Novel 2-phenoxyanilide congeners derived from a hit structure of the TCAMS: Synthesis and evaluation of their in vitro activity against plasmodium falciparum

Article abstract of DOI:10.3390/molecules21020223

The Tres Cantos Antimalarial Compound Set (TCAMS) is a publicly available compound library which contains 13533 hit structures with confirmed activity against Plasmodium falciparum, the infective agent responsible for malaria tropica. The TCAMS provides a variety of starting points for the investigation of new antiplasmodial drug leads. One of the promising compounds is TCMDC-137332, which seemed to be a good starting point due to its antiplasmodial potency and its predicted physicochemical properties. Several new analogues based on a 2-phenoxyanilide scaffold were synthesized by standard amide coupling reactions and were fully characterized regarding their identity and purity by spectroscopic and chromatographic methods. Furthermore, the results of the biological evaluation of all congeners against Plasmodium falciparum NF54 strains are presented. The findings of our in vitro screening could not confirm the presumed nanomolar antiplasmodial activity of TCMDC-137332 and its derivatives.

Full text of DOI:10.3390/molecules21020223

molecules
Article
Novel 2-Phenoxyanilide Congeners Derived from a
Hit Structure of the TCAMS: Synthesis and
Evaluation of Their in Vitro Activity against
Plasmodium falciparum
Thomas Weidner 1,†, Abed Nasereddin 2,†, Lutz Preu 1, Johann Grünefeld 1, Ron Dzikowski 2
and Conrad Kunick 1,3,
*
1
Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig,
Beethovenstraße 55, 38106 Braunschweig, Germany; t.weidner@tu-braunschweig.de (T.W.);
l.preu@tu-braunschweig.de (L.P.); j.gruenefeld@tu-braunschweig.de (J.G.)
Department of Microbiology and Molecular Genetics, IMRIC, The Kuvin Center for the Study of Infectious
and Tropical Diseases, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel;
abedn@ekmd.huji.ac.il (A.N.); rond@ekmd.huji.ac.il (R.D.)
2
3
Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Franz-Liszt-Straße 35A,
38106 Braunschweig, Germany
*
†
Correspondence: c.kunick@tu-braunschweig.de; Tel.: +49-531-391-2754; Fax: +49-531-391-2799
These authors contributed equally to this work.
Academic Editor: Derek J. McPhee
Received: 18 December 2015 ; Accepted: 2 February 2016 ; Published: 17 February 2016
Abstract: The Tres Cantos Antimalarial Compound Set (TCAMS) is a publicly available compound
library which contains 13533 hit structures with confirmed activity against Plasmodium falciparum, the
infective agent responsible for malaria tropica. The TCAMS provides a variety of starting points for the
investigation of new antiplasmodial drug leads. One of the promising compounds is TCMDC-137332,
which seemed to be a good starting point due to its antiplasmodial potency and its predicted
physicochemical properties. Several new analogues based on a 2-phenoxyanilide scaffold were
synthesized by standard amide coupling reactions and were fully characterized regarding their
identity and purity by spectroscopic and chromatographic methods. Furthermore, the results of the
biological evaluation of all congeners against Plasmodium falciparum NF54 strains are presented. The
findings of our in vitro screening could not confirm the presumed nanomolar antiplasmodial activity
of TCMDC-137332 and its derivatives.
Keywords: diarylether; luciferase; Malaria; 2-phenoxyanilide; Plasmodium falciparum; TCAMS;
TCMDC-137332
1. Introduction
Malaria is still one of the most severe infectious diseases. Approximately 3.2 billion people are at
risk of being infected. Despite a decreasing number of mortal cases in the last decade due to better
vector control and artemisinine-based combination therapy (ACT), still 584,000 deaths caused by
malaria infection were reported in 2013 [
treatment of malaria, the discovery of new drugs is important due to the increasing resistances against
available drugs [ ]. A good strategy to circumvent the problem of resistance is to develop compounds
1]. Although there are effective chemotherapeutics for the
2,3
acting with new mechanisms of action. Hence there is an urgent need for compounds with novel
chemotypes which differ from scaffolds of existing drugs.
Molecules 2016, 21, 223; doi:10.3390/molecules21020223
www.mdpi.com/journal/molecules

