Identification of Eukaryotic Translation Elongation Factor 1-α 1 Gamendazole-Binding Site for Binding of 3-Hydroxy-4(1 H)-quinolinones as Novel Ligands with Anticancer Activity

Article abstract of DOI:10.1021/acs.jmedchem.8b00078

Here, we have identified the interaction site of the contraceptive drug gamendazole using computational modeling. The drug was previously described as a ligand for eukaryotic translation elongation factor 1-α 1 (eEF1A1) and found to be a potential target site for derivatives of 2-phenyl-3-hydroxy-4(1H)-quinolinones (3-HQs), which exhibit anticancer activity. The interaction of this class of derivatives of 3-HQs with eEF1A1 inside cancer cells was confirmed via pull-down assay. We designed and synthesized a new family of 3-HQs and subsequently applied isothermal titration calorimetry to show that these compounds strongly bind to eEF1A1. Further, we found that some of these derivatives possess significant in vitro anticancer activity.

Full text of DOI:10.1021/acs.jmedchem.8b00078

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Article
Identification of eEF1A1 Gamendazole-Binding Site for Binding of 3-
Hydroxy-4(1H)-Quinolinones as Novel Ligands with Anticancer Activity
Kristyna Burglova, Gabriela Rylova, Athanasios Markos, Hana Prichystalova,
Miroslav Soural, Marek Petracek, Martina Medvedikova, Gracian Tejral,
Bruno Sopko, Pavel Hradil, Petr Dzubak, Marian Hajduch, and Jan Hlavac
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00078 • Publication Date (Web): 02 Mar 2018
Downloaded from http://pubs.acs.org on March 2, 2018
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Identification of eEF1A1 GamendazoleꢀBinding Site
for Binding of 3ꢀHydroxyꢀ4(1H)ꢀQuinolinones as
Novel Ligands with Anticancer Activity
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Kristyna Burglová,1‡ Gabriela Rylová,1‡Athanasios Markos,1 Hana Prichystalova,1Miroslav
Soural,1Marek Petracek,1 Martina Medvedikova,1Gracian Tejral,2,4,5 Bruno Sopko,3,4 Pavel
Hradil,1Petr Dzubak,1 Marian Hajduch1*and Jan Hlavac6*
1Institute of Molecular and Translation Medicine, Faculty of Medicine and Dentistry, Palacký
University, Hněvotínská 5, 779 00 Olomouc, Czech Republic
2Department of Biophysics, Second Faculty of Medicine, Charles University, V Úvalu 84, 150
06 Praha 5, Czech Republic
3Department of Medical Chemistry and Clinical Biochemistry, Second Faculty of Medicine,
Charles University and Motol University Hospital, V Úvalu 84, 150 06 Praha 5, Czech Republic
4Department of Tissue Engineering, The Czech Academy of Sciences, Institute of Experimental
Medicine, Vídeňská 1083, 142 20 Praha 4, Czech Republic
5University Center for Energy Efficient Buildings (UCEEB), The Czech Technical University in
Prague, Třinecká 1024, 273 43 Bustehrad, Czech Republic
6Department of Organic Chemistry, Faculty of Science, Palacký University, Tř. 17. listopadu 12,
771 46 Olomouc, Czech Republic
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KEYWORDS. translation elongation factor, 3ꢀhydroxyꢀ4(1H)ꢀquinolinones, molecular docking,
affinity chromatography, isothermal titration calorimetry
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ABSTRACT. Here, we have identified the interaction site of the contraceptive drug gamendazole
using computational modeling. The drug was previously described as a ligand for eukaryotic
translation elongation factor 1ꢀalpha 1 (eEF1A1) and found to be a potential target site for
derivatives of 2ꢀphenylꢀ3ꢀhydroxyꢀ4(1H)ꢀquinolinones (3ꢀHQs) which exhibit anticancer
activity. The interaction of this class of derivatives of 3HQs with eEF1A1inside the cancer cells
was confirmed via pullꢀdown assay. We designed and synthesized a new family of 3ꢀHQs and
subsequently applied isothermal titration calorimetry to show that these compounds strongly
bind to eEF1A1. Further, we found some of these derivatives possess significant in vitro
anticancer activity.
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Introduction
eEF1A1), the second most abundant eukaryotic protein, is responsible for ribosomal protein
synthesis. It is also involved in cytoskeletal organization and other physiological activities.1
Given its multifunctional property, eEF1A1 is considered as a moonlighting protein.2 Moreover,
eEF1A1 is associated with the development and progression of various cancers,3 6 and has thus
-
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been proposed as a target for anticancer therapy.7
The overexpression and depletion of
,
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eEF1A1 have been shown to influence the cell apoptosis rate.9
Although eEF1A1was
,
identified as a target a long time ago, only a few biological active compounds have been found to
interact with eEF1A1. The male contraceptive agent gamendazole (Figure 1) binds directly to
eEF1A1 but does not affect protein synthesis.11 The phosphodiesterase inhibitor cilostazol
(Figure 1) influences neurite outgrowth by binding to eEF1A1.12 Narciclasine (Figure 1) and its
analogs were reported to bind to eEF1A1 and overcome drug resistance by melanoma.13
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However, the authors were not successful in the validation of the binding site. eEF1A1 has been
described as a target for two natural flavonoid derivatives (Figure 1)14, which exhibit high
efficacy against breast cancer cells through their interaction with eEF1A1. However, despite this
fact, a binding site has not been elucidated. Although an anticancer activity of such flavonoids
caused by interaction with eEF1A1 has been shown (13,14), the systematic study of the
description of binding site, in silico design, and synthesis of a set of compounds followed by
verification of their biological activity against various types of cancer needs to be performed.
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Several studies have reported that flavonoid azaꢀanalogs – derivatives of 2ꢀphenylꢀ3ꢀhydroxyꢀ
4(1H)ꢀquinolinone (3ꢀHQ)– exhibit significant anticancer activities.15ꢀ18 However, until now, no
study has described their molecular target. We hypothesized that3ꢀHQs due to its structural
similarity to other flavonoid derivatives may serve as good ligands for interaction with eEF1A1.
In this report, we provide evidence of interaction of 3ꢀHQ derivatives with eEF1A1 and the
results of the rational design, synthesis, and in vitro biological evaluation of the new 3ꢀHQ
derivatives.
Figure 1. Structures targeting eEF1A1.11ꢀ14
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Results and discussion
Interaction of 3ꢀHQs with eEF1A1. The possible interaction of 3ꢀHQs with eEF1A1 was first
studied via docking experiments. Three ligands were chosen from the group of previously
published active derivatives substituted by an amine at position 4´ and a nitro group at position
3´ of the 2ꢀphenyl ring (Figure 2).15
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Figure 2. The reported IC50 (µM) of selected 3ꢀHQs.19
Comp.
