiv) and pyridine (414 μL, 5.13 mmol, 2.0 equiv) were
dissolved in DCM (10 mL) and cooled to 0 °C with an ice-bath.
Acetic anhydride (485 μL, 5.13 mmol, 2.0 equiv) was added, the
reaction mixture was stirred overnight, and the solution could warm
to room temperature. The solvent was removed in vacuo, and the
crude product was purified by column chromatography (hexane/
EtOAC 1:1) to give the product (590 mg, 2.36 mmol, 92%) as a
colorless solid. 1H NMR (300 MHz, CDCl3) δ = 8.02 (d, J = 2.0 Hz,
1H), 7.97 (dd, J = 2.0/8.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 2.60−
2.55 (m, 2H), 2.35 (s, 3H), 1.61 (non, J = 6.6 Hz, 1H), 1.52−1.44
(m, 2H), 0.95 (d, J = 6.6 Hz, 6H) ppm; MS (ESI−) m/z: 248.95 [M
− H]−.
Benzyl 4-Fluoro-3-formylbenzoate (42). 4-Fluoro-3-formylben-
zoic acid (41, 1.0 g, 6.0 mmol, 1.0 equiv) and NaHCO3 (0.6 g, 7.2
mmol, 1.2 equiv) were suspended in dry DMF (15 mL) and stirred
for 1 h at room temperature. Then benzyl bromide (871 μL, 7.2
mmol, 1.2 equiv) was added dropwise and stirred for an additional 4
h. The reaction mixture was treated with sat. NaHCO3 solution (10
mL), and the aqueous phase was extracted three times with DCM (3
× 20 mL). The combined organic phase was dried over anhydrous
MgSO4, filtered, and evaporated under reduced pressure. The residue
was purified through column chromatography (hexane/EtOAc 9:1) to
obtain the desired product (1.51 g, 5.85 mmol, 98%) as a colorless oil.
1H NMR (250 MHz, CDCl3) δ = 10.36 (s, 1H), 8.58 (dd, J = 2.3/6.7
Hz, 1H), 8.36−8.29 (m, 1H), 7.46−7.35 (m, 5H), 7.26 (t, J = 9.2 Hz,
1H), 5.38 (s, 2H) ppm; MS (ESI+) m/z: 258.90 [M + H]+.
Methyl 3-Isopentyl-4-methoxybenzoate (52). 49 (262 mg, 1.18
mmol, 1.0 equiv) was dissolved in acetone (10 mL), and K2CO3 (326
mg, 2.36 mmol, 2.0 equiv) was added. The suspension was stirred for
30 min at 80 °C before dimethyl sulfate (134 μL, 1.42 mmol, 1.2
equiv) was added, and the reaction mixture was stirred for additional
2 h at 80 °C. The suspension was filtered, and the filtrate was washed
with sat. NaHCO3 solution (2 × 30 mL), dried over MgSO4, and
filtered. The organic phase was evaporated in vacuo, and the resulting
residue was purified by column chromatography (hexane/EtOAc 9:1)
to obtain the desired product (255 mg, 1.08 mmol, 91%) as a
Cloning. SNAP is a mutant of the DNA repair protein O6-
alkylguanine-DNA alkyltransferase. It reacts specifically and rapidly
with benzylguanine (BG) derivatives. Thereby, the introduced
mutations prevent completion of the enzymatic reaction which allows
irreversible covalent labeling of fusion proteins via the 20 kDa SNAP-
tag (New England Biolabs; NEB).