Molecules 2016, 21, 223
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Major sources for drug discovery are phenotypic screening libraries, providing a huge number of
new starting points. In the last few years extensive libraries with compounds showing antimalarial
activity have been published by Novartis [
4], St. Jude’s Children Research Hospital [
5], the Medical
Research Council Technology [ ] and GlaxoSmithKline (GSK) [
6
7
]. The latter was derived from a high
throughput screening (HTS) of nearly 2 million substances of the GSK corporate collection. 13,533
of these, known as the Tres Cantos Antimalarial Compound Set (TCAMS), were active against the
malaria parasite Plasmodium falciparum and inhibited the parasite growth by at least 80% at 2
concentration. In silico clustering and filtering of the TCAMS set performed by Calderon et al. resulted
in 552 compounds which were declared as “quality starting points from the TCAMS” [ ]. Since
],
µM
8
then, a few of the compound classes have been further examined, e.g., cyclopropylcarboxamides [
2-amino-1-phenylethanols [10], aminohydantoines [11] or carbamoyltriazoles [12].
9
One of the 552 promising compounds is TCMDC-137332 (
7 nM and therefore seemed to be one of the most potent structures of this collection [7,8
1
), which has an estimated IC50 value of
]. We decided
to further investigate this compound which matches nearly all criteria of the Lipinski rules for orally
available drugs [13] (Figure 1) and is structurally dissimilar to currently known antimalarials [14].
Figure 1. Molecular structure of TCMDC-137332 and its properties concerning Lipinski’s rules for
orally available drugs; chemicalize.org was used for calculation of logP [15].
2. Results and Discussion
2.1. Chemistry
To proof its activity, we resynthesized TCMDC-137332. We also prepared a series of congeners,
all having a 2-phenoxyanilide scaffold (Figure 2). The first group of ten compounds was substituted
with chlorine in para position of the phenoxy residue, like in the scaffold of TCMDC-137332 (1–10).
Another equivalent series was synthesized, in which the chlorine was replaced by a more polar
methoxy group (11–20).
Figure 2. Structures of TCMDC-137332 (1) and its congeners (2–19).

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The synthesis of phenoxyanilides
1–
8
and 11
–18 was achieved by standard amide coupling
reactions of commercially available 2-(4-chlorophenoxy)aniline
hydrochloride II with acid chlorides in presence of triethylamine, as outlined in Scheme 1A.
Compounds and 19 were synthesized by the reaction of 2-(4-substituted)phenoxyanilines and
I or 2-(4-methoxyphenoxy)aniline
9
Boc-protected glycine in the presence of PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate) together with DIPEA (Diisopropylethylamine). Cleavage of the protecting group
was accomplished by treatment of
9 and 19, respectively, with trifluoroacetic acid and subsequent
precipitation of the hydrochloride salts 10 and 20 with a hydrochloric acid solution in propan-2-ol
(Scheme 1B). The compounds were synthesized in satisfactory to excellent yields (53%–93%) and the
purity (determined with elemental analyses and HPLC) of most of the products was sufficient for
biological evaluation even before the final purification step (recrystallization, flash chromatography).
Z
Z
O
O
(a)
A
NH2
HN
R
+
Cl
R
O
O
I (Z = Cl)
II (Hydrochloride, Z = OCH3)
1 - 8 (Z = Cl)
11 - 18 (Z = OCH3)
Z
Z
O
NHBoc
O
(b)
B
HN
O
NH2
+
NHBoc
O
HO
I (Z = Cl)
9 (Z = Cl)
II (Hydrochloride, Z = OCH3)
19 (Z = OCH3)
Z
Z
O
O
NHBoc
NH3+Cl-
(c)
HN
HN
O
O
9 (Z = Cl)
10 (Z = Cl)
19 (Z = OCH3)
20 (Z = OCH3)
Scheme 1.
(
A
Ñ
) Synthesis of 2-phenoxyanilides
1
–
8
and 11
–
18. Reaction conditions: (a) triethylamine,
10 19 and 20. Reaction
rt; (c) 1. Trifluoroacetic acid, dichloromethane,
toluene, 0 ˝C
rt. Residues R: see Figure 2; (
B
) Synthesis of 2-phenoxyanilides
9,
,
conditions: (b) PyBOB, DIPEA, dichloromethane, 0 ˝C
rt, 2. Hydrochloric acid (37%)/propan-2-ol (1:1).
Ñ
2.2. Calculations of Rule-of-Five-properties
We determined the properties concerning Lipinski’s Rule of Five (RO5) for orally active drugs for
all derivatives. The RO5 can be a useful reference to predict the quality of a structure to be an orally
available drug-like compound [13
Considering the molecular weight (<380 g/mol), the amount of H-bond acceptors (
donors ( 3), the values of every compound in this series comply with the proposed values of
,
16], which was outlined by Calderon et al. for TCMDC-137332 [
8].
ď
6) and H-bond
ď
Lipinski et al. [13]. Estimation of lipophilicity has been carried out using chemicalize.org for calculation
of logP [15]. According to the RO5 the logP should have a maximum value of 5. The calculated logP
compared to TCMDC-137332 was reduced by replacement of the tert-butyl residue by smaller residues,

