Reversible small molecule inhibitors of MAO A and MAO B with anilide motifs

Article abstract of DOI:10.2147/DDDT.S236586

Background: Ligands consisting of two aryl moieties connected via a short spacer were shown to be potent inhibitors of monoamine oxidases (MAO) A and B, which are known as suitable targets in treatment of neurological diseases. Based on this general blueprint, we synthesized a series of 66 small aromatic amide derivatives as novel MAO A/B inhibitors. Methods: The compounds were synthesized, purified and structurally confirmed by spectroscopic methods. Fluorimetric enzymological assays were performed to determine MAO A/B inhibition properties. Mode and reversibility of inhibition was determined for the most potent MAO B inhibitor. Docking poses and pharmacophore models were generated to confirm the in vitro results. Results: N-(2,4-Dinitrophenyl)benzo[d][1,3]dioxole-5-carboxamide (55, ST-2043) was found to be a reversible competitive moderately selective MAO B inhibitor (IC₅₀ = 56 nM, K_i = 6.3 nM), while N-(2,4-dinitrophenyl)benzamide (7, ST-2023) showed higher preference for MAO A (IC₅₀ = 126 nM). Computational analysis confirmed in vitro binding properties, where the anilides examined possessed high surface complementarity to MAO A/B active sites. Conclusion: The small molecule anilides with different substitution patterns were identified as potent MAO A/B inhibitors, which were active in nanomolar concentrations ranges. These small and easily accessible molecules are promising motifs, especially for newly designed multitargeted ligands taking advantage of these fragments.

Full text of DOI:10.2147/DDDT.S236586

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O R I G I N A L R E S E A R C H
Reversible Small Molecule Inhibitors of MAO A and
MAO B with Anilide Motifs
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
Jens Hagenow1
Background: Ligands consisting of two aryl moieties connected via a short spacer were
1
shown to be potent inhibitors of monoamine oxidases (MAO) A and B, which are known
Stefanie Hagenow
Kathrin Grau1
as suitable targets in treatment of neurological diseases. Based on this general blueprint,
Mohammad Khanfar1–3
we synthesized a series of 66 small aromatic amide derivatives as novel MAO A/B
inhibitors.
Lena Hefke4
Methods: The compounds were synthesized, purified and structurally confirmed by spectroscopic
Ewgenij Proschak4
methods. Fluorimetric enzymological assays were performed to determine MAO A/B inhibition
1
Holger Stark
1Heinrich Heine University Düsseldorf,
Institute of Pharmaceutical and Medicinal
Chemistry, Duesseldorf 40225, Germany;
properties. Mode and reversibility of inhibition was determined for the most potent MAO
B inhibitor. Docking poses and pharmacophore models were generated to confirm the in vitro
results.
2Faculty of Pharmacy, The University of
Jordan, Amman 11942, Jordan; 3College
of Pharmacy, Alfaisal University, Riyadh
11533, Saudi Arabia; 4Goethe University
Frankfurt, Institute of Pharmaceutical
Chemistry, Frankfurt 60438, Germany
Results: N-(2,4-Dinitrophenyl)benzo[d][1,3]dioxole-5-carboxamide (55, ST-2043) was
found to be a reversible competitive moderately selective MAO B inhibitor (IC50 = 56 nM,
Ki = 6.3 nM), while N-(2,4-dinitrophenyl)benzamide (7, ST-2023) showed higher preference
for MAO A (IC50 = 126 nM). Computational analysis confirmed in vitro binding properties,
where the anilides examined possessed high surface complementarity to MAO A/B active
sites.
Conclusion: The small molecule anilides with different substitution patterns were identified
as potent MAO A/B inhibitors, which were active in nanomolar concentrations ranges. These
small and easily accessible molecules are promising motifs, especially for newly designed
multitargeted ligands taking advantage of these fragments.
Keywords: salicylic acid derivatives, molecular modeling, Parkinson’s disease, enzyme
inhibitor, pharmacophore, structure-activity relationships
Plain Language Summary
Monoamine oxidases (MAO) A and B are neurotransmitter-catabolizing enzymes, which
play a role in the pathophysiology of neurological diseases such as Parkinson’s disease,
depression or schizophrenia. Small molecules consisting of two aryl moieties connected via
a short spacer were shown to be potent MAO A/B inhibitors. In this study, aromatic amide
derivatives with different structural variations and substitution pattern were demonstrated
to have MAO A/B inhibition properties in a nanomolar concentration range. Compound 55
was found to be a reversible competitive MAO B preferring inhibitor (IC50 = 56 nM, Ki =
6.3 nM), while compound 7 showed a higher preference for MAO A (IC50 = 126 nM).
Computational analysis confirmed in vitro binding properties as the respective anilides
possessed high surface complementarity to MAO A/B active sites. These results suggest
that the herein described anilides are small and easily accessible molecules, which may
serve as promising precursors for the design of selective or multitargeting MAO A/B
inhibitors.
Correspondence: Holger Stark
Heinrich Heine University Duesseldorf,
Institute of Pharmaceutical and Medicinal
Chemistry, Universitaetsstr. 1,
Duesseldorf 40225, Germany
Tel +49 211 81-10478
Fax +49 211 81-13359
Email stark@hhu.de
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or fused with other pharmacophores with minor influences on
molecular size.
Introduction
The neurotransmitter-catabolizing monoamine oxidases
(MAO) are localized in the outer mitochondrial membrane
and are classified into the A and B isoforms. MAO A is
mainly involved in the degradation of serotonin, melatonin,
norepinephrine, and epinephrine, and is expressed nearly
ubiquitously in the human body. MAO B breaks down phe-
nethylamine and benzylamine and it is highly expressed in
the central nervous system (CNS). Dopamine, tyramine and
tryptamine can be metabolized by both isoforms but with
individual metabolic activity for each substrate.
Consequently, inhibitors of both enzymes are established in
the pharmacotherapy for neurological diseases.1–3
Moclobemide was the first marketed MAO inhibitor and
entered the Swedish market in 1989.4 Slowing down the
degradation of neurotransmitters like dopamine and norepi-
nephrine via MAO A inhibition, this drug exhibits mood-
lifting properties facilitating its use as an antidepressant.4
Since then the irreversible nonselective MAO inhibitor tra-
nylcypromine as well as the irreversible MAO B inhibitors
rasagiline and selegiline were approved for the treatment of
depression and Parkinson’s disease (PD), respectively.
Recently, safinamide as first reversible MAO B inhibitor in
PD therapy was approved.5–7 As the imminent therapeutic
benefits of MAO inhibitors are nowadays clear, multiple
efforts have been made to develop new reversible and irre-
versible inhibitors. Due to long-term enzyme inactivation by
irreversible MAO inhibitors, these can be associated with
serious side effects or drug–drug interactions, eg leading to
a serotonin syndrome.8 Reversible MAO inhibitors have
a more favorable side effect profile; however, they can be
displaced from the active site in case of high levels of the
endogenous ligands. To maintain pharmacological efficacy,
they are aimed to show a high affinity towards the MAOs
and/or tight binding behavior.9 The design of such reversible
and selective inhibitors of MAO A and B became a strongly
researched area to identify suitable drug candidates for the
treatment of neurological diseases, particularly of PD.
Having in mind that such diseases usually derive from
multifactorial disorders, the development of sophisticated
multitargeted ligands (MTLs) became of major exploration
to treat these CNS disorders.10,11 The combination of different
biological active moieties in one molecule is challenging since
the affinity for each target needs to be preserved together with
maintaining drug-likeness properties. Identification of small
Accordingly, we synthesized a series of 66 anilides
with diverse substitution patterns. The presented anilides
are small-sized and have low molecular weights. The
ligands show structural similarities to previously described
potent MAO inhibitors assuring potential MAO inhibition
activities. Multiple publications identified two aromatic
moieties linked by a short bridging element as mutual
MAO scaffold.8,12–16 Due to their high stability under
physiological conditions, amides were used as linkers in
our series. Aromatic moieties bearing different substitu-
ents were chosen to elucidate electronic and steric effects
on the binding affinities towards both MAO isoforms. The
presented ligands were characterized in vitro. For a better
understanding of the binding modes, computational dock-
ing experiments were performed and a ligand-based phar-
macophore-model was established.
Materials and Methods
Reagents and Instrumentation
Reagents and solvents for synthesis were purchased from
Sigma-Aldrich, VWR Chemicals, Fisher Scientific,
Panreac AppliChem, Alfa Aesar and Chemsolute and
were used without further purifications (unless stated
otherwise). 1H NMR and 13C NMR were recorded on
a Bruker AMX spectrometer (Bruker, Germany) at 300
and 75 MHz respectively, where either CDCl3 or DMSO-
d6 was used as a solvent. Tetramethylsilane was used as
standard and chemical shifts are reported in parts
per million (ppm). Elementary analyses (C, H, N) were
measured on a CHN-Rapid (Heraeus, Germany) and were
within 0.4% of the theoretical values for final compounds.
LC-MS analysis was performed on a Bruker Elute SP;
Column: Intensity Solo C18 RP; column dimensions: 100
x 2.1 mm; eluent: acetonitrile (1–40%) in water, 0.1%
formic acid; flow: 0.3 mL/min) combined with mass spec-
trometric detection (Bruker amazon speed with ESI; detec-
tion: ion-trap; ion-polarity: positive; scan: 100–600 m/z).
Data are given as retention time (tR), mass number ([M
+H]+), and normalized peak area (%) as approximated
purity. Melting points (m.p., uncorrected) were determined
on a M-564 Büchi melting point apparatus (Büchi,
Germany). Thin-layer chromatography (TLC) was carried
out using pre-coated silica gel 60 with fluorescence indi-
chemical entities with a promising target affinity are of great cator at UV 254 nm (Macherey-Nagel, Germany). The
value in MTL drug design, as they might be easily combined structure and purity of each compound were confirmed
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Hagenow et al
using 1H NMR, 13C NMR, LC-MS and/or elemental
analysis.
m.p.: 165°C (lit: 165–166°C).38
Human recombinant monoamine oxidase A (MAO A, N-(4-(Trifluoromethoxy)phenyl)benzamide (4)39
E.C. 1.4.3.4), human recombinant monoamine oxidase Using Method A the title compound was isolated as
B (MAO B, E.C. 1.4.3.4), kynuramine dihydrobromide a white solid. Yield: 62%
(KYN) and dimethylsulfoxide (DMSO) were purchased
1H NMR (300 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.-
from Sigma Aldrich. For biological evaluation tests, com- 14–7.79 (m, 4H), 7.57 (ddd, J = 14.4, 7.8, 6.0 Hz, 3H),
pounds were dissolved in 100% DMSO (max. 10−3 M). 7.37 (d, J = 8.6 Hz, 2H).
Fluorescence intensity measurements were performed with
an infinite M1000 Pro multimode reader (Tecan, 138.36, 134.61, 131.68, 128.37, 127.65, 121.60, 121.41.
13C NMR (75 MHz, DMSO) δ 165.66, 143.82, 143.79,
Switzerland). Assay pipetting was partly automated using
a Freedom EVO pipetting robot (Tecan, Switzerland).
Elemental Analysis: calc C 59.79%, H 3.58%,
N 4.98%; found C 59.76%, H 3.42%, N 4.90%.
m.p.: 179°C (lit: 176–178°C).40
Experimental Procedures
N-Phenylbenzamid (1)34
N-(4-Amino-2-methylphenyl)benzamide (5)41
Using Method A the title compound was isolated as
Using Method D the title compound was isolated as
a white amorphous powder. Yield: 53%
a white solid. Yield 89%
1H NMR (300 MHz, DMSO-d6) δ 10.25 (s, 1H), 8.-
00–7.93 (m, 2H), 7.82–7.75 (m, 2H), 7.65–7.49 (m, 3H),
7.41–7.29 (m, 2H), 7.15–7.06 (m, 1H). 13C NMR (75
MHz, DMSO) δ 165.50, 139.13, 134.95, 131.49, 128.55,
128.33, 127.60, 123.60, 120.31.