For the expression of LC3 proteins with N-terminal fusion to
SNAP, an expression construct based on the plasmid vector pET29b
was prepared. The entire section between the original NdeI site and
the fourth base pair following the His-Tag coding sequence of
pET29b was replaced, hence essentially leaving only the vector
backbone unmodified. First, the section was replaced by a codon
optimized sequence encoding a restriction site for NcoI (overlapping
with the start codon) and an open reading frame for Met-Gly-[His10-
tag]-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a cleavage site for
Tobacco Etch Virus protease [TEV site] and restriction sites for KpnI
(in frame; first triplet is Gly codon of TEV site) and XhoI, resulting in
the pET29b derivative pET29bh4. Next, we amplified by PCR the
SNAP open reading frame (residues 2−180) using the plasmid
pSNAP-tag(T7)2 (NEB) as the template. Thereby, restriction sites
for KpnI (5′ end) and BamHI + XhoI (3′ end) were attached. SNAP
was cloned in between KpnI and XhoI restriction sites of the pET29b
derivative. The DNA sequences encoding for LC3A (uniprot
identifier: Q9H492-1; residues 4−119; preceded by Met) and
LC3B (uniprot identifier: Q9GZQ8-1; residues 5−119; preceded by
Met-Gly), each immediately followed by the DNA sequence
TAAATGATTAGA for stop codons in all reading frames, were
then introduced in the frame between the restriction sites for BamHI
and XhoI, respectively.
For the HTRF-based LIR binding assay, we generated a fusion
protein of LIR peptide with N-terminal fusion to superfolder GFP
(sGFP), which served as the FRET acceptor. Cloning followed a
comparable strategy starting with pET29bh4. Instead of the SNAP-tag
sequence, we introduced the sequence encoding sGFP (without start
Met) followed by the BamHI restriction site (in frame), a codon for
Gly, and the previously described sequence with stop codons. The
resulting plasmid was later also used to generate free sGFP. For sGFP-
LIR, we then cloned the LC3-interacting region (LIR) of
sequestosome 1 (p62; uniprot identifier Q13501-1; residues 322−
347) between the BamHI site and the stop codons. The cysteine
within this segment, which corresponds to residue 331 in p62, was
mutated to serine. This LIR sequence encompasses all residues that in
the crystal structure (PDB entry 2k6q) are in close contact with
LC3B.
1
colorless oil. H NMR (300 MHz, CDCl3) δ = 7.88 (dd, J = 2.2/8.5
Hz, 1H), 7.82 (d, J = 2.2 Hz, 1H), 6.84 (d, J = 8.6 Hz, 1H), 3.88 (s,
3H), 3.87 (s, 3H), 2.65−2.59 (m, 2H), 1.60 (non, J = 6.5 Hz, 1H),
1.50−1.42 (m, 2H), 0.94 (d, J = 6.5 Hz, 6H) ppm; MS (ESI+) m/z:
236.95 [M + H]+.
Methyl 3-Formyl-4-hydroxybenzoate (46). Methyl para-hydrox-
ybenzoate (45, 3.00 g, 20.0 mmol, 1.0 equiv) and MgCl2 (9.52 g, 100
mmol, 5.0 equiv) were suspended in 1,2-dichloroethane (80 mL) and
triethylamine (17 mL, 120 mmol, 6.0 equiv). The suspension was
stirred at 40 °C for 1 h before paraformaldehyde (6.00 g, 200 mmol,
10.0 equiv) was added. The reaction mixture was stirred overnight at
70 °C. The reaction was quenched by the addition of 1 M
hydrochloric acid (100 mL). The aqueous phase was extracted with
DCM (3 × 100 mL), and the combined organic phases were washed
with brine, dried over MgSO4, and filtered, and the solvent was
removed in vacuo. The residue was purified by column chromatog-
raphy (hexane/EtOAc 4:1) to obtain the product (1.09 g, 6.07 mmol,
30%) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ = 11.39 (s,
1H), 9.96 (s, 1H), 8.32 (t, J = 2.0 Hz, 1H), 8.19 (dd, J = 2.0/8.7 Hz,
1H), 7.04 (d, J = 8.7 Hz, 1H), 3.93 (s, 3H) ppm; MS (ESI−) m/z:
178.70 [M − H]−.