No
CCRFꢀ
CEM
CEMꢀDNRꢀ
BULK
K562ꢀ
Tax
R
U2OS
K562
A549
BJ
1.55
1.72
1.42
3.00
6.75
3.01
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8.57
9.98
0.75
0.75
0.66
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2.71
1.96
1.62
1.89
0.94
9.83
9.73
9.39
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CCRFꢀCEM, acute lymphoblastic leukemia; CEMꢀDNRꢀBULK, CCRFꢀCEM cells, daunorubicin resistant; K562, acute myeloid
leukemia; K562ꢀTAX, K562 cells paclitaxel resistant; A549, lung adenocarcinoma; BJ, normal cycling fibroblasts cell line.
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Since the crystallographic structure of human eEF1A1 is unavailable, a homology model was
built using its known sequence (UniProt database ID: P6810420) and crystal structures of
homologous eEF1A1from yeast and rabbit. The eEF1A1 protein structure consists of three
domains connected by two coiled links in sequential order. The first domain is a combination of
αꢀhelices and βꢀsheets. The second and third domains consist primarily of βꢀsheets. First, we
tried to verify the chosen docking method by using the known ligand, gamendazole as a
standard, for which the protein binding site was previously identified by MALDIꢀTOF.11 The
results of this docking experiment presented in Figure 3ꢀA and Table 2 show positive interaction
of our model with gamendazole. Moreover, by comparing these results with the abovementioned
binding site, we determined the amino acid sequence of the MALDIꢀTOF identified domain.
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To better understand the binding site, we performed the docking experiments with the previously
characterized crystal structures of the homologous proteins from yeast (1F60, 1G7C, 1IJE, 1IJF,
2B7B, and 2B7C21ꢀ25). All these docking experiments revealed the interaction of gamendazole
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with eEF1A1 with comparable docking energy in aaꢀtRNA binding site.26
A verification of
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binding site using known crystal structures of rabbit origin (4C0S21ꢀ25) could not be performed
because of absence of crucial amino acids together with one artificially modified residue in the
identified aaꢀtRNA binding site.21ꢀ25Among all the used crystal structures, 2B7C and 2B7B
exhibited interaction with eEF1A1 also in proximity to the domain identified by MALDIꢀTOF
and not confirmed by our docking to human eEF1A1 model (see Figure 3). The docking energy
is comparable to the docking in the aaꢀtRNA binding site (see SI, Table S1). The onlyꢀMALDIꢀ
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TOF identified domain 11 is known to be highly flexible.26
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Figure 3. The results of docking of gamendazole (A) and compounds 1ꢀ3 with the homology
model of human eEF1A1 described in Figure 2 (B, C, and D, respectively). Ligands in opaque
red denote more favorable docking energy. The parts of the structures in green show the
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interaction sites identified by both MALDIꢀTOF and our docking; those in yellow indicate
sites identified by MALDIꢀTOF 11 only.
We found that the overall RMS between structures 2B7C and 1F60 is 1.13 Å (0.26 pm per
residue), while RMS between the onlyꢀMALDIꢀTOF identified domains of these structures is
2.18 Å (4.24 pm per residue). The RMS between rest of the molecules in both protein structures
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is 0.89 Å (0.24 pm per residue). We reasoned that due to the reason, no interaction of
gamendazole in the onlyꢀMALDIꢀTOF domain was found with protein crystal structures 1G7C
or 1IJF. Interaction in this domain is also less favorable in the case of 1IJE and 1F60 (see SI,
Table S1). These facts led to the conclusion that our model for interaction with human eEF1A1
is valid. The 3ꢀHQ derivatives 1ꢀ3 (Figure 2) were subjected to the docking study and these
compounds docked into the same site as gamendazole (Figure 3ꢀB,C,D). The conformation of the
protein is less favorable for interaction with ligands in the highly flexible onlyꢀMALDIꢀTOF
identified domain. This discrepancy is likely because our model does not address the loop and
connecting domain flexibility.11
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To provide evidence about the interaction of 3ꢀHQs with eEF1A1, we performed pullꢀdown
assay with ligands A and B (Figure 4), derived from original compound 3 and immobilized on
magnetic beads via streptavidinꢀbiotin interaction.28 Ligand A was attached to biotin via
quinolinone benzene ring to enable interaction of proteins with 2ꢀphenyl moiety, whereas ligand
B was biotinylated via 2ꢀphenyl ring to enable interaction of proteins with the opposite part of
the 3ꢀHQ scaffold. The synthesis of the biotinylated 3ꢀHQs was performed using polystyrene
resin with the combination of previously published protocols (see SI).29
Figure 4. Ligand A and B derived from active compound 3 used for pullꢀdown assay.
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OH
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Pullꢀdown assay for both derivatives was performed with total protein lysate prepared from
CCRFꢀCEM or U2OS cells. Proteins released from the ligands were analyzed using SDSꢀPAGE
(Figure 5) and then subjected to mass spectrometry characterization. The specific ligandꢀprotein
interaction was confirmed only for derivative A, suggesting the 2ꢀphenyl moiety is the part of
pharmacophore. In addition to the expected presence of eEF1A1 in the mixture of isolated
proteins, we confirmed the presence of pyruvate kinase M2 (PKM2) and βꢀactin.
Figure 5. SDSꢀPAGE analysis of proteins isolated from pullꢀdown assay of derivatives A and B
incubated with CCRFꢀCEM protein lysate. The blank sample is represented by immobilization of
biotin to streptavidinꢀcoated magnetic beads.
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To validate identified targets, we performed western blot to visualize isolated proteins. We
identified specific bands for PKM2, eEF1A1, and βꢀactin only for proteins captured by
derivative A; while no signal for these proteins was detected for derivative B or biotinylated
beads (Figure 6).
Figure 6. Characterization of proteins isolated from pullꢀdown assay of derivatives A and B by
western blot.
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Structure design. Structural motifs of the compounds 1ꢀ3 were used to design a chemical analog
set of potentially improved 3HQ inhibitors. The toxicity of these compounds against normal cells
could be diminished by replacing the nitro group with its isosters such as carboxy or
carboxamide motifs (R1 in Figure 8). The amines substituted at position 4´ of the 2ꢀphenyl ring
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were selected with respect to previous studies (Figure 2) and were extended to a morpholine
ring (R2 in Figure 8). Moreover, the substitution of the benzene ring of the quinolinone skeleton
by hydrophobic moieties was previously reported to positively influence its cytotoxicity and
selectivity.18 According to the proved interaction of derivative A with EF1A1, there is no
negative effect on interaction when the moiety is bound via amidic bond at position 7. Therefore,
the pentyl and benzylamide groups in this position were selected as representatives. By
combining the three diverse positions, R1, R2 and R3, an analog set of 3ꢀHQs was compiled
(Figure 8) and subjected to molecular docking simulations with eEF1A1.
Figure 8. Analog set of 3ꢀHQs suggested for molecular docking.
The analog set of 3ꢀHQ derivatives (Figure 8) was constructed using ab initio methods and then
subjected to virtual screening. The derivatives with the most favorable docking energy (docking
energy lower than ꢀ35589.5 J.molꢀ1) were selected. The final selection of 3ꢀHQ derivatives was
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performed based on the ease of synthesis. As a result, seven 3ꢀHQ derivatives were selected and
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synthesized (see Table 1).
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Table 1. Synthesized 3ꢀHQ derivatives.