1H NMR (300 MHz, DMSO-d6) δ 9.57 (s, 1H), 8.06–-
7.86 (m, 2H), 7.62–7.43 (m, 3H), 6.91 (d, J = 8.2 Hz, 1H),
6.47 (d, J = 2.5 Hz, 1H), 6.42 (dd, J = 8.3, 2.6 Hz, 1H),
2.08 (s, 3H).
13C NMR (75 MHz, DMSO) δ 165.29, 146.96, 134.81,
134.59, 131.13, 128.26, 127.78, 127.40, 124.87, 115.19,
111.43, 17.99.
Elemental Analysis: calc
C 79.17%, H 5.62%,
N 7.10%; found C 78.91%, H 5.53%, N 6.89%.
m.p.: 163°C (lit.163–164°C).35
Elemental Analysis: calc
C 74.31%, H 6.05%,
N 12.38%; found C 74.22%, H 6.05%, N 12.08%.
N-(Pyridin-2-yl)benzamide (2)36
m.p.: 201°C (lit: 199–201°C).42
Using Method C the title compound was isolated as
a white amorphous powder. Yield: 83%
1H NMR (300 MHz, DMSO-d6) δ 11.95 (s, 1H), 8.51
(dt, J = 5.6, 1.3 Hz, 1H), 8.28–8.24 (m, 2H), 8.20–8.12 (m,
2H), 7.73–7.64 (m, 1H), 7.62–7.54 (m, 2H), 7.47 (td, J =
5.7, 2.7 Hz, 1H).
N-(2-Methyl-4-nitrophenyl)benzamide (6)41
Using Method A the title compound was isolated as a faint
yellow solid. 63%
1H NMR (300 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.19
(dd, J = 2.6, 0.9 Hz, 1H), 8.12 (dd, J = 8.8, 2.8 Hz, 1H),
8.05–7.98 (m, 2H), 7.84 (d, J = 8.8 Hz, 1H), 7.68–7.52 (m,
3H), 2.41 (s, 3H).
13C NMR (75 MHz, DMSO-d6) δ 166.89, 149.65,
143.30, 142.29, 132.94, 132.55, 128.60, 128.32, 120.34,
116.28.
13C NMR (75 MHz, DMSO) δ 165.59, 144.14, 143.00,
133.99, 133.88, 131.99, 128.47, 127.86, 125.57, 125.35,
121.49, 17.90.
LC-MS tR = 16.8 min; [M+H]+ = 198.9; 100%.
m.p.: 78°C (lit: 75–80°C).
Elemental Analysis: calc
C 65.62%, H 4.72%,
N-(4-Cyanophenyl)benzamide (3)37
N 10.93%; found C 65.38%, H 4.42%, N 10.74%.
Using Method A the title compound was isolated as
m.p.: 186°C (lit:181–186°C).42
a white solid. Yield: 57%
1H NMR (300 MHz, DMSO-d6) δ 10.65 (s, 1H), 8.- N-(2,4-Dinitrophenyl)benzamide (7)43
06–7.92 (m, 4H), 7.87–7.78 (m, 2H), 7.68–7.49 (m, 3H).
13C NMR (75 MHz, DMSO) δ 166.15, 143.46, 134.34, a yellow solid. Yield 23%
133.07, 131.99, 128.44, 127.80, 120.12, 119.04, 105.30.
1H NMR (300 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.76
Using Method A the title compound was isolated as
Elemental Analysis: calc
C
75.60%,
H
4.54%, (d, J = 2.6 Hz, 1H), 8.59 (dd, J = 9.1, 2.7 Hz, 1H), 8.15 (d,
N 12.60%; found C 75.44%, H 4.24%, N 12.33%.
J = 9.1 Hz, 1H), 8.05–7.91 (m, 2H), 7.78–7.53 (m, 3H).
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13C NMR (75 MHz, DMSO) δ 164.49, 156.60, 141.19,
1H NMR (300 MHz, DMSO-d6) δ 10.80 (s, 1H), 8.75
139.12, 137.12, 134.71, 131.32, 129.31, 123.56, 121.58, (ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 8.17 (dt, J = 7.8, 1.2 Hz,
119.84, 117.83, 116.95.
1H), 8.07 (td, J = 7.7, 1.7 Hz, 1H), 7.96–7.88 (m, 2H),
7.69 (ddd, J = 7.5, 4.8, 1.4 Hz, 1H), 7.59–7.52 (m, 2H).
13C NMR (75 MHz, DMSO-d6) δ 162.65, 149.66, 148.41,
138.12, 137.77, 131.41, 127.00, 122.46, 122.24, 115.64.
Elemental Analysis: calc
C 54.36%, H 3.16%,
N 14.63%; found C 54.21%, H 3.48%, N 14.80%.
m.p.: 199°C (lit: 199–201°C).44
Elemental Analysis: calc
C 52.01%, H 3.27%,
N-(4-Amino-2-methoxyphenyl)benzamide (8)45
N 10.11%; found C 51.74%, H 3.06%, N 10.10%.
m.p.: 148°C (lit: 147–148°C).48
Using Method D the title compound was isolated as an off-
white solid. Yield 90%
1H NMR (300 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.01–
7.88 (m, 2H), 7.63–7.40 (m, 3H), 7.18 (d, J = 8.3 Hz, 1H),
6.31 (d, J = 2.3 Hz, 1H), 6.15 (dd, J = 8.4, 2.3 Hz, 1H),
5.08 (s, 2H), 3.71 (s, 3H).
13C NMR (75 MHz, DMSO) δ 153.40, 147.70, 134.82,
131.12, 128.28, 127.29, 126.68, 115.16, 105.13, 97.61, 55.12.
2-Hydroxy-N-phenylbenzamide (12)49
Using Method A the title compound was isolated as
a white solid. Yield 61%
1H NMR (300 MHz, DMSO-d6) δ 11.83 (s, 1H), 10.40
(s, 1H), 7.99 (dd, J = 7.9, 1.7 Hz, 1H), 7.75–7.69 (m, 2H),
7.51–7.33 (m, 3H), 7.18–7.09 (m, 1H), 7.03–6.93 (m, 2H).
13C NMR (75 MHz, DMSO) δ 166.57, 158.48, 138.10,
133.62, 128.99, 128.69, 124.15, 120.96, 118.98, 117.42,
117.19.
Elemental Analysis: calc
C 69.41%, H 5.81%,
N 11.56%; found C 69.49%, H 5.82%, N 11.38%.
m.p.: 115°C (lit: 112–113°C).
Elemental Analysis: calc
C 73.23%, H 5.20%,
N-(2-Methoxy-4-nitrophenyl)benzamide (9)45
N 6.57%; found C 73.13%, H 5.21%, N 6.46%.
Using Method A the title compound was isolated as
m.p.: 132°C (lit: 127–129°C).50
a yellow solid. Yield 60%
1H NMR (300 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.31
(d, J = 8.9 Hz, 1H), 8.03–7.88 (m, 3H), 7.85 (d, J = 2.5 Hz,
1H), 7.69–7.49 (m, 3H), 4.00 (s, 3H).
13C NMR (75 MHz, DMSO) δ 165.32, 149.81, 143.47,
133.76, 132.16, 128.57, 127.62, 121.16, 116.53, 105.97, 56.55.
N-(4-Cyanophenyl)-2-hydroxybenzamide (13)51
Using Method A the title compound was isolated as
a white solid. Yield 47%
1H NMR (300 MHz, DMSO-d6) δ 11.41 (s, 1H), 10.66
(s, 1H), 7.94 (d, J = 8.8 Hz, 2H), 7.88 (dd, J = 7.9, 1.7 Hz,
1H), 7.83 (d, J = 8.8 Hz, 2H), 7.45 (ddd, J = 8.2, 7.2, 1.7
Hz, 1H), 7.05–6.92 (m, 2H).
Elemental Analysis: calc
C 61.76%, H 4.44%,
N 10.29%; found C 61.71%, H 4.53%, N 10.33%.
m.p.: 149°C (lit: 148–151°C).
13C NMR (75 MHz, DMSO) δ 166.45, 157.49, 142.68,
133.67, 133.15, 129.48, 120.40, 119.18, 118.96, 118.47,
117.05, 105.57.
N-(2-Chloro-4-nitrophenyl)benzamide (10)46
Using Method A the title compound was isolated as a pale-
Elemental Analysis: calc
C 70.58%, H 4.23%,
yellow solid. Yield 59%
N 11.76%; found C 70.40%, H 4.11%, N 11.67%.
1H NMR (300 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.41 (d,
J = 2.6 Hz, 1H), 8.27 (dd, J = 9.0, 2.6 Hz, 1H), 8.09 (d, J = 9.0
Hz, 1H), 8.05–7.95 (m, 2H), 7.73–7.50 (m, 3H).
13C NMR (75 MHz, DMSO) δ 165.49, 144.45, 141.31,
133.37, 132.36, 128.59, 128.01, 127.89, 126.58, 124.85,
122.89.
m.p.: 176°C (lit: 175–176.5°C).52
N-(2,4-Dinitrophenyl)-2-hydroxybenzamide (14)53
Using Method A the title compound was isolated as
a yellow solid. Yield 22%
1H NMR (300 MHz, DMSO-d6) δ 12.41 (s, 1H), 11.96
(s, 1H), 9.01 (d, J = 9.4 Hz, 1H), 8.88 (d, J = 2.7 Hz, 1H),
8.59 (dd, J = 9.4, 2.8 Hz, 1H), 8.01 (dd, J = 8.0, 1.8 Hz,
1H), 7.50 (ddd, J = 8.6, 7.2, 1.8 Hz, 1H), 7.11–6.97
(m, 2H).
13C NMR (75 MHz, DMSO) δ 165.50, 142.73, 137.31,
132.90, 132.80, 128.81, 128.61, 127.91, 125.49, 121.16.
Elemental Analysis: calc
C 56.44%, H 3.28%,
N 10.13%; found C 56.14%, H 3.38%, N 10.18%.
m.p.: 161°C (lit: 160°C).
N-(4-Bromophenyl)picolinamide (11)47
Using Method B the title compound was isolated as
a white solid. Yield 75%
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Elemental Analysis: calc
C
51.49%,
H
2.99%,
m.p.: 144°C.
N 13.86%; found C 51.40%, H 2.77%, N 13.97%.
m.p.: 212°C (lit: 213–214°C).54
4-Chloro-N-phenylbenzamide (18)57
Using Method A the title compound was isolated as
2-Hydroxy-N-(2-methoxy-4-nitrophenyl)benzamide (15)55
Using Method A the title compound was isolated as
a yellow solid. Yield 45%
1H NMR (300 MHz, DMSO-d6) δ 11.86 (s, 1H), 11.28
(s, 1H), 8.73 (d, J = 9.0 Hz, 1H), 8.03 (dd, J = 7.9, 1.8 Hz,
1H), 7.95 (dd, J = 9.0, 2.5 Hz, 1H), 7.85 (d, J = 2.5 Hz,
1H), 7.45 (ddd, J = 8.2, 7.2, 1.8 Hz, 1H), 7.09–6.96 (m,
2H), 4.04 (s, 3H).
a white solid. Yield 63%
1H NMR (300 MHz, DMSO-d6) δ 10.32 (s, 1H), 8.-
04–7.95 (m, 2H), 7.83–7.74 (m, 2H), 7.65–7.55 (m, 2H),
7.36 (dd, J = 8.5, 7.3 Hz, 2H), 7.16–7.07 (m, 1H).
13C NMR (75 MHz, DMSO) δ 164.38, 138.92, 136.34,
133.61, 129.57, 128.58, 128.40, 123.77, 120.38.
Elemental Analysis: calc
C 67.40%, H 4.35%,
N 6.05%; found C 67.37%, H 4.21%, N 5.93%.