Molecules 2016, 21, 223
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cyclic aliphatic residues like cyclopropyl- or cyclobutyl-groups, and more polar groups. Introducing a
methoxy group instead of the chlorine residue also reduced the calculated logP value. Some examples
of the calculated octanol/water partition coefficients are outlined in Table 1.
Table 1. Calculated logP values, the number of H-bond acceptors (Hacc) and donors (Hdon) and the
molecular weight (MW) of selected compounds.
ID
Z
R
logP
Hacc
Hdon
MW
1
11
4
14
5
15
7
17
10
20
Cl
OCH3
Cl
OCH3
Cl
OCH3
Cl
OCH3
Cl
5.11
4.35
4.02
3.25
4.10
3.33
3.29
2.52
2.39
1.63
3
4
3
4
3
4
5
6
3
4
1
1
1
1
1
1
1
1
3
3
303.79
299.37
275.73
271.32
287.74
283.33
333.77
329.35
276.72
272.30
OCH3
2.3. In Vitro Assay
After the full spectroscopic characterization of all congeners and validation of their purity by
elemental analyses and chromatographic methods in vitro assays were carried out using P. falciparum
NF54-luc erythrocyte stage parasites expressing luciferase. The derivatives
3 µM concentration performing viability assays measuring the bioluminescence in a luciferase assay
system as described earlier [17 20]. A first screening, in which the cultures were incubated for 48 h,
gave surprisingly low inhibition rates of the parasite growth (see Figure 3). With 22% reduction of the
parasite viability, was the most effective compound in this screening, followed by 13 and 15 with about
18% inhibition. The resynthesized TCMDC-137332 ( ) failed to show any antiplasmodial activity, just
1–20 were examined in
–
5
1
as compounds 10 and 20 which contain polar aminomethyl residues. Due to the unexpected outcome
of the first screening session, we performed another assay in which the cultures were incubated for a
longer period (96 h), to identify slow acting compounds. However, the prolongation of the incubation
time did not improve the inhibitory effect of the derivatives on the parasite growth significantly, the
only exception being compound 16, which diminished the proliferation of P. falciparum NF54 by 31%.
Compared to the first screening, 16 showed a 3-fold better inhibition rate, indicating that it is probably
a rather slow acting inhibitor. However, the results of most of the derivatives were slightly inferior
compared to the first screening. The assumed high antiplasmodial activity of TCMDC-137332 (
not be confirmed by these assays.
1) could
Figure 3. Results of the in vitro assay of derivatives 1–20 in 3 µM concentration against P. falciparum
NF54-Luc parasite line with 48 h (red) and 96 h (blue) incubation time. NOT: no treatment; Blasticidin
S: antibiotic used as antiplasmodial standard.