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Comp. No
R1
R2
R3
ꢀCO2H
ꢀCO2H
ꢀCONH2
ꢀCONH2
ꢀCONH2
ꢀCONH2
ꢀH
ꢀH
4
5
ꢀH
ꢀH
ꢀH
6
7
ꢀCONHꢀPent
ꢀCONHꢀBn
ꢀH
8
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ꢀH
10
The selected compounds included two cyclic amines (piperidine and morpholine) as R2
substituents and substitution at position R1 by the carboxylate and carboxamide functionalities.
Further, unsubstituted derivative 10 was included as a control; it was previously described as
inactive in cytotoxic assay.16 In addition, molecular docking simulation also classified this
compound as unsuitable for the target (Figure 9ꢀB). The results of the molecular docking of the
selected compounds to eEF1A1 are presented in Table 2. Most of the compounds bind to the
same site as gamendazole, with binding energies ranging from ꢀ38520.4 to ꢀ41032.6 J.molꢀ1
(Table 2, Figure 9ꢀA and SI). The results of compound 10 are in accordance with the expectation
because it binds at a different location on the protein surface with the energy of ꢀ34752.1 J.molꢀ1
(Figure 9ꢀB). Some weak interactions of compound 10 with the gamendazole docking site with
the energy of ꢀ31821.2 J.molꢀ1 were also revealed. We identified that all studied compounds
share same binding site which is located at the center of eEF1A (Table 2). The compounds
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interacted with the loop between the first and second domains and was identified as part of the
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aaꢀtRNA binding site.26 27. Moreover, this site is positioned opposite to that of the GTP, GDP,
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and GMP binding pockets as observed in the known PDB structures.21 22 These results suggest
,
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that the interaction between our compounds and eEF1A1 would not reduce eEF1A1´s activity by
inhibiting the GTP binding site.
Figure 9. Docking of unsubstituted derivative 10 (B) compared with active derivative 6 (A) to an
eEF1A1 model. Ligands shown in opaque red denote more favorable docking energy. The parts
of the structures in green show the interactions identified by both MALDIꢀTOF11 and our
experiment; those in yellow indicate the interactions identified by MALDIꢀTOF.11
Synthesis. Derivatives 1ꢀ3 were synthesized according to a published protocol.19 The synthesis
of carboxylate and carboxamide analogs 4ꢀ7 is displayed in Scheme 1. Phenacyl ester 12was
prepared by the reaction of anthranilic acid with bromoketone11. The cyclization to 3ꢀHQ 13was
inspired by a wellꢀestablished method described previously.30 31Hydrolysis of the methyl ester
,
afforded carboxylate 3ꢀHQ 14 and its following amidation, carboxamide 3ꢀHQ 15. These
derivatives were subsequently transformed to the desired 3ꢀHQ 4ꢀ5 and 6ꢀ7 by nucleophilic
substitution with secondary amines (piperidine or morpholine) in a microwave reactor.
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Introduction of carboxamide moiety into position 7 of the 3ꢀHQ 8 and 9 was achieved in the first
step of the synthesis (Scheme 2).
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Scheme 1.Synthesis of selected 3ꢀHQs.
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Starting 2ꢀaminoꢀ4ꢀmethylterephthalate was converted to amide 16or 17 by reaction with pentylꢀ
or benzylamine, respectively, in pyridine. Hydrolysis of ester group by sodium hydroxide to
obtain the first building blocks 18 and 19 followed. Second building block 23 was gradually built
up from ester 20 by its successive hydrolysis, chlorination, and amidation to form amide 21
followed by substitution of chlorine with piperidine and finally αꢀbromination (Scheme 2).
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Scheme 2. Synthesis of designed 3ꢀHQs substituted by 7ꢀcarboxamide group.
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Biological evaluation. The interaction between the prepared compounds and eEF1A1 was
studied using isothermal titration calorimetry (ITC). ITC measures the changes in heat during
interaction and provides thermodynamic information about the binding affinity of a ligand to a
protein. The measured enthalpies for the interactions of the synthesized 3ꢀHQ derivatives with
the protein and the Ka values suggest high affinities (Table 2). The only exception within the
series is the nonꢀsubstituted derivative 10, used here as a negative control, which exhibited no
interaction with the protein. This result is in accordance with the poor molecular docking results
32
of the compound 10. Similarly, gamendazole in the form of analytical standard was subjected
to the interaction studies as a positive control. As expected, binding of gamendazole with eEF1A
was confirmed, although the interaction is much weaker than those of 3ꢀHQs (see the titration
profile in SI).
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Table 2. Thermodynamic and binding parameters for the interaction of the 3ꢀHQ compounds
8
9
ө
with eEF1A1 (ꢀrHm = enthalpy change of binding; Ka= association constant; n = stoichiometry;
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ө
ө
ө
ꢀrSm = entropy change, ꢀrGm = Gibbs energy change, Docking ꢀrGm = Gibbs energy change
determined by molecular modeling).
Compound
ꢀrHmө(kJ molꢀ
ꢀ11272
ꢀ1872
ꢀrSmө(J molꢀ1 Kꢀ
ꢀ37686
ꢀrGmө(J molꢀ Docking ꢀrGmө(J molꢀ
Ka (Mꢀ1)
2.536 x
1.029 x
2.899 x
6.796 x
5.297 x
1.809 x
n
ө
0.798
1.640
0.932
0.746
1.129
1.694
no
ꢀ35919
ꢀ39272
ꢀ36347
ꢀ38777
ꢀ37990
ꢀ40713
ꢀ38911
ꢀ38911
ꢀ38492
ꢀ38911
ꢀ38911
ꢀ43932
4
5
6
7
8
9
10
ꢀ6147
ꢀ53539
ꢀ26474
ꢀ31454
ꢀ15999
ꢀ7932
ꢀ9416
ꢀ9213
ꢀ30764
1,268 x
ꢀ1209
1,777
ꢀ3919
ꢀ40550
ꢀ36401
Gamendazole
The value of the stoichiometric binding number (n) is approximately 1 for all the compounds.
The negative changes in free energy and enthalpy confirm that the binding of the ligands to
eEF1A1 is spontaneous and exothermic. An example of a calorimetric titration profile showing
the ligandꢀprotein interaction, i.e., that of derivative 6 in comparison with the nonꢀinteracting
derivative10, is given in Figure 6 (for the titration profiles of other compounds, see SI).
Figure 6. Calorimetric titration profile for the interaction of 3ꢀHQ 6 (left) and 10 (right) with
eEF1A1.
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9
Finally, we tested the in vitro activity of the prepared derivatives against cancer cell lines (Table
3). Compared to original compound 1, the replacement of nitro group by carboxy moiety
(derivatives 4 and 5) caused the loss of activity. This could be caused by the carboxy group
itself, which apparently restricts the penetration of the compounds through the cell membrane.