13C NMR (75 MHz, DMSO) δ 163.54, 156.01, 147.85,
142.27, 134.60, 133.98, 131.09, 119.89, 118.33, 118.17,
117.34, 116.95, 105.64, 56.78.
m.p.: 198°C (lit: 200–201°C).
4-Chloro-N-(pyridin-2-yl)benzamide (19)58
Elemental Analysis: calc
C 58.33%, H 4.20%,
Using Method C the title compound was isolated as
N 9.72%; found C 58.15%, H 4.05%, N 9.54%.
a white solid. Yield 78%
1H NMR (300 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.51
(ddd, J = 5.7, 1.7, 0.9 Hz, 1H), 8.36–8.14 (m, 4H), 7.73–
7.60 (m, 2H), 7.47 (ddd, J = 7.1, 5.6, 1.5 Hz, 1H).
13C NMR (75 MHz, DMSO-d6) δ 165.86, 149.58, 143.24,
142.44, 137.80, 131.38, 130.33, 128.66, 120.45, 116.30.
LC-MS tR = 22.3 min; [M+H]+ = 232.9; 100%.
m.p.: 138°C (lit: 137–139°C).59
m.p.: 207°C (lit: 205–206°C).
N-(2-Chloro-4-nitrophenyl)-2-hydroxybenzamide (16)56
Using Method A the title compound was isolated as a pale-
yellow solid. Yield 55%
1H NMR (300 MHz, DMSO-d6) δ 12.18 (s, 1H), 11.42
(s, 1H), 8.89 (d, J = 9.3 Hz, 1H), 8.44 (d, J = 2.6 Hz, 1H),
8.31 (dd, J = 9.3, 2.7 Hz, 1H), 8.09 (dd, J = 7.9, 1.8 Hz,
1H), 7.53 (ddd, J = 8.2, 7.2, 1.8 Hz, 1H), 7.16–7.02
(m, 2H).
4-Chloro-N-(4-cyanophenyl)benzamide (20)
Using Method A the title compound was isolated as
13C NMR (75 MHz, DMSO) δ 163.74, 156.15, 142.21,
141.45, 134.41, 131.14, 124.67, 123.78, 122.09, 120.45,
120.03, 117.82, 116.98.
a white solid. Yield 49%
1H NMR (300 MHz, DMSO-d6) δ 10.67 (s, 1H), 8.00
(d, J = 1.6 Hz, 2H), 7.97 (d, J = 1.8 Hz, 2H), 7.81 (d, J =
8.8 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H).
Elemental Analysis: calc
C 53.35%, H 3.10%,
13C NMR (75 MHz, DMSO) δ 164.97, 143.25, 136.87,
133.04, 132.97, 129.75, 128.49, 120.18, 118.98, 105.48.
N 9.57%; found C 53.05%, H 3.32%, N 9.27%.
m.p.: 219°C (lit: 218–220°C).
Elemental Analysis: calc
C 65.51%, H 3.53%,
N 10.91%; found C 65.62%, H 3.51%, N 10.95%.
m.p.: 208°C.
4-Methyl-N-(4-nitro-2-(trifluoromethyl)phenyl)benzamide
(17)
Using Method A the title compound was isolated as
a paleyellow solid. Yield 12%
4-Chloro-N-(2-methyl-4-nitrophenyl)benzamide (21)60
1H NMR (300 MHz, DMSO-d6) δ 10.31 (s, 1H), 8.56 Using Method A the title compound was isolated as
(dd, J = 8.7, 2.7 Hz, 1H), 8.52 (d, J = 2.6 Hz, 1H), 7.96 (d, J = a yellow solid. Yield 52%
8.8 Hz, 1H), 7.92–7.86 (m, 2H), 7.38 (d, J = 7.9 Hz, 2H), 2.40
1H NMR (300 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.19
(s, 3H).
(d, J = 2.7 Hz, 1H), 8.11 (dd, J = 8.8, 2.7 Hz, 1H), 8.02 (d,
13C NMR (75 MHz, DMSO) δ 166.12, 145.09, 142.45, J = 8.6 Hz, 2H), 7.81 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.6
141.88, 141.86, 141.84, 131.48, 130.41, 129.09, 127.87, Hz, 2H), 2.40 (s, 3H).
127.80, 126.04, 125.63, 124.29, 122.32, 122.25, 120.66, 20.99.
Elemental Analysis: calc 55.56%,
13C NMR (75 MHz, DMSO) δ 164.57, 144.26, 142.76,
C
H
3.42%, 136.83, 134.00, 132.70, 129.81, 128.53, 125.72, 125.36,
121.48, 17.89.
N 8.64%; found C 55.46%, H 3.25%, N 8.39%.
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Elemental Analysis: calc
C
57.84%,
H
3.81%,
1H NMR (300 MHz, DMSO-d6) δ 10.47 (s, 1H),
7.69 (dd, J = 8.3, 1.3 Hz, 2H), 7.36 (t, J = 7.9 Hz,
2H), 7.21–7.15 (m, 1H), 7.12 (d, J = 7.4 Hz, 1H), 6.73
(d, J = 2.0 Hz, 2H), 4.72 (s, 2H).
N 9.64%; found C 58.03%, H 3.99%, N 9.64%.
m.p.: 193°C.
13C NMR (75 MHz, DMSO) δ 165.96, 148.64, 140.98,
138.50, 128.72, 123.71, 120.37, 120.29, 118.02, 117.50,
113.68.
4-Chloro-N-(2-methoxy-4-nitrophenyl)benzamide (22)
Using Method A the title compound was isolated as
a yellow solid. Yield 54%
1H NMR (300 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.24
(d, J = 8.9 Hz, 1H), 8.03–7.91 (m, 3H), 7.88 (d, J = 2.5 Hz,
1H), 7.67–7.58 (m, 2H), 4.00 (s, 3H).
Elemental Analysis: calc
C 68.41%, H 5.30%,
N 12.27%; found C 68.01%, H 4.96%, N 12.38%.
m.p.: 176°C (lit: 174–176°C).62
13C NMR (75 MHz, DMSO) δ 164.51, 150.30, 143.83,
136.97, 133.55, 132.59, 129.72, 128.60, 122.03, 116.45,
106.18, 56.56.
2-Hydroxy-5-nitro-N-phenylbenzamide (26)61
Using Method A the title compound was isolated as
Elemental Analysis: calc
C 54.83%, H 3.62%,
a white solid. Yield 61%
1H NMR (300 MHz, DMSO-d6) δ 12.78 (s, 1H),
10.57 (s, 1H), 8.79 (d, J = 2.9 Hz, 1H), 8.29 (dd, J =
9.1, 2.9 Hz, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.39 (t, J =
7.7 Hz, 2H), 7.16 (dd, J = 8.4, 5.5 Hz, 2H).
13C NMR (75 MHz, DMSO) δ 164.08, 163.27,
139.34, 137.89, 128.75, 128.33, 125.71, 124.39,
120.77, 119.33, 117.97.
N 9.13%; found C 54.67%, H 3.57%, N 8.86%.
m.p.: 200°C (decomposition).
4-Chloro-N-(2-chloro-4-nitrophenyl)benzamide (23)
Using Method A the title compound was isolated as
a yellow solid. Yield 17%
1H NMR (300 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.43
(d, J = 2.6 Hz, 1H), 8.27 (dd, J = 8.9, 2.6 Hz, 1H),
8.09–7.98 (m, 3H), 7.66 (d, J = 8.6 Hz, 2H).
13C NMR (75 MHz, DMSO) δ 164.56, 144.69, 141.12,
137.19, 132.13, 129.87, 128.69, 128.32, 127.00, 124.90,
122.89.
Elemental Analysis: calc
C 60.47%, H 3.90%,
N 10.85%; found C 60.20%, H 3.85%, N 10.65%.
m.p.: 223°C (lit: 219–221°C).
5-Chloro-2-hydroxy-N-phenylbenzamide (27)56
Elemental Analysis: calc
C 50.19%, H 2.59%,
Using Method A the title compound was isolated as
N 9.00%; found C 50.44%, H 2.30%, N 8.96%.
m.p.: 219°C.
a white solid. Yield 62%
1H NMR (300 MHz, DMSO-d6) δ 11.87 (s, 1H), 10.41
(s, 1H), 7.98 (d, J = 2.7 Hz, 1H), 7.77–7.64 (m, 2H), 7.47
(dd, J = 8.8, 2.7 Hz, 1H), 7.43–7.32 (m, 2H), 7.21–7.10
(m, 1H), 7.02 (d, J = 8.8 Hz, 1H).
4-Bromo-N-(2-chloro-4-nitrophenyl)benzamide (24)
Using Method A the title compound was isolated as an off-
white solid. Yield 20%
1H NMR (300 MHz, DMSO-d6) δ 10.41 (s, 1H),
8.41 (d, J = 2.6 Hz, 1H), 8.27 (dd, J = 9.0, 2.6 Hz,
1H), 8.04 (d, J = 9.0 Hz, 1H), 7.99–7.92 (m, 2H),
7.83–7.76 (m, 2H).
13C NMR (75 MHz, DMSO) δ 164.70, 144.65, 141.10,
132.49, 131.62, 130.00, 128.25, 126.92, 126.19, 124.88,
122.88.
13C NMR (75 MHz, DMSO) δ 164.96, 156.87,
137.95, 132.99, 128.72, 128.32, 124.29, 122.68,
120.81, 119.45, 119.04.
Elemental Analysis: calc
C 63.04%, H 4.07%,
N 5.66%; found C 62.83%, H 4.17%, N 5.55%.
m.p.: 209°C (lit: 211–212°C).63
5-Chloro-N-(2-chlorophenyl)-2-hydroxybenzamide (28)64
Using Method A the title compound was isolated as a pale-
yellow solid. Yield 46%
1H NMR (300 MHz, DMSO-d6) δ 12.26 (s, 1H), 10.88
(s, 1H), 8.40 (dd, J = 8.3, 1.6 Hz, 1H), 7.99 (d, J = 2.8 Hz,
1H), 7.54 (dd, J = 8.0, 1.5 Hz, 1H), 7.48 (dd, J = 8.7, 2.8
Elemental Analysis: calc
C 43.91%, H 2.27%,
N 7.88%; found C 43.65%, H 1.90%, N 7.53%.
m.p.: 205°C.
5-Amino-2-hydroxy-N-phenylbenzamide (25)61
Using Method D the title compound was isolated as an off- Hz, 1H), 7.43–7.34 (m, 1H), 7.17 (ddd, J = 8.0, 7.4, 1.6
white solid. Yield 90% Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H).
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13C NMR (75 MHz, DMSO-d6) δ 162.59, 155.35,
Elemental Analysis: calc C 53.27%, H 2.87%,
134.98, 133.33, 129.72, 129.29, 127.78, 125.32, 123.46, N 4.44%; found C 53.09%, H 2.63%, N 4.38%.
m.p.: 221°C (lit: 222–223°C).63
122.76, 119.58, 118.99.
Elemental Analysis: calc
C 55.35%, H 3.22%,
N 4.96%; found C 55.35%, H 3.31%, N 4.75%.
5-Chloro-2-hydroxy-N-(4-nitrophenyl)benzamide (32)65
m.p.: 189°C (lit:184–186°C).62
Using Method A the title compound was isolated as
a yellow solid. Yield 9%
1H NMR (300 MHz, DMSO-d6) δ 11.46 (s, 1H), 10.84 (s,
1H), 8.28 (d, J = 9.2 Hz, 2H), 7.99 (d, J = 9.2 Hz, 2H), 7.84 (d,
J = 2.8 Hz, 1H), 7.48 (dd, J = 8.8, 2.8 Hz, 1H), 7.04 (d, J = 8.8
Hz, 1H).
13C NMR (75 MHz, DMSO) δ 164.98, 155.83, 144.50,
142.70, 133.00, 128.73, 124.85, 122.77, 120.88, 120.01,
118.90.