Molecules 2016, 21, 223
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3. Experimental Section
3.1. Apparatus and Materials
Starting materials were purchased from the suppliers indicated below and were used
without further purification. Pivaloyl chloride was from Merck-Schuchardt (Hohenbrunn,
Germany), 2,2-dimethylbutyryl chloride, 3-methylbutyryl chloride and 4-methoxybenzoyl chloride
were from Alfa-Aesar (Karlsruhe, Germany), propyl chloride, cyclopropanecarbonyl chloride,
cyclobutanecarbonyl chloride and methyl succinyl chloride were from Acros (Geel, Belgium),
Boc-glycine was from Aldrich (Steinheim, Germany), 2-(4-chlorophenoxy)aniline was from Enamine
(Monmouth Jct., NJ, USA), 2-(4-methoxyphenoxy)aniline hydrochloride was from Fluorochem
(Hadfield, UK). Toluene and dichloromethane were dried by published methods before usage [21].
Melting points were determined on an electric variable heater (Electrothermal IA9200, Bibby Scientific,
Stone, UK) in open glass capillaries and are uncorrected. IR spectra were recorded as KBr disks on
a Thermo Nicolet FT-IR 200 (Thermo Nicolet, Madison, WI, USA). 1H-NMR spectra and 13C-NMR
spectra were recorded on BrukerAvance DRX-400 and BrukerAvance II-600 instruments (Bruker
Corporation, Billerica, MA, USA) (at the NMR laboratories of the Chemical Institutes of the Technische
Universität Braunschweig). Chemical shifts were recorded as
δ values in ppm and are referenced
to an internal standard tetramethylsilane. Signals in 13C spectra were assigned based on the result
of 13C DEPT135 experiments. Elemental analyses were determined on a CE Instruments FlashEA
1112 elemental analyzer (Thermo Quest, San Jose, CA, USA). Mass spectra were recorded on
a Finnigan-MAT 95 (Thermo Finnigan MAT, Bremen, Germany). Accurate measurements were
conducted according to the peak match method using perfluorokerosene (PFK) as an internal mass
reference. (EI) MS: ionization energy 70 eV (Department of Mass Spectrometry of the Chemical
Institutes of the Technische Universität Braunschweig). TLC: Polygram Sil G/UV254 (Macherey-Nagel,
Düren, Germany), 40 mm ˆ 80 mm, visualization by UV illumination (254 and 366 nm). Purity was
determined by HPLC using isocratic and gradient elution performed on Merck Hitachi Elite LaChrom
systems (Hitachi High Technologies Inc., San Jose, CA, USA): pump L-2130, autosampler L-2200, diode
array detector L-2450 (isocratic elution) or UV detector L-2400 (gradient elution), organizer box L-2000;
column, Merck LiChroCART 125-4, LiChrosphere 100, RP 18, 5 µm (Merck, Darmstadt, Germany); flow
rate 1.000 mL/min; detection wavelength: 254 and 280 nm (isocratic elution) and 254 nm (gradient
elution); AUC, % method; time of detection 15 min (isocratic elution) or 20 min (gradient elution),
retention time (tR); dead time (tM) related to DMSO. For isocratic runs, mixtures of ACN and water or
mixtures of ACN and buffer were used. For all gradient runs, mixtures of ACN and water were used
(gradient: 0–2 min: 10% ACN, 2–12 min: 10%
H2O + (Et3NH)2SO4 buffer (pH 2.7) for isocratic HPLC: triethylamine (20.0 mL) and sodium hydroxide
(242 mg) were dissolved in water to 1 L. The solution was adjusted to pH 2.7 by addition of sulfuric
Ñ
90% ACN, linear, 12–20 min 90% ACN). Preparation of
acid. All compounds which were biologically tested were of >95% purity. Absorption maxima (λ
)
max
were extracted from the spectra recorded by the DAD in the HPLC peak maxima in isocratic runs
(software, EZ Chrom Elite Client/server, version 3.1.3., Scientific Software Inc., Pleasanton, CA, USA).
3.2. Chemical Synthesis and Characterization of Compounds 1–20
General Procedure for the Synthesis of Compounds 1–8
To a stirred and cooled solution of 2-(4-chlorophenoxy)aniline (330 mg, 1.50 mmol) and
triethylamine (230 µL, 1.65 mmol) in toluene (5 mL), the appropriate acid chloride (pivaloyl chloride,
2,2-dimethylbutyryl chloride, 3-methylbutyryl chloride, propyl chloride, cyclopropanecarbonyl
chloride, cyclobutanecarbonyl chloride, methyl succinyl chloride, 4-methoxybenzoyl chloride)
(1.65 mmol) was added. Subsequently the reaction mixture was allowed to warm to room temperature.
The progress of the reaction was monitored by TLC. After 2–9 h the reaction mixture was extracted
with a saturated sodium hydrogen carbonate solution, with a hydrogen chloride solution (10%), with