Methyl 4-(Benzyloxy)-3-formylbenzoate (47). 46 (970 mg, 5.38
mmol, 1.0 equiv) was dissolved in dry DFM (10 mL), and K2CO3
(1.12 g, 8.07 mmol, 1.5 equiv) was added. The suspension was stirred
for 30 min at 85 °C. Thereafter, benzyl bromide (960 μL, 8.07 mmol,
1.1 equiv) was added and the reaction mixture was stirred for an
For all these fusion proteins expression, lysis, initial purification by
immobilized metal affinity chromatography (IMAC) followed by
cleavage of the His-tag by TEV protease, reverse IMAC, and
concentration of the product prior to size exclusion chromatography
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(SEC) was all performed following the protocol that we published
before for expression and purification of free PPARγ LBD.
nm (acceptor) and 620 nm (donor reference) after excitation at 340
nm were recorded on a Tecan Infinite F200 instrument (Tecan
̈
As a final purification step, volumes of 5 mL of concentrated
protein were separated on a HiLoad 16/600 Superdex 75 pg gel
filtration column (Ge Healthcare) equilibrated and run in SEC buffer
[25 mM HEPES pH 7.5 adjusted with KOH, 150 mM KF, 10% (w/v)
glycerol, 5 mM DTT] at 1 mL/min. Fractions from the middle of the
peak corresponding to monomeric protein were collected and used
for assays.
Group Ltd., Mannedorf, Switzerland). FI520 nm was divided by
FI620 nm and multiplied by 10 000 to give a dimensionless HTRF
signal. Displacement of the LIR-LC3 interaction by test compound
separates FRET donor and acceptor resulting in a loss in FRET signal.
Expression and Purification. The expression and purification of
LC3A and LC3B used in the TSA, ITC, and crystallization
experiments were performed in a similar manner for both proteins.
In brief, TB medium (1.2% (w/v) tryptone, 2.4% (w/v) yeast
extract, 0.5% glycerol, and 89 mM phosphate buffer) with kanamycin
(100 μg/mL) was inoculated with 1% (v/v) of a preculture of E. coli
BL21-(DE3) transformed with either the LC3A or LC3B plasmid.
Cultures were incubated by shaking at 37 °C and 180 rpm until an
OD600 of 1.5 was reached, before the incubation temperature and
shaking speed were reduced to 20 °C as well as 170 rpm, respectively.
Cultures were induced by the addition of 500 μM IPTG at an OD600
of 2.5. Cells were harvested by centrifugation (10 866g, 20 min) after
approximately 16 h. Cell pellets were either stored at −20 °C or
directly used in the following protein purification.
Screening of Libraries of Compounds That Affect the
Interaction between LC3b and LIRtide. The AlphaScreen assay
was performed with the GST-detection kit (Streptavidin Donor beads
and Anti-GST conjugated AlphaScreen Acceptor beads) essentially as
described by the manufacturer (PerkinElmer). The assay (25 μL)
contained 50 mM Tris (pH 7.5), 100 mM NaCl, 0.01% Tween 20, 2
mM DTT, 1% DMSO and 2−30 nM GST-LC3B and biotin-LIRtide
2−30 nM. The alphascreen signal was measured in an Envision
multiplate reader (PerkinElmer). GST-LC3B was obtained by
transient transfection of HEK293 cells and purified in batch on
gluthathione beads.22 LIRtide (SGGDDDWTHLS) and Biotin-
LIRtide (biotin-Ahx-GNSSGGDDDWTHLS) were synthesized by
Genescript. The concentrations of GST-LC3B and biotin-LIRtide
were experimentally chosen from cross-titration assays exploring the
different combinations of for each batch of protein used and when a
new batch of buffer was prepared. LIRtide inhibited the GST-LC3b
interaction with an IC50 of 1.6 μM and was used as a control. For the
screening, conditions were chosen that could have identified
enhancers or inhibitors of the interaction. The signal/background
was >35; the estimated Z′ was 0.87. We present the data on the
screening of the Prestwick Chemical Library (Prestwick Chemical,
Illkirch, France) that contains 1280 mostly approved drugs (FDA,
EMA and other agencies (http://www.prestwickchemical.com/
prestwick-chemical-library.html). The library was screened at 50
μM. Compounds inhibiting <65% of remaining signal or increasing
the signal by >120% were identified. Those initial hits from the
screening were compared with hits on previous screening campaigns,
and hits that appeared previously were eliminated. Eighteen
compounds (nine potential inhibitors and nine potential enhancers
of the interaction) were selected and reordered. IC50’s were obtained
on reordered compounds, and the ability to affect the signal from the
LC3b-LIRtide assay was compared with the ability to affect the signal
obtained with biotinylated-GST (PerkinElmer). “Hit” compounds
that affected the control biotnylated-GST were presumed to affect the
alphascreen assay and were discarded. Novobiocin was the only
confirmed hit identified from the screening of the Prestwick library.