5-Chloro-N-(3-chlorophenyl)-2-hydroxybenzamide (29)56
Using Method A the title compound was isolated as a pale-
yellow solid. Yield 41%
1H NMR (300 MHz, DMSO-d6) δ 11.65 (s, 1H), 10.49
(s, 1H), 7.92 (t, J = 2.1 Hz, 1H), 7.90 (d, J = 2.7 Hz, 1H),
7.61 (ddd, J = 8.3, 2.1, 1.0 Hz, 1H), 7.47 (dd, J = 8.8, 2.7
Hz, 1H), 7.40 (t, J = 8.1 Hz, 1H), 7.20 (ddd, J = 8.0, 2.1,
1.0 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H).
Elemental Analysis: calc
C 53.35%, H 3.10%,
N 9.57%; found C 53.37%, H 3.02%, N 9.36%.
13C NMR (75 MHz, DMSO-d6) δ 164.96, 156.41,
139.55, 133.01, 130.39, 128.44, 123.87, 122.72, 120.05,
119.94, 118.99.
m.p.: 259°C (li: 260–262°C).62
5-Chloro-2-hydroxy-N-(4-(trifluoromethoxy)phenyl)ben-
zamide (33)63
Elemental Analysis: calc
C 55.35%, H 3.22%,
N 4.96%; found C 55.12%, H 3.25%, N 4.86%.
Using Method A the title compound was isolated as
m.p.: 219°C (lit: 217–218°C).62
a white solid. Yield 60%
1H NMR (300 MHz, DMSO-d6) δ 11.76 (s, 1H), 10.54 (s,
1H), 7.94 (d, J = 2.7 Hz, 1H), 7.83 (d, J = 9.0 Hz, 2H), 7.46
(dd, J = 8.8, 2.7 Hz, 1H), 7.41–7.30 (m, 2H), 7.02 (d, J = 8.8
Hz, 1H).
13C NMR (75 MHz, DMSO) δ 165.04, 156.73, 144.25 (m),
137.21, 133.02, 128.36, 122.71, 122.12, 121.46, 119.51,
118.99.
Ethyl 4-(5-chloro-2-hydroxybenzamido)benzoate (30)65
Using Method A the title compound was isolated as a pale-
yellow solid. Yield 37%
1H NMR (300 MHz, DMSO-d6) δ 11.63 (s, 1H), 10.65
(s, 1H), 8.03–7.93 (m, 2H), 7.92–7.83 (m, 3H), 7.47 (dd,
J = 8.8, 2.7 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H), 4.30 (q, J =
7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H).
13C NMR (75 MHz, DMSO) δ 165.22, 164.87, 156.25,
142.53, 132.99, 130.11, 128.61, 125.00, 122.74, 120.28,
119.80, 118.96, 60.49, 14.17.
Elemental Analysis: calc
C 50.70%, H 2.74%,
N 4.22%; found C 50.30%, H 2.65%, N 4.06%.
m.p.: 200°C (lit: 200–202°C).
Elemental Analysis: calc
C 60.10%, H 4.41%,
N-(4-Bromophenyl)-5-chloro-2-hydroxybenzamide (34)67
Using Method A the title compound was isolated as
a yellow solid. Yield 55%
N 4.38%; found C 60.46%, H 4.38%, N 4.32%.
m.p.: 214°C (lit: 212–214°C).
1H NMR (300 MHz, DMSO-d6) δ 11.75 (s, 1H), 7.93 (d,
J = 2.7 Hz, 1H), 7.76–7.65 (m, 2H), 7.59–7.50 (m, 2H), 7.45
(dd, J = 8.8, 2.7 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H).
13C NMR (75 MHz, DMSO) δ 164.92, 156.63, 137.43,
133.00, 131.52, 128.38, 122.71, 122.57, 119.64, 119.00,
115.97.
5-Chloro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benza-
mide (31)66
Using Method A the title compound was isolated as a pale-
yellow solid. Yield 62%
1H NMR (300 MHz, DMSO-d6) δ 11.65 (s, 1H), 10.65
(s, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 2.7 Hz, 1H),
7.72 (dd, J = 8.7, 0.9 Hz, 2H), 7.46 (dd, J = 8.8, 2.7 Hz,
1H), 7.03 (d, J = 8.8 Hz, 1H).
Elemental Analysis: calc
C 47.81%, H 2.78%,
N 4.29%; found C 47.49%, H 2.47%, N 3.94%.
m.p.: 237°C (lit: 240–241.5°C).68
13C NMR (75 MHz, DMSO) δ 165.02, 156.30, 141.75,
141.73, 133.02, 128.57, 125.97, 125.92, 122.78, 120.39,
120.07, 118.95.
5-Chloro-2-hydroxy-N-(2-methyl-4-nitrophenyl)benza-
mide (35)69
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Using Method A the title compound was isolated as
a yellow solid. Yield 16%
13C NMR (75 MHz, DMSO) δ 162.21, 154.92, 147.93,
142.54, 134.19, 133.54, 130.00, 123.62, 119.85, 119.04,
1H NMR (300 MHz, DMSO-d6) δ 12.33 (s, 1H), 10.73 118.34, 117.31, 105.72, 56.84.
(s, 1H), 8.52 (d, J = 9.0 Hz, 1H), 8.25–8.11 (m, 2H), 7.98
(d, J = 2.8 Hz, 1H), 7.53 (dd, J = 8.7, 2.8 Hz, 1H), 7.10 (d,
J = 8.7 Hz, 1H), 2.45 (s, 3H).
Elemental Analysis: calc C 52.10%, H 3.28%,
N 8.68%; found C 52.10%, H 3.28%, N 8.65%.
m.p.: 230°C (lit: 233–235°C).72
13C NMR not performed due to poor solubility.
Dimethyl 2-(5-chloro-2-hydroxybenzamido)terephthalate
(39)
Elemental Analysis: calc
C 54.83%, H 3.62%,
N 9.13%; found C 54.78%, H 3.32%, N 8.96%.
Using Method A the title compound was isolated as a pale-
m.p.: 229°C (lit: 225–227).
yellow solid. Yield 57%
1H NMR (300 MHz, DMSO-d6) δ 12.05 (s, 1H), 11.89
(s, 1H), 9.23 (d, J = 1.6 Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H),
7.92 (d, J = 2.8 Hz, 1H), 7.77 (dd, J = 8.2, 1.7 Hz, 1H), 7.49
(dd, J = 8.8, 2.8 Hz, 1H), 7.06 (d, J = 8.8 Hz, 1H), 3.91 (s,
3H), 3.90 (s, 3H).
13C NMR (75 MHz, DMSO) δ 166.13, 165.35, 163.21,
155.43, 139.41, 133.74, 133.34, 131.12, 129.96, 123.60,
123.07, 122.67, 121.54, 120.19, 118.83, 52.78, 52.58.
Elemental Analysis: calc C 56.13%, H 3.88%, N 3.85%;
found C 56.53%, H 3.64%, N 4.00%.
N-(2,4-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxy-
benzamide (36)70
Using Method A the title compound was isolated as an off-
white solid. Yield 51%
1H NMR (300 MHz, DMSO-d6) δ 11.42 (s, 1H), 10.76
(s, 1H), 8.36 (d, J = 1.8 Hz, 2H), 7.91–7.63 (m, 2H), 7.39
(dd, J = 8.8, 2.7 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H).
13C NMR (75 MHz, DMSO) δ 165.46, 156.39, 140.14,
133.21, 130.64 (q, J = 32.9 Hz), 128.47, 124.94, 122.73,
121.33, 120.22, 119.74, 118.97,116.70 (q, J = 7.8, 4.0 Hz).
m.p.: 193°C.
Elemental Analysis: calc
C 46.96%, H 2.10%,
N 3.65%; found C 46.73%, H 1.70%, N 3.61%.
m.p.: 171°C.
5-Chloro-2-hydroxy-N-(5-fluoro-2-nitrophenyl)benzamide
(40)
Using Method A the title compound was isolated as
5-Chloro-2-hydroxy-N-(4-nitro-2-(trifluoromethyl)phenyl)
benzamide (37)
a yellow solid. Yield 11%
1H NMR (300 MHz, DMSO-d6) δ 12.24 (s, 1H), 12.20
(s, 1H), 8.58 (dd, J = 11.9, 2.9 Hz, 1H), 8.30 (dd, J = 9.2,
5.8 Hz, 1H), 7.90 (d, J = 3.0 Hz, 1H), 7.49 (dd, J = 8.6, 2.9
Hz, 1H), 7.20 (ddd, J = 9.8, 7.3, 3.0 Hz, 1H), 7.05 (d, J =
8.7 Hz, 1H).
Using Method A the title compound was isolated as a pale-
yellow solid. Yield 63%
1H NMR (300 MHz, DMSO-d6) δ 12.51 (s, 1H), 11.24
(s, 1H), 8.77 (d, J = 9.2 Hz, 1H), 8.53 (dd, J = 9.2, 2.7 Hz,
1H), 8.43 (d, J = 2.7 Hz, 1H), 7.91 (d, J = 2.8 Hz, 1H),
7.51 (dd, J = 8.8, 2.8 Hz, 1H), 7.06 (d, J = 8.8 Hz, 1H).
13C NMR (75 MHz, DMSO) δ 162.50, 155.11, 142.28,
141.10, 134.09, 130.06, 128.68, 123.81, 123.74, 122.11,
122.02, 118.98, 118.95, 118.26.
13C NMR (75 MHz, DMSO-d6) δ 164.87 (d, J = 252.5
Hz), 163.08, 155.34, 136.01 (d, J = 13.7 Hz), 134.52 (d,
J = 2.7 Hz), 133.83, 130.12, 128.75 (d, J = 11.4 Hz),
123.36, 119.48, 118.92, 111.09 (d, J = 24.2 Hz), 109.40
(d, J = 29.8 Hz).
Elemental Analysis: calc
C 46.62%, H 2.24%,
Elemental Analysis: calc
C 50.26%, H 2.60%,
N 7.77%; found C 46.32%, H 1.94%, N 7.64%.
m.p.: 191°C.
N 9.02%; found C 50.19%, H 2.73%, N 9.02%.
m.p.: 178°C.
5-Chloro-2-hydroxy-N-(2-methoxy-4-nitrophenyl)benza-
mide (38)71
5-Chloro-2-hydroxy-N-(4-methyl-3-nitrophenyl)benza-
mide (41)
Using Method A the title compound was isolated as an off-
Using Method A the title compound was isolated as
white solid. Yield 13%
a brown solid. Yield 37%
1H NMR (300 MHz, DMSO-d6) δ 12.25 (s, 1H), 11.24
(s, 1H), 8.69 (d, J = 9.0 Hz, 1H), 8.04–7.82 (m, 3H), 7.50 (dd,
J = 8.7, 2.9 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H), 4.04 (s, 3H).
1H NMR (300 MHz, DMSO-d6) δ 11.61 (s, 1H), 10.65 (s,
1H), 8.50 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.1, 2.5 Hz, 2H),
7.58–7.44 (m, 2H), 7.03 (d, J = 8.8 Hz, 1H), 3.33 (s, 3H).
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13C NMR (75 MHz, DMSO) δ 165.18, 156.50, 148.51,
137.04, 133.10, 133.04, 128.39, 128.15, 125.13, 122.69, N 9.00%; found C 50.11%, H 2.70%, N 8.81%.
Elemental Analysis: calc C 50.19%, H 2.59%,
119.80, 119.02, 115.79, 19.19.
Elemental Analysis: calc
m.p.: 171°C.
C
54.83%, H 3.62%,
N 9.13%; found C 55.21%, H 3.50%, N 8.73%.
m.p.: 278°C.