Transformation of carboxy group to carboxamide (derivative 6) significantly improved the
activity in comparison with derivatives 4 and 5 and reached the similar values as observed for
derivative 1, except for CEMꢀDNR and U2OScell lines. The toxicity to normal cell line BJ was
improved by one order. When the piperidine ring was replaced with morpholine (compare
derivatives 6 and 7), the activity was lost. As predicted, the improvement of toxicity against
majority of the cancer cell lines was achieved by insertion of pentylamide moiety to position 7
(derivative 8). In the case of improved toxicity,submicromolar IC50values were achieved for two
lines. Unfortunately, the toxicity against normal BJ cells was similar to that of original
compound 1. Replacement of pentylamide with benzylamide group (derivative 9) led to IC50
values at micromolar range for adenocarcinoma A549 line, leukemia cell lines K562, CCRFꢀ
CEM, and colorectal tumor HCTꢀ116. Similarly, the compound displayed submicromolarIC50
values for osteosarcoma cell lines (U2OS), whereas it did not exhibit activity against cancer cell
lines (CEMꢀDNR and K562ꢀTAX) resistant to chemotherapy. Most importantly, toxicity of 9
against normal cell line (BJ) was significantly higher than that of original compounds 1ꢀ3.
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Table 3. Cytotoxic activity of prepared derivatives against cancer cell lines.
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ꢀCO2H
ꢀCO2H
1ꢀpiperidinyl
4ꢀmorpholinyl
1ꢀpiperidinyl
Hꢀ
50
50
50
50
49.56
49.56
50
50
50
50
50
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50
4
5
Hꢀ
ꢀCONH2
Hꢀ
Hꢀ
9.96 26.02 3.34 21.43 4.46
7.1
50
6.41
50
48.92
50
6
ꢀCONH2 4ꢀmorpholinyl
44.37
1.89
50
16.24
1.73
1.73
50
30.6
0.64
1.01
7
ꢀCONH2
ꢀCONH2
ꢀH
1ꢀpiperidinyl
1ꢀpiperidinyl
ꢀH
C5H11CONHꢀ
0.56
0.58
8.03
>50
2.39
8.30
32.71
>50
10.24
>50
8
PhCH2CONHꢀ 3.77
9
Hꢀ
>50
46.96 26.74 22.49 44.20 47.96
27.44
>50
10
Conclusion
We suggested previously reported cytotoxic derivatives of 2ꢀphenylꢀ3ꢀhydroxyꢀ4(1H)ꢀ
quinolinone as the ligands for eEF1A1 and via docking experiments we provided evidence of
their interaction with this target. We further described in detail the binding site of these
compounds which are similar to contraceptive agent gamendazole. The results obtained via
computational study were corroborated by pullꢀdown assay. The knowledge was used for the
design of advanced derivatives of 3ꢀHQs and their interaction with eEF1A1 was confirmed using
micro calorimetric measurement. Some of these derivatives possess remarkable inhibitory
activity against selected cancer cell lines and low toxicity against normal cell line, namely
fibroblasts BJ. In summary, this study is the first report describing rational design and synthesis
of anticancer compounds acting as eEF1A1 ligands using in silico technique and demonstrating
the molecular target for 3ꢀHQ derivatives, which have been reported as potent anticancer agents.
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Detailed elucidation of binding site can serve for future design of new eEF1A1 ligands to inhibit
this relatively unexplored molecular target and set up new strategy in anticancer treatment.
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Experimental Section
Materials and Methods
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For preparation and characterization of the compounds, LC/MS analyses were performed using
UHPLC/MS with UHPLC chromatograph (Acquity) attached with a PDA detector, a single
quadrupole mass spectrometer (Waters), an XꢀSelect C18 column at 30 °C with a flow rate of
600 µl/min. The mobile phase consisted of (A) 0.01 M ammonium acetate in water and (B)
acetonitrile, with a linear gradient over the course of 2.5 min; the final ratio was maintained for
1.5 min. The column was reꢀequilibrated with 10% B for 1 min. The APCI ionization operated at
a discharge current of 5 µA, a vaporizer temperature of 350 °C, and a capillary temperature of
200 °C. Purity of the compounds was determined using the ratio of the appropriate peak area to
sum of areas of all peaks of the mixture. Areas were determined by integration of the peaks from
the PDA detector response. Purification was performed using semiꢀpreparative HPLC with a
Waters 1500 series HPLC equipped with a 2707 Autosampler, a 1525 binary HPLC pump, a
2998 Waters Photodiode Array Detector, and a Waters Fraction Collector III with a YMC C18
reverse phase column (20 × 100 mm, 5 µm particle size). The mobile phase consisted of
acetonitrile and a 10 mM aqueous ammonium acetate gradient over 6 min. The purity of the
tested compounds was determined by HPLC, which in all the cases was ≥96%.
NMR spectra were measured in DMSOꢀd6 using a Jeol ECXꢀ500 (500 MHz) spectrometer or a
Bruker Avance (300 MHz) instrument. Chemical shifts (δ) are reported in parts per million
(ppm) and coupling constants (J) are reported in Hertz (Hz).
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Solvents and chemicals were purchased from SigmaꢀAldrich (Milwaukee, IL,
www.sigmaaldrich.com).
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All compounds tested on the biological activity were successfully filtered for PAINS.
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Synthesis of studied compounds
2ꢀ(2ꢀ(2ꢀ(4ꢀ(Ethylamino)ꢀ3ꢀnitrophenyl)ꢀ3ꢀhydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ7ꢀ
carboxamido)ethoxy)ethyl 2ꢀ(2ꢀoxohexahydroꢀ1Hꢀthieno[3,4ꢀd]imidazolꢀ4ꢀyl)acetate(A)
Ligand A was prepared according to the following scheme:
The compound 26was prepared on solid support (BAL linker) according to the published
procedure.33 The resin 26was then reacted with solution of biotin (0.24 g, 1.0 mmol), HOBt (135
mg, 1.0 mmol), N,Nꢀdiisopropylethylamine (DIEA) (0.05 ml, 0.3 mmol), and DIC (0.15 ml, 0.3
mmol) in DMF (2 ml) for 16 h at room temperature. The obtained resin 27was then washed three
times with DMF (3× 2 ml) and DCM (3 ×2 ml) and the compound was cleaved from the resin by
50% trifluoracetic acid (TFA) in DCMand dried undera stream of nitrogen. Final cyclization was
performed by refluxing the cleaved compound in acetic acid (AcOH) for 2 h. The obtained
ligand A was purified by HPLC, which resulted in~25%yield. MS (APCI) exact mass calculated
for C16H11ClNO4 [M+H]+: 683.25; found: 683.70. H NMR (300 MHz, DMSOꢀd6) δ ppm 11.70
1
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(br, 1 H), 8.60 ꢀ 8.76 (m, 2 H), 8.39 (t, J = 5.58 Hz, 1 H), 8.24 (br, 1 H), 8.18 (d, J = 8.42 Hz, 1
H), 8.05 (d, J = 8.42 Hz, 1 H), 7.67 (d, J = 8.60 Hz, 1 H), 7.25 (d, J = 9.15 Hz, 1 H), 6.38 (d, J =