The hit was validated by obtaining the crystal structure of LC3A in
complex with Novobiocin.
Labeling of SNAP-LC3s with Terbium Cryptate. For covalent
coupling of terbium cryptate, we utilized SNAP-Lumi4-Tb (Cisbio
Bioassays, Codolet, France). A volume of 20 μL with 12 μM protein
(0,24 pmol) in SEC buffer was diluted with additional 360 μL of SEC
buffer and supplemented with 20 μL of a 10 μM dilution of SNAP-
Lumi4-Tb in DMSO. This resulted in a 20% molar excess of SNAP-
tag relative to the substrate and prevented remnants of free label. The
reaction was allowed to complete overnight at 4 °C. The product had
a protein concentration of 600 nM with approximately 80% being
labeled. Aliquots were stored at −80 °C.
Displacement Assay. Displacement of LIR-LC3 interaction was
studied by homogeneous time-resolved fluorescence resonance energy
transfer (HT-FRET or HTRF). The SNAP fused LC3 proteins were
incubated with SNAP-Lumi4-Tb labeling reagent (Cisbio assays,
France) overnight in a fridge.
Solutions containing 1 mM recombinant LIR-sequence fused to N-
terminal sGFP or sGFP alone as FRET acceptor and 10 nM FRET
donor complex with either LC3A or LC3B with N-terminal SNAP-tag
covalently coupled to terbium cryptate as well as 1% DMSO with test
compound at varying concentrations or DMSO alone were prepared
in HTRF buffer [25 mM HEPES pH 7.5 adjusted with KOH, 150
mM KF, 10% (w/v) glycerol, 0.1% (w/v) CHAPS, 5 mM DTT].
After 2 h incubation at RT, the fluorescence intensities (FI) at 520
For the purification of LC3A and LC3B, cell pellets containing the
expressed protein were suspended in buffer A (50 mM Tris pH 8, 500
mM NaCl, 20 mM imidazole, 5 mM β-mercaphtoethanol, and 5% (v/
v) glycerol) which was supplemented with one tablet of Complete
EDTA free protease inhibitor mix (Roche, Basel, Switzerland) and a
trace amount of DNase I (Applichem, Darmstadt, Germany). The
resuspended cells were lysed by using a liquid homogenizer
(Zelldisruptor, Constant Systems Ltd.), and cell debris were removed
by centrifugation (51 632g, 60 min, 4 °C). Nickel affinity
chromatography (GE Healthcare, Solingen, Germany) was used to
further purify the supernatant. Chromatography was performed as a
step gradient protocol using buffer A as the running buffer and buffer
B (identical to buffer A with an imidazole concentration of 400 mM)
as the elution buffer. The fractions containing the target protein were
pooled, before 1 mg of TEV protease per 10 mg of LC3 was added
and samples were dialyzed overnight against a 100-fold excess of
buffer A at 4 °C using a 3500 kDa membrane. Contaminations,
uncleaved protein, tags, as well as the TEV protease were removed by
reverse nickel affinity chromatography using buffers A and B. The flow
through was concentrated by ultrafiltration (3000 Da cutoff
membrane) and loaded onto a Superdex 75 HiLoad 16/600 column
(GE Healthcare, Germany) equilibrated with either HTRF buffer
(150 mM KF, 25 mM HEPES pH 7.5, 10% (w/v) glycerol, 5 mM
DTT) or crystallization buffer (20 mM Tris pH 8, 100 mM NaCl)
and run at 1 mL/min. The concentrations of the pooled protein
stocks were determined by using a Nanodrop spectrophotometer
(Implen, Muenchen, Germany), before flash freezing aliquots in liquid
nitrogen. Aliquots were stored at −80 °C.