2-Chloro-4-nitro-N-(3-chlorophenyl)benzamide (45)
Using Method A the title compound was isolated as an off-
white solid. Yield 51%
5-Chloro-2-hydroxy-N-(3,4-dimethoxyphenyl)benzamide
(42)73
1H NMR (300 MHz, DMSO-d6) δ 10.95 (s, 1H), 8.44
(d, J = 2.2 Hz, 1H), 8.32 (dd, J = 8.4, 2.2 Hz, 1H), 7.99–7.87
Using Method A the title compound was isolated as (m, 2H), 7.58 (ddd, J = 8.3, 2.1, 1.0 Hz, 1H), 7.42 (t, J = 8.1
a yellow solid. Yield 29%
Hz, 1H), 7.22 (ddd, J = 8.0, 2.1, 1.0 Hz, 1H).
1H NMR (300 MHz, DMSO-d6) δ 12.04 (s, 1H), 10.30
13C NMR (75 MHz, DMSO-d6) δ 163.57, 148.44,
(s, 1H), 8.01 (d, J = 2.7 Hz, 1H), 7.46 (dd, J = 8.8, 2.7 Hz, 141.85, 139.78, 133.18, 131.09, 130.62, 130.06, 124.63,
1H), 7.39 (d, J = 2.4 Hz, 1H), 7.25 (dd, J = 8.7, 2.4 Hz, 123.98, 122.49, 119.14, 118.09.
1H), 7.01 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H),
Elemental Analysis: calc C 50.19%, H 2.59%,
3.77 (s, 3H), 3.75 (s, 3H).
N 9.00%; found C 50.26%, H 2.64%, N 8.92%.
m.p.: 163°C.
13C NMR (75 MHz, DMSO) δ 165.02, 157.36, 148.46,
145.70, 133.03, 131.21, 127.99, 122.58, 119.10, 118.81,
113.05, 111.77, 106.00, 55.63, 55.42.
2-Chloro-4-nitro-N-(4-nitrophenyl)benzamide (46)74
Elemental Analysis: calc
C
58.55%,
H
4.59%, Using Method A the title compound was isolated as a pale-
N 4.55%; found C 58.53%, H 4.68%, N 4.46%.
m.p.: 185°C.
yellow solid. Yield 39%
1H NMR (300 MHz, DMSO-d6) δ 11.36 (s, 1H), 8.45
(d, J = 2.2 Hz, 1H), 8.36–8.25 (m, 3H), 7.97 (dd, J = 8.7,
5-Chloro-2-hydroxy-N-(3,5-dichlorophenyl)benzamide
(43)63
5.3 Hz, 3H).
13C NMR (75 MHz, DMSO-d6) δ 164.04, 148.58, 144.35,
Using Method A the title compound was isolated as a pale- 142.96, 141.45, 131.09, 130.15, 125.03, 124.67, 122.54, 119.54.
yellow solid. Yield 56%
Elemental Analysis: calc 48.54%, 2.51%,
1H NMR (300 MHz, DMSO-d6) δ 11.75–11.27 (s, 1H), N 13.06%; found C 48.16%, H 2.63%, N 12.81%.
C
H
10.56 (s, 1H), 7.90–7.76 (m, 2H), 7.47 (dd, J = 8.8, 2.7 Hz,
m.p.: 197°C (lit: 196–198°C).
1H), 7.34 (t, J = 1.9 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H).
13C NMR (75 MHz, DMSO) δ 164.99, 156.11, 140.56, 2-Chloro-4-nitro-N-(5-chloro-2-hydroxyphenyl)benzamide
133.99, 133.05, 128.53, 123.23, 122.74, 120.21, 118.93, (47)
118.54.
Elemental Analysis: calc
Using Method B the title compound was isolated as
C
49.32%, H 2.55%, a yellow solid. Yield 20%
N 4.42%; found C 49.70%, H 2.20%, N 4.36%.
1H NMR (300 MHz, DMSO-d6) δ 10.26 (s, 1H), 10.11
(s, 1H), 8.38 (d, J = 2.2 Hz, 1H), 8.27 (dd, J = 8.4, 2.2 Hz,
1H), 8.01 (d, J = 2.6 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H),
7.08 (dd, J = 8.6, 2.6 Hz, 1H), 6.92 (d, J = 8.6 Hz, 1H).
13C NMR (75 MHz, DMSO-d6) δ 163.88, 148.22,
147.47, 142.16, 131.16, 130.13, 126.51, 124.96, 124.35,
m.p.: 247 °C (lit: 247–249 °C).
2-Chloro-4-nitro-N-(2-chlorophenyl)benzamide (44)
Using Method A the title compound was isolated as an off-
white solid. Yield 50%
1H NMR (300 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.42 122.24, 121.98, 116.59.
(d, J = 2.2 Hz, 1H), 8.32 (dd, J = 8.4, 2.2 Hz, 1H), 7.92 (d,
Elemental Analysis: calc
J = 8.4 Hz, 1H), 7.74 (dd, J = 8.0, 1.7 Hz, 1H), 7.58 (dd, N 8.56%; found C 47.68%, H 2.48%, N 8.51%.
C
47.79%, H 2.47%,
J = 8.0, 1.5 Hz, 1H), 7.43 (td, J = 7.6, 1.6 Hz, 1H), 7.32
(td, J = 7.7, 1.7 Hz, 1H).
m.p.: 223°C.
13C NMR (75 MHz, DMSO-d6) δ 163.90, 148.38, 3,4-Diethoxy-N-(4-(trifluoromethoxy)phenyl)benzamide (48)
141.98, 133.87, 131.20, 130.11, 129.72, 128.35, 127.69, Using Method A the title compound was isolated as
127.56, 124.57, 122.40.
a white solid. Yield 66%
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1H NMR (300 MHz, DMSO-d6) δ 10.22 (s, 1H), 7.- J = 9.0 Hz, 1H), 7.64 (dd, J = 8.4, 2.1 Hz, 1H), 7.56 (d, J =
95–7.81 (m, 2H), 7.60 (dd, J = 8.4, 2.1 Hz, 1H), 7.53 (d,
J = 2.1 Hz, 1H), 7.41–7.30 (m, 2H), 7.08 (d, J = 8.5 Hz,
1H), 4.11 (qd, J = 7.0, 1.7 Hz, 4H), 1.36 (td, J = 6.9, 1.1
Hz, 6H).
13C NMR (75 MHz, DMSO) δ 165.01, 151.23, 147.59,
143.67, 143.64, 138.50, 126.46, 121.63, 121.35, 121.16,
112.52, 111.99, 63.96, 63.82, 14.67, 14.55.
2.1 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H), 4.12 (qd, J = 6.9, 5.4
Hz, 4H), 1.37 (t, J = 6.9 Hz, 6H).
13C NMR (75 MHz, DMSO) δ 164.82, 151.71, 147.70,
144.28, 141.56, 127.89, 126.54, 125.22, 124.84, 122.86,
121.51, 112.43, 112.10, 63.94, 63.89, 14.65, 14.54.
Elemental Analysis: calc
C 55.98%, H 4.70%,
N 7.68%; found C 56.00%, H 4.69%, N 7.54%.
m.p.: 168°C.
Elemental Analysis: calc
C 58.54%, H 4.91%,
N 3.79%; found C 58.64%, H 5.16%, N 3.66%.
m.p.: 173°C.
N-Phenylbenzo[d][1,3]dioxole-5-carboxamide (52)75
Using Method A the title compound was isolated as
a white solid. Yield 64%
1H NMR (300 MHz, DMSO-d6) δ 10.06 (s, 1H), 7.83–-
7.72 (m, 2H), 7.59 (dd, J = 8.2, 1.8 Hz, 1H), 7.53 (d, J = 1.8
Hz, 1H), 7.34 (dd, J = 8.5, 7.3 Hz, 2H), 7.15–7.02 (m, 2H),
6.14 (s, 2H).
13C NMR (75 MHz, DMSO) δ 164.44, 149.98, 147.32,
139.18, 128.70, 128.50, 123.46, 122.79, 120.31, 107.87,
107.68, 101.76.
3,4-Diethoxy-N-(4-bromophenyl)benzamide (49)
Using Method A the title compound was isolated as
a white solid. Yield 57%
1H NMR (300 MHz, DMSO-d6) δ 10.16 (s, 1H), 7.-
79–7.71 (m, 2H), 7.59 (dd, J = 8.4, 2.1 Hz, 1H), 7.56–7.49
(m, 3H), 7.07 (d, J = 8.5 Hz, 1H), 4.11 (qd, J = 7.0, 1.3 Hz,
4H), 1.36 (td, J = 7.0, 1.1 Hz, 6H).
13C NMR (75 MHz, DMSO) δ 164.96, 151.19, 147.57,
138.67, 131.31, 126.53, 122.22, 121.14, 115.03, 112.48,
111.99, 63.96, 63.82, 14.69, 14.57.
Elemental Analysis: calc
C 69.70%, H 4.60%,
N 5.81%; found C 69.54%, H 4.65%, N 5.77%.
Elemental Analysis: calc
C 56.06%, H 4.98%,
m.p.: 142°C (lit: 138–139°C).76
N 3.85%; found C 56.19%, H 5.26%, N 3.77%.
m.p.: 198°C.
N-(4-Cyanophenyl)benzo[d][1,3]dioxole-5-carboxamide
(53)
3,4-Diethoxy-N-(4-nitro-2-(trifluoromethyl)phenyl)benza-
mide (50)
Using Method A the title compound was isolated as
a white solid. Yield 48%
Using Method A the title compound was isolated as
1H NMR (300 MHz, DMSO-d6) δ 10.44 (s, 1H), 7.97 (d,
J = 8.8 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.59 (dd, J = 8.1, 1.8
Hz, 1H), 7.52 (d, J = 1.8 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.15
(s, 2H).
a white solid. Yield 64%
1H NMR (300 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.56
(dd, J = 8.7, 2.7 Hz, 1H), 8.52 (d, J = 2.7 Hz, 1H), 7.93 (d,
J = 8.8 Hz, 1H), 7.60 (dd, J = 8.4, 2.1 Hz, 1H), 7.53 (d, J =
2.1 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 4.24–4.01 (m, 4H),
1.37 (t, J = 6.9 Hz, 6H).
13C NMR (75 MHz, DMSO) δ 165.02, 150.42, 147.41,
143.54, 133.02, 128.01, 123.21, 120.06, 119.06, 107.96,
107.80, 105.10, 101.91.
13C NMR (75 MHz, DMSO) δ 165.66, 151.64, 147.68,
145.02, 142.03, 131.43, 127.87, 125.97, 125.56, 125.10,
122.36, 121.35, 112.38, 112.13, 63.92, 63.88, 14.63,
14.53.
Elemental Analysis: calc
C 67.67%, H 3.79%,
N 10.52%; found C 67.62%, H 3.87%, N 10.67%.
m.p.: 193°C.
Elemental Analysis: calc
C 54.27%, H 4.30%,
N-(2-Methyl-4-nitrophenyl)benzo[d][1,3]dioxole-5-car-
boxamide (54)
N 7.03%; found C 54.18%, H 4.22%, N 6.96%.
m.p.: 158°C.
Using Method A the title compound was isolated as an off-
white solid. Yield 18%
3,4-Diethoxy-N-(2-chloro-4-nitrophenyl)benzamide (51)
Using Method A the title compound was isolated as an off-
white solid. Yield 65%
1H NMR (300 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.19 (d,
J = 2.7 Hz, 1H), 8.10 (dd, J = 8.8, 2.8 Hz, 1H), 7.78 (d, J =
8.8 Hz, 1H), 7.61 (dd, J = 8.1, 1.8 Hz, 1H), 7.53 (d, J = 1.8
1H NMR (300 MHz, DMSO-d6) δ 10.08 (s, 1H), 8.41
(d, J = 2.6 Hz, 1H), 8.26 (dd, J = 9.0, 2.6 Hz, 1H), 8.04 (d, Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 6.15 (s, 2H), 2.39 (s, 3H).
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13C NMR (75 MHz, DMSO) δ 164.56, 150.40, 147.43,
144.05, 143.11, 133.85, 127.74, 125.59, 125.33, 123.20, N 8.74%; found C 52.20%, H 2.85%, N 8.65%.
Elemental Analysis: calc C 52.44%, H 2.83%,
121.47, 108.02, 107.83, 101.88, 17.89.