18.66 Hz, 2 H), 4.04 ꢀ 4.19 (m, 3 H), 3.18 ꢀ 3.69 (m, 15 H), 2.79 (dd, J1= 12.26 Hz, J2 = 4.94 Hz,
1 H), 2.55 (d, J = 12.44 Hz, 1 H), 2.25 (t, J = 7.41 Hz, 2 H), 1.37 ꢀ 1.65 (m, 3 H), 1.27 (t, J =
7.14 Hz, 3 H).
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2ꢀ(2ꢀ((4ꢀ(3ꢀHydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolinꢀ2ꢀyl)ꢀ2ꢀnitrophenyl)amino)ethoxy)ethyl 2ꢀ
(2ꢀoxohexahydroꢀ1Hꢀthieno[3,4ꢀd]imidazolꢀ4ꢀyl)acetate (B)
Ligand B was prepared according to the following scheme:
First, the polymerꢀsupported biotinylated spacer 28was prepared according to previously
published procedure.34 The resin 28(300 mg) was then shaken in the solution of 2ꢀ(4ꢀchloroꢀ3ꢀ
nitro)phenylꢀ3ꢀhydroxyquinolinꢀ4(1H)ꢀone (3 mmol) and DIEA (0.15 ml, 1 mmol) in DMSO (9
ml) for 16 h at room temperature. Acidꢀmediated cleavage (50% TFA in DCM, 30 min, room
temperature) and HPLC purification were performed to obtain final ligand B.
General procedure for preparation of compounds 4ꢀ7: Derivative 14 or 15 (0.28 mmol) was
dissolved in corresponding amine (3 ml) and the mixture was stirred in microwave reactor for 6 h
at 200 °C and 200 W. The precipitate formed was filtered, washed with water, and dried under
vacuum.
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5ꢀ(3ꢀHydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolinꢀ2ꢀyl)ꢀ2ꢀ(piperidinꢀ1ꢀyl)benzoic acid (4): Yellow
5
6
1
powder. HPLC purity: 98%. Yield: 0.14 g (40%). H NMR (500 MHz, DMSOꢀd6) δ ppm 11.64
7
8
9
(br, 1H), 8.43 (s, 1 H), 8.15 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 8.5 Hz,
1H), 7.71 (d, J = 8.5 Hz, 1H), 7.60 (t, J = 7.0 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 3.12 (t, J = 5.0
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13
Hz, 4H), 1.81 – 1.74 (m, 4H), 1.68 – 1.60 (m, 2H). C NMR (126 MHz, DMSOꢀd6) δ ppm
170.1, 166.6, 151.1, 138.1, 138.0, 134.2, 131.5, 130.7, 130.3, 130.0, 124.7, 124.5, 122.6, 121.9
(2C), 118.4, 53.5, 25.6, 22.3. HRMS (ESI) calculated for [M+H]+ C21H20N2O4 365.1496; found:
365.1495.
5ꢀ(3ꢀHydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolinꢀ2ꢀyl)ꢀ2ꢀmorpholinobenzoic
acid
(5):Yellow
1
powder. HPLC purity: 98%. Yield: 0.05 g (47%). H NMR (500 MHz, DMSOꢀd6) δ ppm 11.58
(s, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.00 (dd, J1= 8.5 Hz, J2 = 2.0 Hz, 1H),
7.71 (d, J = 8.5 Hz, 1H), 7.62 ꢀ 7.58 (m, 2H) 7.27 (t, J = 7.5 Hz, 1H), 3.81 (t, J = 4.5 Hz, 4 H),
13
3.13 (t, J = 4.5 Hz, 4H). C NMR (126 MHz, DMSOꢀd6) δ ppm 172.0, 170.0, 167.3, 151.2,
138.1, 137.9, 133.9, 131.7, 130.6, 130.2, 127.8, 124.5, 124.3, 121.9, 121.8, 120.9, 119.5, 118.4,
66.2, 52.2. HRMS (ESI) calculated for [M+H]+ C20H19N2O5 367.1288; found: 367.1288.
5ꢀ(3ꢀHydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolinꢀ2ꢀyl)ꢀ2ꢀ(piperidinꢀ1ꢀyl)benzamide (6): Yellow
powder. HPLC purity: 98%. Yield: 0.14 g (48%);1H NMR (500 MHz, DMSOꢀd6) δ ppm 8.49
(br, 1H), 8.18 (s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.88 (br, 1H), 7.72 (br, 1H), 7.66 (s, 1H), 7.58 (t,
J = 7.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 2.97 (t, J = 4.5 Hz, 4H), 1.73 –
13
1.66 (m, 4H), 1.57 – 1.50 (m, 2H). C NMR (126 MHz, DMSOꢀd6) δ ppm 169.8, 167.9, 152.5,
138.0, 137.9, 132.3, 131.0, 130.9, 130.4, 128.1, 126.3, 124.4, 121.8, 119.3, 118.4, 53.6, 25.8,
23.3. HRMS (ESI) calcd for [M+H]+ C21H22N3O3 364.1656; found: 364.1654.
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5ꢀ(3ꢀHydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolinꢀ2ꢀyl)ꢀ2ꢀmorpholinobenzamide (7): Yellow powder.
HPLC purity: 99%. Yield: 0.10 g (56%);1H NMR (500 MHz, DMSOꢀd6) δ ppm 11.53 (br, 1H),
8.26 (s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 2.5 Hz, 1H), 7.88 (dd, J1= 2.0 Hz, J2 = 8.0 Hz,
1H), 7.72 (d, J = 8.6 Hz, 1H), 7.63 (s, 1H), 7.58 (td, J1= 1.5 Hz, J2 = 7.0 Hz, 1H), 7.32 – 7.23 (m,
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2H), 3.78 (t, J = 3.5 Hz, 4H), 3.03 (t, J = 3.5 Hz, 4H). C NMR (126 MHz, DMSOꢀd6) δ ppm
169.9, 168.4, 151.0, 138.0, 137.8, 132.2, 130.9, 130.8, 130.5, 128.6, 126.1, 124.4, 121.8 (2C),
118.6, 118.4, 66.2, 52.3. HRMS (ESI) calculated for [M+H]+ C20H20N3O4 366.1448; found:
366.1447.
2ꢀ(3ꢀCarbamoylꢀ4ꢀ(piperidinꢀ1ꢀyl)phenyl)ꢀ3ꢀhydroxyꢀ4ꢀoxoꢀNꢀpentylꢀ1,4ꢀdihydroquinolineꢀ
7ꢀcarboxamide (8)
The phenacylester24 (0.08 g, 0.2 mmol) was dissolved in glacial acetic acid (3 ml) and the
mixture was refluxed for 2 h. After cooling down to room temperature, the mixture was poured
into iceꢀcold water. The precipitate was filtered off, washed with 10% solution of NaHCO3 and
1
dried under the vacuum. Green powder. HPLC purity: 99%. Yield: 0.07 g (92%). H NMR (500
MHz, DMSOꢀd6) δ ppm 11.71 (s, 1H), 8.71ꢀ8.53 (m, 1H), 8.48 (s, 1H), 8.27ꢀ8.08 (m, 3H), 7.89
(d, J = 7.4 Hz, 1H), 7.72ꢀ7.52 (m, 2H), 7.33 (d, J = 8.6 Hz, 1H), 3.33ꢀ3.21 (m, 2H), 3.03ꢀ2.92
(m, 4H), 1.75ꢀ1.62 (m, 4H), 1.62ꢀ1.48 (m, 4H), 1.37ꢀ 1.26 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H). 13C
NMR (126 MHz, DMSOꢀd6) δ ppm 169.5, 168.0, 165.8, 152.6, 138.4, 137.5, 136.6, 132.3,
131.6, 131.1, 128.1, 125.8, 124.6, 122.8, 119.7, 119.2, 118.4, 53.6, 40.0 (overlay with solvent),
28.7 (2C), 25.8, 23.3, 21.9, 13.9. HRMS (APCI) calculated for [M+H]+ C27H32N4O4 477.2496;
found: 477.2503.