For ITC and NMR experiments, all six human LC3 and GABARAP
proteins were cloned and expressed under a modified Ub tag23 and
purified as described previously.24 Prior to experiments, all proteins
were equilibrated against a buffer containing 50 mM Na2HPO4, 100
mM NaCl, and 5% DMSO, pH 7.0. Solutions of 1 were prepared in
the same buffer.
Differential Scanning Fluorimetry (DSF). DSF experiments of
LC3A and LC3B were performed in transparent 96-well PCR plates
(MicroAmp, Applied Biosystems). Protein/SYPRO orange solution in
HTRF buffer was mixed with and inhibitor solved in DMSO (or pure
DMSO in case of the control) in a final assay volume of 40 μL
containing 30 μM protein, 0,1% (w/v) CHAPS, 2.5× SYPRO orange,
300 μM inhibitor, as well as 1% (v/v) DMSO. Background
fluorescence was measured without enzyme to exclude inhibitor/
SYPRO orange interactions. A temperature-dependent increase of
fluorescence was measured from 25.0 to 79.8 °C in steps of 1 °C/1
min, using an Icycler IQ single color real time PCR system (BioRad).
The fluorescence was measured at an excitation wavelength of 490 nm
as well as an emission wavelength of 570 nm. The measurements were
performed as triplicate in three independent experiments. The first
derivatives of the measured melting curves were automatically
calculated with the MyIQ 1.0 software of the Icyler. Data was further
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analyzed in Microsoft Excel, were the respective maxima were
determined, which were considered as the melting point. For DSF
experiments with LC3C, GABARAP, GABARAPL1, and GABAR-
APL2, recombinant proteins at 2 μM in 10 mM HEPES, pH 7.5 and
100 mM NaCl were mixed with 20 μM inhibitors. The reaction was
incubated at room temperature for 10 min. SYPRO orange was added
at 1:1000 dilution, and the fluorescence signals corresponding to
temperature-dependent protein unfolding were measured using a real-
time PCR Mx3005p machine (Stratagene) at a heating rate of 1 °C/
min. Melting temperature shifts and the corresponding ΔTm values
were calculated using the previously described method.25
ITC Measurements. The ITC experiments were performed at 25
°C using a MicroCal VP-ITC microcalorimeter (Malvern Instruments
Ltd., UK). The 1 at concentrations of 0.4 mM were titrated into 0.020
mM LC3 and GABARAP proteins in 21 steps (10 μL per injection).
The proteins and 1 concentrations were calculated from the UV
absorption at 280 nm with a Nanodrop spectrophotometer (Thermo
Fisher Scientific, Wilmington, DE, USA). The ITC data was analyzed
with the MicroCal ITC software implemented in Origin 7.0 and fitted
based on a “one-side” binding reaction.
ITC experiments were performed on a low volume Affinity ITC
instrument (TA Instruments) with the protein solution in the sample
cell and the inhibitor solution in the syringe. All measurements were
performed at 25 °C and a stirring speed of 75 rpm. The protein and
inhibitor were diluted in HTRF buffer from the same batch used in
the purification. Samples were supplemented with CHAPS as well as
DMSO at final concentrations of 0.1% and 1% respectively. The
protein concentration used was 80 μM while the inhibitor
concentration was either 500 μM or 1 mM (inhibitor stocks were
dissolved in pure DMSO). The number of injections, the delay
between the injections, as well as the injection volume were optimized
for each compound individually. The linearity of the dilution heat was
tested in separate buffer in protein as well as inhibitor in buffer
titrations. ITC data was analyzed using NanoAnalyze Data Analysis
software (version 3.5.0) software. Baseline subtraction was performed
based on observed change in enthalpy in the last data points in each
measurement in case of linear reference blank experiments.