Elemental Analysis: calc 60.00%,
m.p.: 227°C.
C
H 4.03%,
N 9.33%; found C 59.85%, H 3.76%, N 9.22%.
m.p.: 230°C.
N-(2-Chloro-4-nitrophenyl)-2-naphthamide (58)
Using Method A the title compound was isolated as a pale-
yellow solid. Yield 52%
N-(2,4-Dinitrophenyl)benzo[d][1,3]dioxole-5-carboxamide
(55)
1H NMR (300 MHz, Chloroform-d) δ 8.86 (d, J = 9.2
Hz, 1H), 8.79 (s, 1H), 8.43–8.37 (m, 1H), 8.31 (d, J = 2.5
Using Method A the title compound was isolated as Hz, 1H), 8.19 (ddd, J = 9.3, 2.6, 0.5 Hz, 1H), 7.99–7.82
a yellow solid. Yield 10%
(m, 4H), 7.63–7.49 (m, 2H).
1H NMR (300 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.74
13C NMR (75 MHz, CDCl3) δ 165.44, 143.07, 140.51,
(d, J = 2.6 Hz, 1H), 8.57 (dd, J = 9.0, 2.7 Hz, 1H), 8.12 (d, 135.31, 132.57, 130.76, 129.30, 129.24, 128.64, 128.32,
J = 9.1 Hz, 1H), 7.59 (dd, J = 8.2, 1.8 Hz, 1H), 7.47 (d, J = 127.92, 127.37, 124.76, 123.83, 123.06, 122.59, 120.24.
1.8 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 6.17 (s, 2H).
Elemental Analysis: calc C 62.49%, H 3.39%,
13C NMR (75 MHz, DMSO) δ 164.49, 151.20, 147.73, N 8.57%; found C 62.72%, H 3.09%, N 8.71%.
142.52, 140.60, 137.48, 128.56, 126.54, 125.30, 123.62,
121.14, 108.26, 107.68, 102.18.
m.p.: 225°C.
Elemental Analysis: calc
C
50.77%,
H
2.74%, 1-Hydroxy-N-(2-chloro-4-nitrophenyl)-2-naphthamide
(59)56
N 12.69%; found C 50.84%, H 2.46%, N 12.68%.
m.p.: 200°C.
Using Method A the title compound was isolated as
a yellow solid. Yield 10%
N-(2-Methoxy-4-nitrophenyl)benzo[d][1,3]dioxole-5-car-
boxamide (56)
1H NMR (300 MHz, DMSO-d6) δ 12.21 (s, 1H), 11.64
(s, 1H), 8.91 (d, J = 9.2 Hz, 1H), 8.75 (s, 1H), 8.46 (d, J =
Using Method A the title compound was isolated as 2.6 Hz, 1H), 8.34 (dd, J = 9.2, 2.7 Hz, 1H), 8.03 (d, J = 8.2
a yellow solid. Yield 33% Hz, 1H), 7.81 (dd, J = 8.5, 1.2 Hz, 1H), 7.56 (ddd, J = 8.2,
1H NMR (300 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.24 (d, 6.9, 1.3 Hz, 1H), 7.46–7.35 (m, 2H).
J = 8.9 Hz, 1H), 7.94 (dd, J = 8.9, 2.5 Hz, 1H), 7.87 (d, J =
13C NMR (75 MHz, DMSO) δ 163.56, 152.25, 142.46,
2.5 Hz, 1H), 7.59 (dd, J = 8.2, 1.8 Hz, 1H), 7.52 (d, J = 1.8 141.41, 136.28, 133.41, 129.23, 128.86, 127.21, 125.73,
Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.15 (s, 2H), 4.01 (s, 3H). 124.81, 124.14, 123.93, 122.35, 120.73, 120.10, 110.95.
13C NMR (75 MHz, DMSO) δ 164.40, 150.58, 150.01,
147.55, 143.50, 133.87, 127.62, 123.09, 121.56, 116.53, N 8.17%; found C 58.97%, H 3.20%, N 7.67%.
108.09, 107.67, 106.07, 101.96, 56.57.
m.p.: 230°C (lit: 233°C).77
Elemental Analysis: calc 56.97%,
Elemental Analysis: calc C 59.58%, H 3.24%,
C
H 3.82%,
N 8.86%; found C 56.67%, H 3.71%, N 8.64%.
m.p.: 237°C.
3-Hydroxy-N-(4-(trifluoromethyl)phenyl)-2-naphthamide
(60)78
Using Method A the title compound was isolated as an off-
N-(2-Chloro-4-nitrophenyl)benzo[d][1,3]dioxole-5-car-
boxamide (57)
white solid. Yield 26%
1H NMR (300 MHz, DMSO-d6) δ 11.20 (s, 1H), 10.89
Using Method A the title compound was isolated as a pale- (s, 1H), 8.48 (s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.94 (d, J =
yellow solid. Yield 53% 8.2 Hz, 1H), 7.76 (dd, J = 8.7, 4.6 Hz, 3H), 7.51 (ddd, J =
1H NMR (300 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.41 8.3, 6.9, 1.3 Hz, 1H), 7.42–7.31 (m, 2H).
(d, J = 2.6 Hz, 1H), 8.26 (dd, J = 9.0, 2.6 Hz, 1H), 8.04 (d,
13C NMR (75 MHz, DMSO) δ 165.84, 153.25, 142.22,
J = 9.0 Hz, 1H), 7.63 (dd, J = 8.2, 1.8 Hz, 1H), 7.53 (d, J = 135.71, 130.63, 128.66, 128.11, 126.83, 126.12, 126.06,
1.8 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 6.16 (s, 2H).
126.01, 125.75, 123.72, 122.51, 120.11, 110.47.
Elemental Analysis: calc 65.26%,
13C NMR (75 MHz, DMSO) δ 164.49, 150.72, 147.55,
C
H
3.65%,
144.38, 141.41, 127.99, 127.12, 126.65, 124.85, 123.39, N 4.23%; found C 64.43%, H 3.69%, N 3.93%.
122.86, 108.12, 107.78, 101.99.
m.p.: 280°C (lit: 276–278°C).
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3-Hydroxy-N-(4-bromophenyl)-2-naphthamide (61)78
1H NMR (300 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.82
Using Method A the title compound was isolated as an off- (d, J = 2.6 Hz, 1H), 8.63 (dd, J = 9.1, 2.7 Hz, 1H), 8.37 (d,
white solid. Yield 29%
J = 9.1 Hz, 1H), 7.94–7.84 (m, 2H), 7.78 (dd, J = 8.4, 1.0
1H NMR (300 MHz, DMSO-d6) δ 11.22 (s, 1H), 10.68 Hz, 1H), 7.58 (ddd, J = 8.4, 7.2, 1.3 Hz, 1H), 7.42 (td, J =
(s, 1H), 8.47 (s, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.83–7.71 7.6, 1.0 Hz, 1H).
13C NMR not performed due to poor solubility.
Elemental Analysis: calc 55.05%, 2.77%,
(m, 4H), 7.63–7.45 (m, 4H), 7.42–7.30 (m, 2H).
13C NMR (75 MHz, DMSO) δ 165.64, 153.46, 137.91,
135.70, 131.57, 130.44, 128.65, 128.09, 126.82, 125.75,
123.72, 122.26, 122.12, 115.62, 110.48.
C
H
N 12.84%; found C 54.77%, H 2.67%, N 12.85%.
m.p.: 237°C.
Elemental Analysis: calc
C 59.67%, H 3.53%,
N-(2-Chloro-4-nitrophenyl)benzofuran-2-carboxamide (65)
Using Method A the title compound was isolated as
a yellow solid. Yield 54%
N 4.09%; found C 59.37%, H 3.56%, N 3.98%.
m.p.: 250°C (lit: 248–250°C).
1H NMR (300 MHz, Chloroform-d) δ 9.13 (s, 1H),
8.79 (d, J = 9.2 Hz, 1H), 8.29 (d, J = 2.6 Hz, 1H), 8.15
(ddd, J = 9.2, 2.6, 0.6 Hz, 1H), 7.71–7.65 (m, 1H), 7.62 (d,
J = 1.0 Hz, 1H), 7.54 (dq, J = 8.5, 1.0 Hz, 1H), 7.44 (ddd,
J = 8.4, 7.2, 1.3 Hz, 1H), 7.29 (ddd, J = 8.0, 7.2, 1.1
Hz, 1H).
13C NMR (75 MHz, CDCl3) δ 156.56, 155.05, 147.26,
143.26, 139.86, 128.12, 127.41, 124.84, 124.33, 123.71,
123.13, 122.73, 120.23, 113.21, 112.13.
3-Hydroxy-N-(2-chloro-4-nitrophenyl)-2-naphthamide
(62)79
Using Method A the title compound was isolated as
a yellow solid. Yield 13%
1H NMR (300 MHz, DMSO-d6) δ 13.06 (s, 1H), 11.-
92–11.53 (m, 1H), 8.50–8.42 (m, 2H), 8.38 (d, J = 8.2 Hz,
1H), 8.31 (dd, J = 9.1, 2.6 Hz, 1H), 8.07 (d, J = 8.8 Hz,
1H), 7.92 (d, J = 8.1 Hz, 1H), 7.72–7.54 (m, 2H), 7.48 (d,
J = 8.8 Hz, 1H).
Elemental Analysis: calc
C 56.89%, H 2.86%,
13C NMR (75 MHz, DMSO) δ 167.42, 143.79, 141.30,
136.40, 129.02, 127.62, 126.20, 125.80, 124.85, 124.75,
124.52, 123.42, 123.35, 109.88.
N 8.85%; found C 56.99%, H 2.78%, N 8.64%.
m.p.: 203°C.
Elemental Analysis: calc
C 59.58%, H 3.24%,
N-(2-Chloro-4-nitrophenyl)benzo[b]thiophene-2-carboxa-
mide (66)80
N 8.17%; found C 59.36%, H 3.25%, N 8.05%.
m.p.: 238°C.
Using Method A the title compound was isolated as an off-
white solid. Yield 21%
1H NMR (300 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.-
49–8.39 (m, 2H), 8.28 (dd, J = 8.9, 2.6 Hz, 1H), 8.13–7.99
(m, 3H), 7.58–7.45 (m, 2H).
N-(2-Chloro-4-nitrophenyl)-3-methyl-1H-indene-2-car-
boxamide (63)
Using Method A the title compound was isolated as a pale-
13C NMR (75 MHz, DMSO) δ 160.53, 144.74,
140.72, 140.63, 138.90, 138.05, 128.21, 127.35,
127.03, 126.94, 125.69, 125.20, 124.97, 122.93, 122.90.
yellow solid. Yield 47%
1H NMR (300 MHz, Chloroform-d) δ 8.90 (d, J = 9.2
Hz, 1H), 8.46–8.29 (m, 2H), 8.21 (dd, J = 9.3, 2.6 Hz, 1H),
7.57 (ddt, J = 5.3, 3.8, 1.9 Hz, 2H), 7.50–7.39 (m, 2H),
3.80 (q, J = 2.4 Hz, 2H), 2.69 (t, J = 2.4 Hz, 3H).
13C NMR (75 MHz, CDCl3) δ 163.71, 152.04, 145.10,
142.64, 141.96, 140.74, 130.78, 128.36, 127.23, 124.68,
124.04, 123.76, 122.02, 121.40, 119.77, 38.03, 12.76.
Elemental Analysis: calc
C 54.14%, H 2.73%,
N 8.42%, S 9.63%; found C 54.34%, H 2.46%,
N 8.37%, S 9.49%.
m.p.: 228°C.
Elemental Analysis: calc
C 62.11%, H 3.99%,
Chemistry
N 8.54%; found C 62.23%, H 3.59%, N 8.29%.
m.p.: 216°C.