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NꢀBenzylꢀ2ꢀ(3ꢀcarbamoylꢀ4ꢀ(piperidinꢀ1ꢀyl)phenyl)ꢀ3ꢀhydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ
7ꢀcarboxamide (9)
7
8
The compound was prepared from phenacylester 25 according to the procedure described for
9
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compound 8. Green powder. HPLC purity: 99%. Yield: 0.06 g (88%). H NMR (500 MHz,
DMSOꢀd6) δ ppm 9.26ꢀ9.13 (m, 1H), 8.50 (s, 1H), 8.28 (s, 1H), 8.23ꢀ8.17 (m, 2H), 7.91 (d, J =
8.0 Hz, 1H), 7.72ꢀ7.66 (m, 2H), 7.38ꢀ7.29 (m, 5H) 7.29ꢀ7.55 (m, 1H), 4.51 (d, J = 5.7 Hz, 2H),
13
3.01ꢀ2.94 (m, 4H), 1.76ꢀ1.63 (m, 4H), 1.60ꢀ1.51 (m, 2H). C NMR (126 MHz, DMSOꢀd6) δ
ppm 172.2, 168.0, 166.0, 152.5, 139.5, 138.6, 137.7, 136.0, 132.3, 132.0, 131.1, 128.3, 128.1,
127.2, 126.8, 126.2, 124.6, 123.0, 119.6, 119.2, 119.04, 53.6, 42.7, 25.8, 23.4. HRMS (APCI)
calculated for [MꢀH]ꢀ C29H28N4O4 495.2027; found: 495.2022.
Methyl 5ꢀ(2ꢀ((2ꢀaminobenzoyl)oxy)acetyl)ꢀ2ꢀchlorobenzoate (12): Anthranilic acid (0.97 g,
7.1 mmol) and NaHCO3 (0.78 g, 9.3 mmol) were dissolved in DMF (50 ml) and stirred for 30
min at 100 °C. The mixture was cooled down to room temperature and bromoacetophenone 11
(2.05 g, 7.1 mmol) was added. The reaction mixture was stirred for 16 h at room temperature.
The product was extracted to EtOAc (three times), the organic layer was dried over Na2SO4 and
evaporated to dryness. White powder. LC purity: 99%. Yield: 1.06 g (91%). 1H NMR (500 MHz,
DMSOꢀd6) δ ppm 8.37 (d, J = 2.5 Hz, 1H), 8.18 (dd, J1= 8.5 Hz, J2 = 2.0 Hz, 1H), 7.81 (d, J =
8.5 Hz, 1H), 7.81 (dd, J1= 8.0 Hz, J2 = 1.5 Hz, 1H), 7.31 – 7.28 (m, 1H), 6.80 (d, J = 8.5 Hz,
1H), 6.64 (br, 2H), 6.59 – 6.54 (m, 1H), 5.68 (s, 2H), 3.91 (s, 3H). 13C NMR (126 MHz, DMSOꢀ
d6) δ ppm 192.1, 166.6, 164.8, 151.6, 137.1, 134.5, 132.8, 132.0, 131.6, 130.8, 130.7, 130.2,
166.6, 114.8, 108.0, 66.4, 52.9.
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Methyl 2ꢀchloroꢀ5ꢀ(3ꢀhydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolinꢀ2ꢀyl)benzoate (13): Derivative 12
(1.04 g, 3.0 mmol) was dissolved in AcOH (25 ml) and the mixture was boiled for 4 h. It was
then cooled down to room temperature and poured into iceꢀcold water. The precipitate formed
was filtered off, washed with water and dried under vacuum. Yellow powder. LC purity: 99%.
Yield: 0.86 g (87%). 1H NMR (500 MHz, DMSOꢀd6) δ ppm 11.64 (s, 1H), 8.67 (br, 1H) 8.23 (d,
J = 1.7 Hz, 1H), 8.12 (d, J = 7.4 Hz, 1 H), 8.00 (dd, J1= 8.3 Hz, J2 = 2.0 Hz, 1H), 7.80 (d, J = 8.0
Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1 H), 3.91 (s, 3H).
13C NMR (126 MHz, DMSOꢀd6) δ ppm 170.2, 165.2, 138.1 (2C), 133.7, 132.4, 131.5, 131.4,
130.8, 130.0, 129.1, 124.5, 122.0, 121.9, 118.4, 52.8, 21.1. HRMS (ESI) calculated for [M+H]+
C17H13ClNO4 330.0528; found: 330.0527.
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2ꢀChloroꢀ5ꢀ(3ꢀhydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolinꢀ2ꢀyl)benzoic acid (14): Derivative 13
(0.49 g, 1.5 mmol) was treated with 10% aqueous NaOH (20 ml) for 1 h at 50 °C. The reaction
mixture was then cooled down to room temperature, acidified with 1M HCl and extracted three
times to ethylacetate. The combined organic layers were washed with brine and dried over
MgSO4. The solvent was evaporated and the residue dried under vacuum. Yellow powder. LC
purity: 99%. Yield: 0.25 g (90%). LC/MS analysis: MS (ESI) exact mass calculated for
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C16H11ClNO4 [M+H]+: 316.04; found: 316.35, purity: 99%. H NMR (500 MHz, DMSOꢀd6) δ
ppm 13.64 (br, 1H) 11.64 (s, 1H) 8.23 (d, J = 2.5 Hz, 1H), 8.15 (d, J = 10.5 Hz, 1H), 7.97 (dd,
J1= 2.5 Hz, J2 = 10.5 Hz, 1H), 7.76 – 7.70 (m, 2H), 7.61 (t, J = 10.5 Hz, 1 H), 7.28 (t, J = 9 Hz,
13
1H). C NMR (101 MHz, DMSOꢀd6) δ ppm 170.2, 166.4, 138.2, 138.1, 133.2, 132.4, 131.5,
131.4, 131.2, 130.8, 130.7, 129.3, 124.5, 122.0, 121.9, 118.5.
2ꢀChloroꢀ5ꢀ(3ꢀhydroxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolinꢀ2ꢀyl)benzamide (15): The compound was
prepared from corresponding acid 14(0.49 g, 1.5 mmol) which was stirred in SOCl2 (10 ml) for 2
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h at room temperature. The solvent was evaporated, and the residue dissolved in DCM(10 ml).