NMR Experiments. NMR experiments were performed on a
Bruker Avance spectrometers operating at proton frequencies of 600
and 700 MHz at 298 K. Titrations were performed with 150 μM 15N
LC3B samples to which 1 was added stepwise with increasing molar
ratios to 8 times molar excess according to the recent guidelines.26
[1H−15N] heteronuclear single quantum coherence (HSQC) spectra
were recorded at each step. Spectra were analyzed using the Sparky
3.114 software (University of California, San Francisco, CA, USA).
Crystallization. LC3A in crystallization buffer was concentrated
to a final concentration of 20 mg/mL, before spinning the protein
sample for 15 min at 4 °C and max speed in an Eppendorf bench top
centrifuge. The supernatant was mixed with 2 to a final concentration
of 5 mM and 5% DMSO before incubation on ice for 1 h. The
solution was centrifuged again for 15 min at 4 °C and max speed in an
Eppendorf bench top centrifuge, before the supernatant was used to
set up crystallization experiments. Crystallization experiments were
performed in 96-well sitting drop plates with a drop volume of 150 nL
in the ratios of 2:1, 1:1 and 1:2 with a JCSG7 instrument (MD1-
Custom M96-HT96 BN004). Crystallization plates were incubated at
20 °C, and crystals grew over the course of 3 weeks in the well with
0.2 M ammonium nitrate and 20% w/v PEG 3350. Crystals were
soaked with a mixture of 25% ethylene glycol and 75% precipitation
solution for cryoprotection, before fishing and flash freezing the
crystals in liquid nitrogen.
refinement showed that Rwork and Rfree factors of 0.1860 and
0.2122, respectively, were reached. Statistics of data collection and
structural refinement are summarized in Table S1. Binding of the
compound was validated using a polder omit map with a solvent
exclusion radius of 4 Å and a resolution factor of 0.25. The graphical
representations were made using MOE. The coordinates and
structure-factor amplitudes of the structure have been deposited in
the Protein Data Bank as entry 6TBE.
Lower abundance secondary conformations of amino acids, unclear
electron densities mainly at the surface of the protein, as well as one
density close to the inhibitor have not been modeled. RSZR for some
AS is higher with the most prominent outlines for the first and the last
resolved amino acid due to the low amount of visible density.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01564.
Full [1H−15N]-HSQC spectra of LC3B upon titration
with 1 and chemical shift perturbations of LC3B upon
interaction with 1; data collection and refinement
statistics; thermodynamic parameters of 1 binding to
LC3A and LC3B obtained by ITC (PDF)
Molecular formula strings (CSV)
HPLC-based purity of 6a, 14o, and 2 (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Ewgenij Proschak − Institute of Pharmaceutical Chemistry,
Goethe-University Frankfurt, 60438 Frankfurt, Germany;
Fraunhofer Institute for Translational Medicine and
Pharmacology ITMP, 60596 Frankfurt, Germany; German
translational cancer network (DKTK), 60438 Frankfurt,
Germany; orcid.org/0000-0003-1961-1859;
Email: proschak@pharmchem.uni-frankfurt.de
Authors
Markus Hartmann − Institute of Pharmaceutical Chemistry,
Goethe-University Frankfurt, 60438 Frankfurt, Germany
Jessica Huber − Institute of Biophysical Chemistry and Center
for Biomolecular Magnetic Resonance, Goethe-University
Frankfurt, 60438 Frankfurt, Germany
Jan S. Kramer − Institute of Pharmaceutical Chemistry,
Goethe-University Frankfurt, 60438 Frankfurt, Germany
Jan Heering − Fraunhofer Institute for Translational Medicine
and Pharmacology ITMP, 60596 Frankfurt, Germany;
orcid.org/0000-0002-4922-1993
Larissa Pietsch − Department of Internal Medicine I, Goethe
University Hospital Frankfurt, 60596 Frankfurt, Germany
Holger Stark − German translational cancer network
(DKTK), 60438 Frankfurt, Germany; Institute of
Pharmaceutical and Medicinal Chemistry, Heinrich Heine