General Synthesis Procedures
Method A
The aniline derivative (6 mmol, 1 eq) and the aromatic
acid derivative (6 mmol, 1 eq) were suspended in toluene
(10 mL). After heating to reflux and dissolving of the
precipitate, phosphor trichloride (2.4 mmol, 0.4 eq) was
N-(2,4-Dinitrophenyl)benzofuran-2-carboxamide (64)
Using Method A the title compound was isolated as
a yellow solid. Yield 13%
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added in a dropwise manner. The reaction was monitored 30 µm for MAO A and B, respectively). MAO A (1.25
using TLC (ethyl acetate:n-hexane 1:4). After reaction ng mL−1, 900 units mL−1) or MAO B (1.67 ng mL−1, 375
completion, the hot mixture was filtered and the organic units mL−1), respectively, was added to start the reactions.
solution was stored at 5 °C. The formed precipitate was The reaction mixtures were incubated for 20 min incuba-
filtered off, washed with ice-cold toluene and recrystal- tion time at 37°C. All enzyme assays were conducted in
lized from toluene.17
pre-warmed potassium phosphate buffer (50 mM, pH =
7.4) and a final assay volume of 100 µL. Reactions were
stopped by adding 35 µL sodium hydroxide solution (2 N)
to the assay mixture. The enzyme activity was determined
by measuring the 4-hydroxyquinoline formed during incu-
bation time (expressed as RFU; λEm = 405 20 nm and λEx
= 320 20 nm). Compounds showing a MAO B inhibition
>90% at 10−6 M were evaluated for their IC50-values. The
IC50 curves were fitted (via nonlinear regression) either to
the respective bottom plateau (graphically defined by
RFUs for the at least two highest concentrations used,
see Figure S12 in supplemental material) or set to zero
in case when the bottom plateau was not reached with
highest concentration tested (leading to “IC50 estimates”).
The highest concentrations to be tested had to be identified
considering each compound’s solubility and potential
interfering fluorescence properties under assay conditions.
Reversibility of inhibition was confirmed via prein-
cubation of inhibitor (10 ⨰ IC50 in preincubation set-
ting) with MAO B (10 ng µL−1 in preincubation setting)
for 0, 30, 60 and 90 min (37 °C), followed by 50⨰
dilution in buffer and assayed with an excess of sub-
strate (10 ⨰ KM final concentration) as described
above.21 Data were calculated as percentage of vehicle
control (DMSO; set to 100% enzyme activity remained)
for each time point.
Method B
1,1ʹ-Carbonyldiimidazole (CDI) (9 mmol, 1.5 eq) was
dissolved in THF (10 mL). To this solution the respec-
tive aromatic acid (9 mmol, 1.5 eq) was added dissolved
in THF (10 mL). After 10 min stirring at room tem-
perature, a solution of the aniline derivative (6 mmol, 1
eq) in THF (10 mL) was added. The reaction mixture
was stirred for 10 min at room temperature and then
refluxed overnight. After cooling to room temperature,
the solvent was evaporated under reduced pressure and
the residue dissolved in ethylacetate. The organic phase
was then washed with saturated aqueous sodium hydro-
gen carbonate solution and brine. After drying over
magnesium sulfate, the solvent was evaporated under
reduced pressure and the residue was recrystallized
from toluene.18
Method C
The respective pyridine derivative (6 mmol, 1 eq) and the
benzoyl chloride derivative (6 mmol, 1 eq) were sealed in
a glass vial. The reaction mixture was stirred for 4 min at
160°C under microwave irradiation. The resulting mixture
was recrystallized from ethanol.19
Method D
Mode of MAO B inhibition was determined by substrate-
dependent (seven concentrations, 5 to 400 µm kynuramine)
Michaelis-Menten kinetic analysis without inhibitor and in
the presence of five different inhibitor concentrations (0.25⨰,
0.5⨰, 0.75⨰, 1⨰ and 2 ⨰ IC50) and assayed as described
above. Data were fitted using nonlinear “Competitive
inhibition” fit and transformed into double reciprocal
(Lineweaver–Burk) plots. The slopes of respective
Lineweaver–Burk linearization were plotted against inhibitor
concentration for additional Ki determination.22
The respective nitro derivative (6, 9, 26) (6 mmol, 1 eq)
was dissolved in dried ethanol (20 mL). After adding Pd/C
(0.6 mmol, 0.1 eq) and hydrazine monohydrate (60 mmol,
10 eq), the mixture was refluxed for 2 h and afterwards
stirred for another 45 min at room temperature. Filtration
via a short Celite column and evaporation of the solvent
afforded the desired compounds (5, 8, 25).20
Enzymology
Monoamine oxidase A/B inhibition assays were performed
All data were analyzed with GraphPad Prism 6.
as described previously using the discontinuous fluori-
metric method with kynuramine as MAO substrate.21 For Molecular Modelling
the screening (test concentration: 10−6 M) and IC50 deter- The crystal structure 2Z5X23 was used as the represen-
minations (ten suitable concentrations ranging from 10−11 tative complex structure of MAO A and the crystal
to 10−5 M), the test compounds were pre-mixed with the structure 6FVZ9 for MAO B. Both crystal structures
substrate (2 ⨰ KM final concentration; KM of 20 µm and were chosen due to the lipophilicity of the crystalized
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ligands (HRM for MAO A and E8Z for MAO B) and inactive classification accuracy of these models is good
the presence of a peptide bond of the MAO B ligand. with ROC-AUC values of 0.779 and 0.750 for MAO
The enzyme structures were prepared using the Quick A and MAO B models, respectively. Further theoretical
Prep tool in MOE 2018.0101 (Chemical Computing and experimental methodology of ROC analyses are
Group Inc., Canada) including protonation and energy described in supplemental material.
minimization. Poses were generated using the Dock
tool in MOE 2018.0101. A general docking was con-
Results and Discussion
Chemistry
In 2018 the European Medicines Agency (EMA) recom-
ducted. In case of MAO A, solvent was ignored. The
placement method triangle matcher with the scoring
function London dG was chosen. The refinement
mended 22 biological active small molecules for market
method rigid receptor with the scoring function Alpha
authorization.27 All newly approved small molecules
HB was chosen.
exhibit rather complex structures with the need for mul-
tistep synthesis, often including stereoselective synthesis
Pharmacophore Modeling
steps. Using a one-step synthesis followed by easy pur-
The pharmacophore hypotheses of the MAO A and
ification procedures, we generated a series of promising
B inhibitors were constructed using the HipHop mod-
MAO A/B inhibitors. These small entities might be of
ule of Discovery studio software 2.5.5. A subset of 25
interest for the development of selective or multitarget-
representative compounds was selected and classified
ing MAO A/B inhibitors. The substituted aromatic
as active, moderately active, and inactive (Table S1 in
amides were synthesized in low to good yields via
the supplemental material). In total 16 HipHop runs
activation of the carboxylic acid using either PCl3 or
were separately conducted for MAO A and MAO
CDI. CDI was used as a coupling agent for compound
B ligands that ultimately generate 160 models (10
47 to prevent condensation to a substituted benzoxazole,
from each run) for each target by varying the types
which was the major product using PCl3 method. For
and ranges of pharmacophoric features and the number
compounds 2 and 19, solvent free acylation using ben-
of features (Table S2 in the supplemental material).
zoyl chloride derivatives was performed under micro-
The generated models were allowed to compete in
wave irradiation (Figure 1). The aromatic amines (5, 8,
Receiver Operating Characteristic (ROC) curve analy-
25) were obtained in excellent yields from reduction of
sis to assess their abilities to selectively capture diverse
the respective nitro derivatives (6, 9, 26). Due to their
MAO A or MAO B inhibitors from a large list of
simple structures, many of our compounds have been
decoys. The decoy list was prepared as described by
described previously, but to our knowledge none of
Verdonk et al.24 The testing sets include structurally
them were determined as potent inhibitors for MAO
diverse active inhibitors of 14 MAO A and 18 MAO
B retrieved from literature.14,25,26 36 decoys were
A or B so far.
selected for each active compound in the testing set
retrieved from ZINC-database. The ROC testing set
Biological Activity
was screened by each pharmacophore for ROC analysis MAO A and B Inhibition
employing the “Best rigid search” option, while the An enzyme inhibition screening at a concentration of 1
conformational spaces of the compounds were gener- µm revealed that the synthesized anilides showed the pre-
ated employing the “CAESAR conformation generation sumed MAO inhibition potency. The majority of derivatives
option”. Compounds missing one or more features (32 compounds) were found to be MAO B-preferring inhibi-
were discarded from hit lists. The validity of tors, while 24 compounds were not selective for either of the
a particular pharmacophore is assessed by the area isoforms and only ten compounds exhibited MAO
under the curve (AUC) of the corresponding ROC A preference (Table 1 and see Figure 2 for all compound
curve, as well as accuracy, specificity, true positive structures). The most potent MAO inhibitors were chosen for
rate, and false negative rate of the pharmacophore. further IC50 evaluation. Six compounds (31, 33, 34, 39, 55, and
Table S3 and Figure S10 in the supplemental material 65) showed >90% inhibition of MAO B. The most potent
show the ROC performance of the best pharmacophore MAO A-preferring inhibitor (with MAO B inhibition <50%),
models generated for MAO A and MAO B. The active/ compound 7 was also considered for additional investigations.
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Hagenow et al
Figure 1 Synthetic route to the amide analogs 1–4, 6, 7, 9–24, 26–66. Reagents and conditions: (A) PCl3, toluene, reflux. (B) CDI, THF, reflux. (C) mw, 160 °C. For R1,
R2, and R3 see Table 1.
Compounds 31, 33, 34, 55, and 65 showed the expected pre- A preference for MAO A could be confirmed for compound 7
ference for MAO B with IC50 values in nanomolar concentra- (ST-2023) (MAO A, IC50 = 126 nM) with an IC50 > 1000 nM
tion ranges (IC50 values between 55 nM and 139 nM; Table 2). for MAO B; selectivity index, SI = 0.1). The potent MAO
Table 1 MAO A and B Inhibition Percentages Measured at One-Point Screening of Anilides (Screening Concentration 1 µm)
n
% Inhibition at 1 µm
MAO A
12 8
n
% Inhibition at 1 µm
MAO B
5 3
MAO A
9 3
MAO B
7 5
1
15
16
17
18
19
20
21
22
23
24
25
26
27
28
41
42
43
44
45
46
47
2
11 5
2 4
20 12
10 5
18 5
13 2
72 2
13 1
2 1
14 10
69 28
24 9
11 7
61 4
10 7
14 3
25 4
48 5
11 4
2 3
3
29 3
27 3
29 3
−2 3
7 5
4
24 20
1 2
5
6
16 7
7
82 2
10 4
46 4
1 3
8
9
14 7
14 5
25 3
85 1
5 3
23 3
27 4
25 2
11 2
20 6
8 4
10
11
12
13
14
29
30
31
32
33
34
35
28 8
48 3
28 8
50 4
32 10
18 11
87 5
38 7
104 2
66 5
96 2
96 1
5 10
44 23
5 2
49 5
15 1
8 4
85 3
30 5
0 5
7 3
5 3
82 1
45 5
4 8
34 3
13 2
13 3
35 3
6 2
74 2
36 4
68 2
36 4
(Continued)
71 5
8 1
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Table 1 (Continued).
n
% Inhibition at 1 µm
n
% Inhibition at 1 µm
MAO A
6 4
MAO B
84 12
30 10
−2 2
91 1
6 2
MAO A
5 5
MAO B
1 2
36
37
38
39
40
53
54
55
56
57
58
59
48
49
50
51
52
60
61
62
63
64
65
66
13 4
9 2
3 3
13 6
62 2
34 2
80 4
67 2
72 2
48 5
44 23
80 4
92 4
40 31
6 5
20 8
8 4
3 4
19 4
−12 18
−3 5
−2 20
−3 19
84 2
52 10
11 8
38 6
7 9
87 1
24 26
91 2
47 5
44 4
1 2
9 9
62 7
11 7
−4 10
58 1
28 2
Notes: aData represent mean values standard deviation of at least two independent experiments each performed in duplicates (global fit). Percentage values were
calculated relative to control (set to 100% remained activity). Bold values represent inhibition rates >90 % MAO B and highest MAO A inhibitions.