NH4OH (10 ml) was added and the mixture was stirred 16 h at room temperature. It was then
washed with 1M HCl, water and brine and dried over Na2SO4. The inorganic salt was filtered
off, the solvent evaporated, and the residue dried under vacuum. Yellow powder. LC purity:
99%. Yield: 0.37 g (76%). LC/MS analysis: MS (ESI) exact mass calculated for C16H12ClN2O3
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[M+H]+: 315.73; found: 315.34, purity: 98%. H NMR (500 MHz, DMSOꢀd6) δ ppm 11.61 (br,
1H), 8.15 (d, J = 10.5 Hz, 1H), 7.99 (s, 1H), 7.92 ꢀ 7.89 (m, 2H), 7.74 ꢀ 7.67 (m, 3H), 7.60 (t, J =
13
9.0 Hz, 1H), 7.28 (t, J = 9.0 Hz, 1H). C NMR (101 MHz, DMSOꢀd6) δ ppm 170.2, 167.7,
138.1, 136.9, 131.4, 131.3, 131.0, 130.7, 130.5, 129.6, 129.4, 129.1, 124.5, 122.0, 121.8, 118.5.
Methyl 2ꢀaminoꢀ4ꢀ(pentylcarbamoyl)benzoate (16)
2ꢀaminomethylterephthalate (1.00 g, 5.1 mmol), 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
(EDC) (0.87 g, 7.5 mmol), and pentylamine (0.85 ml, 5.1 mmol) in pyridine (20 ml) were stirred
for 16 h at room temperature. The solvents were then evaporated, the residue dissolved in
EtOAc, and washed three times with 1M HCl and saturated solution of NaHCO3, and dried over
MgSO4. The solvent was evaporated and the residue was dried under the vacuum. Yellow
powder. HPLC purity: 99%. Yield: 1.30 g (97%). 1H NMR(500 MHz, CDCl3) δ ppm 7.85 (d, J =
8.6 Hz, 1H), 7.11 (d, J = 1.1 Hz, 1H), 6.86 (dd, J = 8.0, 1.7 Hz, 1H), 6.24 (s, 1H), 5.85 (s, 2H),
3.87 (s, 3H), 3.44ꢀ3.35 (m, 2H), 1.63ꢀ1.53 (m, 2H), 1.39ꢀ1.28 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H).
13C NMR (126 MHz, DMSOꢀd6)δ ppm 168.1, 167.0, 150.6, 140.0, 131.8, 115.9, 113.6, 112.6,
51.9, 40.3, 29.4, 29.2, 22.5, 14.1. HRMS (APCI) calculated for [M+H]+ C14H20N2O3 265.1547;
found: 265.1548.
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Methyl 2ꢀaminoꢀ4ꢀ(benzylcarbamoyl)benzoate (17)
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The compound was prepared according to the procedure described for compound 16with the use
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of benzylamine. Yellow oil. HPLC purity: 99%. Yield: 1.39 g (96%). H NMR (500 MHz,
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CDCl3) δ ppm 9.02ꢀ8.94 (m, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.37ꢀ7.19 (m, 6H), 6.96 (dd, J = 8.6,
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1.7 Hz, 1H), 6.76 (s, 2H), 4.43 (d, J = 6.3 Hz, 2H), 3.81 (s, 3H). C NMR (126 MHz, DMSOꢀ
d6) δ ppm 167.4, 166.0, 151.0, 139.6, 130.8, 128.3, 127,2, 126.7, 116.0, 112.9, 110.4, 51.6, 42.6.
HRMS (ESI) calculated for [M+H]+ C16H16N2O3 285.1234; found: 285.1235.
2ꢀAminoꢀ4ꢀ(pentylcarbamoyl)benzoic acid (18)
Ester 16(0.75 mg, 2.8 mmol) was dissolved in EtOH (17 ml). Aqueous solution of NaOH (0.55
ml, 1M) was added and the reaction mixture was stirred for 2 h at 40 °C. The solvents were then
removed under the vacuum, water (10 ml) was added and the pH was adjusted to 7 by addition of
AcOH. The precipitate formed was filtered off, washed with water and dried under the vacuum.
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Pink powder. HPLC purity: 99%. Yield: 605 mg (80%). H NMR (500 MHz, DMSOꢀd6) δ ppm
8.37ꢀ8.29 (m, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 1.7 Hz, 1H), 6.86 (dd, J = 8.6, 1.7 Hz,
13
1H), 3.24ꢀ3.14 (m, 2H), 1.54ꢀ1.44 (m, 2H), 1.35ꢀ1.21 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H). C
NMR (126 MHz, DMSOꢀd6) δ ppm 169.5, 166.1, 151.1, 139.4, 131.2, 115.5, 112.7, 112.3, 40.0
(overlay with solvent), 28.7 (2C), 21.9, 13.9. HRMS (APCI) calculated for [M+H]+
C13H18N2O3 251.1390; found: 251.1388.
2ꢀAminoꢀ4ꢀ(benzylcarbamoyl)benzoic acid (19)
The compound was prepared according to the procedure described for compound 18 from
1
derivative 17. Yellow powder. HPLC purity: 99%. Yield: 0.7 g (75%). H NMR (500 MHz,
DMSOꢀd6) δ ppm 9.06ꢀ8.88 (m, 1H), 7.75 (d, J = 8.6 Hz, 1H), 7.42ꢀ7.29 (m, 4H), 7.29ꢀ7.21 (m,
2H), 6.96 (dd, J = 8.6, 1.7 Hz, 1H), 4.46 (d, J = 5.7 Hz, 2H). 13C NMR (126 MHz, DMSOꢀd6) δ
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ppm 169.2, 166.2, 151.3, 139.6, 139.3, 131.3, 128.3, 127.2, 126.7, 115.7, 112.7, 111.4, 52.6.
HRMS: (ESI) calculated for [MꢀH]ꢀ C15H14N2O3 269.0921; found: 269.0920
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5ꢀAcetylꢀ2ꢀchlorobenzamide (21)
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Starting ester 20 (0.68 g, 3.4 mmol), which had been prepared according to the published
procedure,35was stirred in the mixture of concentrated acetic acid and concentrated sulfuric acid
(8.5/1.5, w/v, 32 ml) for 2 h at 80 °C. The reaction mixture was then allowed to cool down to
room temperature and neutralized to pH 3 with saturated solution of K2CO3. The mixture was
then extracted to DCM (3 ×50 ml), the organic layers were washed with saturated solution of
NaHCO3, and dried over Na2SO4. After the filtration and evaporation of the solvent, the obtained
yellow powder was immediately used in the next step. The powder was dissolved in DCM (10
ml) under inert atmosphere. The suspension was stirred at 0 °C and SOCl2 (2 ml, 0.4 mmol) was
added dropꢀwise. The mixture was stirred for 1.5 h at 50 °C. During this time, the insoluble
material was dissolved. It was then allowed to cool down to room temperature and the solvent
was removed under the stream of nitrogen. Resulting brown solid was dissolved in THF (10 ml),
cooled down to 0 °C, and NH4OH solution (25%, 2 ml) was added. This mixture was stirred for
30 min at room temperature, followed by extraction to EtOAc (3 × 50 ml). Combined organic
layers were washed with brine and dried over MgSO4. The solvent was finally evaporated under
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the vacuum yielding amide 21. Yellow powder. HPLC purity: 99%. Yield: 0.53 mg (78%). H
NMR(500 MHz, DMSOꢀd6) δ ppm 8.04 (s, 1H), 8.02ꢀ7.93 (m, 2H), 7.72 (s, 1H), 7.64 (dd, J =
8.6, 1.1, 1H), 2.60 (s, 3H). 13C NMR (126 MHz, DMSOꢀd6)δ ppm 196.8, 167.4, 137.3, 135.3,
134.7, 130.2, 130.0, 128.3, 26.8. HRMS (APCI) calculated for [M+H]+ C9H8ClNO2 198.0316;
found: 198.0319.