University Dusseldorf, 40225 Duesseldorf, Germany;
̈
orcid.org/0000-0003-3336-1710
X-ray diffraction data was collected form a single crystal at the SLS
beamline. Processing of the obtained data was performed using the
XDS software package. The phaser program as part of the PHENIX
software package was used for the initial structure determination. As
starting model for the molecular replacement in internal LC3A, the
structure where coordinates for heteroatoms (water and ligands) were
excluded was used. The initial model was further optimized by several
iterative rounds of model building with Coot and the model
refinement using the PHENIX software package. The final round of
Dalibor Odadzic − German translational cancer network
(DKTK), 60438 Frankfurt, Germany
Iris Bischoff − Institute of Pharmaceutical Biology, Goethe-
University Frankfurt, 60438 Frankfurt, Germany
̈
Robert Furst − Institute of Pharmaceutical Biology, Goethe-
University Frankfurt, 60438 Frankfurt, Germany
Martin Schröder − Institute of Pharmaceutical Chemistry,
Goethe-University Frankfurt, 60438 Frankfurt, Germany
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Masato Akutsu − Buchmann Institute for Molecular Life
Sciences, Goethe-University Frankfurt, 60438 Frankfurt,
Germany
(trimethylsilyl)amide; LIR, LC3-interacting region; PE,
phosphatidyl-ethanolamine; UBL, ubiquitin-like proteins
Apirat Chaikuad − Institute of Pharmaceutical Chemistry,
Goethe-University Frankfurt, 60438 Frankfurt, Germany;
orcid.org/0000-0003-1120-2209
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Volker Dötsch − Institute of Biophysical Chemistry and
Center for Biomolecular Magnetic Resonance, Goethe-
University Frankfurt, 60438 Frankfurt, Germany
Stefan Knapp − Institute of Pharmaceutical Chemistry,
Goethe-University Frankfurt, 60438 Frankfurt, Germany;
German translational cancer network (DKTK), 60438
Frankfurt, Germany; orcid.org/0000-0001-5995-6494
Ricardo M. Biondi − Department of Internal Medicine I,
Goethe University Hospital Frankfurt, 60596 Frankfurt,
Germany
Vladimir V. Rogov − Institute of Pharmaceutical Chemistry,
Goethe-University Frankfurt, 60438 Frankfurt, Germany;
Institute of Biophysical Chemistry and Center for
Biomolecular Magnetic Resonance, Goethe-University
Frankfurt, 60438 Frankfurt, Germany
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01564
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Evelyn Su
̈
ss for technical assistance. E.P. was
supported by the Deutsche Forschungsgemeinschaft (DFG,
Heisenberg-Professur PR1405/7-1). A.C., S.K., and V.V.R. are
grateful for support by the Structural Genomic Consortium
(SGC). The SGC is a registered charity (No.: 1097737) that
receives funds from AbbVie, Bayer AG, Boehringer Ingelheim,
Canada Foundation for Innovation, Eshelman Institute for
Innovation, Genentech, Genome Canada through Ontario
Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/
KTH/Diamond, Innovative Medicines Initiative 2 Joint
Undertaking [EUbOPEN Grant 875510], Janssen, Merck
KGaA (aka EMD in Canada and USA), Merck & Co (aka
MSD outside Canada and USA), Pfizer, Sao Paulo Research
̃
Foundation-FAPESP, Takeda and Wellcome [106169/ZZ14/
Z]. This research was supported by the DFG funded
Collaborative Sonderforschungsbereich 1177 Autophagy
(SFB1177) at Frankfurt University as well as the German
Cancer Consortium (DKTK) and the Frankfurt Cancer Centre
(FCI). We acknowledge the Paul Scherrer Institut, Villigen,
Switzerland for provision of synchrotron radiation beamtime at
beamline Beamline X06SA of the SLS.
ABBREVIATIONS
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AIM, Atg8-interacting motif; Atg, autophagy related proteins;
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homogeneous time-resolved FRET; ITC, isothermal titration
calorimetry; LC3, microtubule-associated proteins 1A/1B light
chain 3; LDS, LIR docking site; LiHMDS, lithium bis-
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