B inhibition capacity of 39, presumed depending on screening involved in the binding of the presented ligands. Compounds
results, could not be verified in IC50 determinations (MAO B, 31, 33, and 34 showed close structural similarities and compar-
IC50 >1000 nM), where assay limitations did not allow the able inhibition properties. All of these ligands are substituted
determination of the bottom plateau due to low solubility and with a hydroxyl group at the 2-position of the benzylic site and
a chloro substituent at the 5-position. The aniline moiety in all
cases is para-substituted with a highly lipophilic residue, ie a
bromo-substituent (34), a trifluoromethoxy group (33), and
a trifluoromethyl group (31) in the para-position. The interac-
tion of these hydrophobic residues with the lipophilic cavity of
the MAO B binding pocket, including the nonpolar amino
acids Pro102 and Leu164, might be responsible for their potent
MAO B inhibition properties. The carbonyl function of the
linking amide and the hydroxyl groups in 2-position are direc-
ted towards Cys172 and interact via polar aprotic forces (see
Figures S5-S7 in the supplemental material). A similar polar
system is present in pyrrolo-pyridinyl derivatives synthesized
by Tzvetkov et alindicating that a central hydrogen bond
donor/acceptor complex is favorable for MAO B binding.28
The docking pose of the MAO A preferring inhibitor 7 (ST-
2023) is inverted by 180° in the binding pocket of the MAO
fluorescence crosstalk. Assuming that the real but
nondetectable bottom plateau might be » 0, the previous
determined percent inhibition value in one-point screening
would have been overestimated (as calculated with bottom
= 0). The most potent compound 55 (ST-2043) showed high
inhibition of MAO B (IC50 = 56 nM), which is in a similar
range as described for the marketed drug safinamide (IC50 = 53
nM; Table 1). Compared to safinamide (MAO A/B SI >940),
55 demonstrated only an about five-fold lower inhibition of
isoform A, thus being a rather balanced MAO inhibitor. The
highest MAO B preference within this series was shown for
compound 31 with an SI >10 (MAO B, IC50 = 92 nM).
The docking experiments revealed a good match of all
compounds in the lipophilic binding pockets of the MAO
A and B with exception of compound 39, where no suitable
docking pose into the MAO A binding pocket could be identi-
fied (see Figures S1-S9 in the supplemental material). B model, where the polar nitro-group is interacted with Cys172
According to the result of the docking experiments, surface (Figure 3). This would enlarge the distance to the FAD and
complementarities influence the binding properties of the ani- could be responsible for the low MAO B activity compared
lides nearly exclusively, whereas direct interactions are not with MAO A. This reversed pose was also observed in
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Figure 2 Compounds with anilide motifs taken to MAO A and B screening.
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Table 2 MAO A and B Inhibition Activities of the Most Potent
Compounds Expressed as IC50 Values Within the 95%CI
a polar interaction with a bridging water molecule and
a tyrosine close to the FAD.29 The most potent inhibitor 55
(ST-2043) in this series is oriented with its piperonylic acid
moiety towards the FAD (Figure 3). Using the same moiety,
Vishnu et al designed MTLs where the piperonylic acid aligned
within the hydrophobic region opposite to the FAD, and the
high inhibition capacities resulted from irreversible binding via
a propargyl amine residue.30
No.
IC50 [nM] [95%CI] (N)a
c
hMAO B
hMAO A
MAO SI
0.1
7 (ST-2023)
1052 [759; 1459] (3)
92.3 [70.3; 121] (3)
128 [54.0; 305] (3)
139 [64.7; 300] (3)
>1000b
126 [101; 158] (4)
>1000b
31
>10.8
>7.8
33
>1000b
>1000b
>7.2
Due to their small size and molecular weight, the presented
structures are useful starting skeletons to design MTLs with
MAO B activity. The docking experiments suggest using the
aniline residue of 55 (ST-2043) as an attachment point to
implement pharmacophores of additional targets. Zhang et al
showed that larger substituents at this position are tolerated,
where a bulky fluorobenzyl group is located further outside of
the enzymes binding pocket than the here presented aniline
groups.31 The hypothesis that substitution at this position
might be suitable for designing MTLs is coherent with the
linear structures synthesized by Pisani et al that combine MAO
B inhibition with nitric oxide releasing precursors and acet-
ylcholine esterase (AChE) inhibition moieties.32
34
39
>5000b
55 (ST-2043)
65
55.5 [31.9; 96.7] (5)
123 [87.6; 172] (5)
53 [20, 141] (4)
289 [193; 433] (5)
293 [203; 424] (3)
>50000 (4)
5.2
2.4
Safinamided
>940
Notes: aData represent mean values within the 95%CI of multiple independent experi-
ments (n) each performed in duplicates. bCompounds did not reach bottom plateau at
highest tested concentrations; Data are estimates extrapolated with bottom was set to
zero. cSelectivity index (SI) = IC50 MAO A/IC50 MAO B. dValues taken from Affini et al.21
compounds 39 and 65. In case of inhibitor 39, the salicylic
moiety is shifted towards the lipophilic cavity at the entrance of
the binding pocket, which might be responsible for its rather
poor inhibition properties (see Figures S8 in the supplemental
material). In contrast, inhibitor 65 maintained the lipophilic
In the MAO A binding pocket, the polar salicylic group
of compounds 31, 33, 34, and 39 did not match the surround-
interaction with its benzofuran residue resulting in an orienta- ing hydrophobic cavity, which might explain the loss of
tion of the polar nitro group towards the FAD (see Figures S9 in inhibition properties for MAO A (see Figures S1-S3 in the
the supplemental material). Carboxamides synthesized by supplemental material). The three compounds, missing the
Tzvetkov et al showed the same orientation resulting from salicylic moiety (7, 55, 65), are aligned well into the MAO
Figure 3 Visualization of 55 (ST-2043, A1, A2) and 7 (ST-2023, B1, B2) in the binding pockets of MAO A and B. Compound 55 (ST-2043) binding to the crystal structure
of A1) hMAO B (PDB: 6FVZ) and A2) hMAO A (PDB: 2Z5X). Compound 7 (ST-2023) binding to the crystal structure of B1) hMAO B (PDB: 6FVZ) and B2) hMAO A (PDB:
2Z5X). Surface coloring: white: neutral, green: lipophilic; magenta: hydrophilic.
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Hagenow et al
Figure 4 ROC-selected pharmacophores of MAO A and MAO B. (A) Overlay of compound 55 (ST-2043) and MAO B pharmacophore model. (B) Overlay of compound 7
(ST-2013) and MAO A pharmacophore model. Color code: green vectored sphere: hydrogen bond acceptor; orange vectored sphere: aromatic feature; blue sphere:
hydrophobic feature.
A active site with respect to surface complementarity, show- CI = [5.0; 7.5] (Michaelis Menten fit “competitive inhibi-
ing that small alteration in the molecular structure can have
a major impact on the modulation of the MAO isoform
selectivity. The modeling results are further supported by
ROC-selected pharmacophore models based on inhibitors
presented here (see Figure 4 and supplemental material).
The essential predicted characteristics for MAO A and
MAO B binding are a central hydrogen bond acceptor
flanked by aromatic or hydrophobic features. This is in
good accordance with the docking poses, with the limitation
that the molecular docking showed no direct hydrogen bond
interactions between the ligands and the amino acids of the
binding pocket.
tion”) and 9.5 nM (slopes from Lineweaver–Burk plots vs
inhibitor concentration) for MAO B, respectively.
Cholinesterase Inhibition
Cholinesterase enzymes are of potential interest in MTL drug
design for the treatment of neurodegenerative diseases.33
Therefore, compound 55 (ST-2043) was screened for inhibi-
tion of acetyl- and butyrylcholine esterases (AChE/BuChE).
At a concentration of 1 µm, 55 (ST-2043) showed only
moderate inhibition capacity with 24 1.8% and 57 4.5%
for AChE and BuChE, respectively (see Table S4 and
Figure S11 in supplemental material). Thus, no further char-
acterization in this direction has been performed.
The most potent MAO B inhibitor 55 (ST-2043) was
further investigated to determine mode and type of inhibition.
Reversibility studies with excess of substrate verified the
expected reversible mode of inhibition. Compared to the
suicide inhibitor L-deprenyl, inhibition by compound 55
(ST-2043) (10-fold IC50 concentrations) preincubated with
MAO B for 30, 60 or even 90 min was completely reversed
after 50-fold dilution in assays mixture (Figure 5). It could be
assumed, that compound 55 was readily displaced from the
MAO B active side, while a more tight-binding interaction, e.
g. as shown for safinamide, is considered to be more favor-
able in terms of pharmacological activity.9
140
0 min (n=2)
120
30 min (n=1)
100
60 min (n=2)
80
60
40
20
0
90 min (n=1)
Inhibition studies using different concentrations of 55
(ST-2043) with seven appropriate substrate concentrations
suggest a competitive inhibition type as demonstrated by
Lineweaver–Burk plots (Figure 6). Compound 55 (ST-
2043) showed promising Ki values of 6.3 nM with 95%
Figure 5 Reversibility of inhibition after preincubation of 55 (ST-2043) and L-deprenyl
with MAO B. Inhibitors (10 ⨰ IC50) were preincubated with enzyme for 0, 30, 60 or 90 min
prior to 50-fold dilution in assays mixture containing kynuramine as substrate (10 ⨰ KM).
Data represent mean values SD of n independent experiments, each performed in
duplicates.
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0.015
0.010
0.005
0.004
0.003
0.002
0.001
0 nM
R2
=
0.9989
12.5 nM
25 nM
-40
-20
20
40
60
80
100
120
I [nM]
37.5 nM
50 nM
100 nM
0.1
0.2
0.3
-0.1
1/S [µM-1]
Figure 6 Lineweaver–Burk plots of compound 55 (ST-2043) (12.5 to 100 nM) with seven suitable concentrations of kynuramine. Data represent mean values SD of one
representative experiment performed in duplicates.
for the design of new selective or multitargeting MAO
Conclusion
inhibitors.
This study presented small anilides as MAO A and
B inhibitors. Out of 66 screened compounds seven showed
promising inhibition properties at a concentration of
Acknowledgments
1 µm (>90% or >80% inhibition for MAO B and A, This work was kindly supported by COST action CM1103,
CA15135 and CA18133 as well as DFG INST 208/664e1
FUGG (Germany). Mohammad Khanfar and Holger Stark
have been supported by the George Forster Research
Fellowship granted by the Alexander von Humboldt-
Foundation.
respectively). Evaluation of their IC50 values revealed six
MAO B preferring (31, 33, 34, 55 (ST-2043), 65) inhibi-
tors and one MAO A preferring (7 (ST-2023)) inhibitor
with activities in low nanomolar concentration ranges. The
MAO B preferring inhibitors showed IC50 values ranging
from 56 nM to 128 nM with selectivity indices between
2.4 and >10. Computational analysis confirmed in vitro
binding properties by demonstrating good surface comple-
mentarity of the inhibitors with the binding-pockets of the
two MAO isoforms. The highest affinity for MAO A was
found for ST-2023 (7, IC50 = 126 nM) with 8.3-fold lower
affinity towards MAO B. ST-2043 (55) was identified as
most potent MAO B inhibitor (IC50 = 56 nM, Ki = 6.3 nM)
within this series, which demonstrated a similar inhibition
capacity as the reference reversible MAO B inhibitor safi-
namide, but with rather low MAO selectivity (SI = 5.2).
Further characterization suggested a competitive mode of
MAO B inhibition for 55, however, with a readily rever-
sible binding rather than tight binding behavior as antici-
pated for pharmacological efficacy. Nevertheless, these
small-sized ligands might be a promising starting point
Disclosure
The authors report no conflicts of interest in this work.
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