5ꢀAcetylꢀ2ꢀ(piperidinꢀ1ꢀyl)benzamide (22)
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Amide 21 (0.70 g, 3.5 mmol) and piperidine (0.79 ml, 8.0 mmol) were dissolved in DMSO (3
ml). The mixture was stirred at 120 °C for 1.5 h. After cooling down to room temperature, the
mixture was poured into water and extracted to EtOAc (3 × 40 ml). Combined organic layers
were then washed with water (3 ×30 ml), brine and dried over Na2SO4. The solvent was removed
under the vacuum and the residue was filtered through silicaꢀgel with EtOAc as the eluent. White
powder. HPLC purity: 99%. Yield: 0.66 g (76%). 1H NMR(500 MHz, DMSOꢀd6) δ ppm 8.08 (d,
J = 2.3 Hz, 1H), 8.01 (s, 1H), 7.92 (dd, J = 8.6, 2.3 Hz, 1H), 7.58 (s, 1H), 7.12 (d, J = 8.6 Hz,
1H), 3.04ꢀ2.02 (m, 4H), 2.51 (s, 3H, overlay with solvent), 1.68ꢀ1.63 (m, 4H), 1.57ꢀ1.49 (m, 2H).
13C NMR (126 MHz, DMSOꢀd6) δ ppm 196.1, 168.9, 154.9, 131.2, 130.5, 129.2, 127.6, 118.1,
52.6, 26.4, 25.4, 23.4. HRMS (APCI) calculated for [MꢀH]ꢀ C14H18N2O2 247.1441; found:
247.1444.
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5ꢀ(2ꢀBromoacetyl)ꢀ2ꢀ(piperidinꢀ1ꢀyl)benzamide (23)
Acetophenone22(0.50 g, 2.0 mmol) was dissolved in THF (10 ml). Catalytic amount of 33% HBr
in AcOH was added followed by dropꢀwise addition of Br2 (0.21 ml, 4.1 mmol) in THF (20 ml).
The reaction was followed by TLC analysis. After the consumption of the starting material, the
precipitate formed was filtered off and washed several times with diethyl ether. White powder.
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HPLC purity: 99%. Yield: 0.43 mg (65%). H NMR(500 MHz, DMSOꢀd6) δ ppm 8.05 (d, J =
2.3 Hz, 1H), 7.99ꢀ7.84 (m, 2H), 7.59 (s, 1H), 7.13 (d, J = 8.6 Hz, 1H), 4.77 (s, 2H), 3.15ꢀ3.05
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(m, 4H), 1.70ꢀ1.61 (m, 4H), 1.58ꢀ1.50 (m, 2H). C NMR (126 MHz, DMSOꢀd6) δ ppm 190.0,
169.0, 155.0, 131.5, 130.7, 129.3, 126.7, 118.9, 52.8, 33.6, 25.2, 23.0. HRMS (APCI) calculated
for [M+H]+ C14H17BrN2O2 325.0546; found: 325.0543.
2ꢀ(3ꢀCarbamoylꢀ4ꢀ(piperidinꢀ1ꢀyl)phenyl)ꢀ2ꢀoxoethyl
(pentylcarbamoyl)benzoate (24)
2ꢀaminoꢀ4ꢀ
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Aminoterephthalate18 (0.70 g, 0.3 mmol) and K2CO3 (0.38 g, 0.3 mmol) were suspended in
DMF (20 ml). The reaction mixture was stirred for 1 h at 90 °C. It was then allowed to cool
down to room temperature and 2ꢀbromacetophenone 23was added. The reaction was stirred for 1
h at room temperature and subsequently poured into the iceꢀcold aqueous solution of NaHCO3
(10%). The precipitate formed was filtered off, washed with water and dried under the vacuum.
Greenꢀyellow powder. HPLC purity: 99%. Yield: 0.11 g (80%). 1H NMR (500 MHz, DMSOꢀd6)
δ ppm 8.43 (t, J = 5.7 Hz, 1H), 8.07 (d, J = 1.7 Hz, 1H), 7.99ꢀ7.94 (m, 2H), 7.84 (d, J = 8.6 Hz,
1H), 7.60 (s, 1H), 7.24 (d, J = 1.1 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 6.95 (dd, J = 8.4, 1.8 Hz,
1H), 6.76 (s, 1H), 5.62 (s, 2H), 3.26ꢀ3.17 (m, 2H), 3.13ꢀ3.02 (m, 4H), 1.68ꢀ1.63 (m, 4H), 1.59ꢀ
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1.44 (m, 4H), 1.36ꢀ1.23 (m, 4H), 0.88 (t, J = 6.9 Hz, 3H). C NMR (126 MHz, DMSOꢀd6)δ
ppm 191.0, 169.0, 166.4, 165.9, 155.0, 151,2, 140.2, 130.9, 130.7, 130.0, 127.5, 125.7, 118.0,
115.9, 112.9, 109.9, 66.3, 52.3, 40.0 (overlay with solvent), 28.7 (2C), 25.3, 23.4, 21.9, 13.9.
HRMS (APCI) calculated for [M+H]+ C27H34N4O5 495.2602; found: 495.2604.
2ꢀ(3ꢀCarbamoylꢀ4ꢀ(piperidinꢀ1ꢀyl)phenyl)ꢀ2ꢀoxoethyl
(benzylcarbamoyl)benzoate (25)
2ꢀaminoꢀ4ꢀ
The compound was prepared according to the same procedure as compound 24 with the use
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ofaminoterephthalate19. Greenꢀyellow powder. HPLC purity: 99%. Yield: 0.79 g (70%). H
NMR (500 MHz, DMSOꢀd6) δ ppm 9.05ꢀ9.01 (m, 1H), 8.07 (d, J = 2.3 Hz, 1H), 7.99ꢀ7.91 (m,
2H), 7.87 (d, J = 8.3 Hz, 1H), 7.59 (s, 1H), 7.37ꢀ7.27 (m, 5H), 7.27ꢀ7.19 (m, 1H), 7.14 (d, J =
8.9 Hz, 1H), 7.01 (dd, J = 8.3, 1.4 Hz, 1H), 6.78 (s, 2H), 5.62 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H),
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3.16ꢀ3.02 (m, 4H), 1.68ꢀ1.63 (m, 4H), 1.59ꢀ1.52 (m, 2H). C NMR (126 MHz, DMSOꢀd6) δ
ppm 191.0, 169.0, 166.4, 166.1, 155.1, 151.3, 139.9, 139.6, 131.1, 130.7, 130.0, 128.3, 127.6,
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