Molecules 2016, 21, 223
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brine and finally with water. Afterwards the organic solution was dried over sodium sulfate and
evaporated under reduced pressure. The residue was further purified by recrystallization or column
chromatography over silica gel.
N-[2-(4-Chlorophenoxy)phenyl]-2,2-dimethylpropanamide (1): Crystallization from ethanol (70%) yielded
slightly brown needles (277 mg, 0.92 mmol, 61%); m.p.: 99–100 ˝C ; IR (KBr):
ν
[cm´1] = 3336 (br,
N-H), 1663 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6)
2H, arom. H), 6.99–7.15 (m, 1H, arom. H), 7.15–7.32 (m, 2H, arom. H), 7.27–7.48 (m, 2H, arom. H),
7.56–7.78 (m, 1H, arom. H), 8.72 (s, 1H, NH); 13C-NMR: (101 MHz, DMSO-d6)
[ppm] = 26.95 (3C,
δ [ppm] = 1.05 (s, 9H, C(CH3)3), 6.80–7.01 (m,
δ
CH3), 118.40 (2C), 120.46, 124.63, 126.13, 126.62, 129.49 (2C) (CH), 38.12, 126.38, 130.28, 147.84, 155.91
(C), 176.13 (C=O); C17H18ClNO2 (303.78): calcd. C 67.21, H 5.97, N 4.61, found C 67.13, H 6.00, N 4.51;
EI-MS: m/z (%): 303.1 [M]+ (28), 176.1 [M
tR = 7.42 min, tM(DMSO) = 1.06 min (ACN/H2O 60:40),
tR = 13.62 min, tM(DMSO) = 1.28 min.
´
127]+ (100); HPLC: 99.7% at 254 nm, 99.9% at 280 nm;
λ
max
[nm] = 230, 275; HPLC-gradient: 99.4%,
N-[2-(4-Chlorphenoxy)phenyl]-2,2-dimethylbutanamide (2): Crystallization from methanol/water (33:10)
˝
yielded a beige solid (332 mg, 1.05 mmol, 70%); m.p.: 69–70 C ; IR (KBr):
N-H), 1657 (s, C=O); H-NMR: (400 MHz, DMSO-d6)
ν
[cm´1] = 3316 (br,
1
δ [ppm] = 0.67 (t, J = 7.4 Hz, 3H, CH3), 1.01
(s, 6H, C(CH3)2), 1.47 (q, J = 7.4 Hz, 2H, CH2), 6.87–6.96 (m, 2H, arom. H), 7.03–7.13 (m, 1H, arom.
H), 7.17–7.27 (m, 2H, arom. H), 7.34–7.43 (m, 2H, arom. H), 7.59–7.68 (m, 1H, arom. H), 8.70
(s, 1H, NH); 13C-NMR: (101 MHz, DMSO-d6)
δ [ppm] = 8.82, 24.54 (2C) (CH3), 32.82 (CH2) 118.49
(2C), 120.46, 124.63, 126.16, 126.74, 129.55 (2C) (CH), 42.42, 126.43, 130.29, 147.98, 155.99 (C), 175.48
(C=O); C18H20ClNO2 (317.81): calcd. C 68.03, H 6.34, N 4.41, found C 68.09, H 6.22, N 4.28; EI-MS:
m/z (%): 317.1 [M]+ (28), 190.1 [M
tR = 9.82 min, tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
97.3%, tR = 14.17 min, tM(DMSO) = 1.28 min.
´
127]+ (100); HPLC: 98.7% at 254 nm, 99.0% at 280 nm;
λmax[nm] = 232, 275; HPLC-Gradient:
N-[2-(4-Chlorophenoxy)phenyl]-3-methylbutanamide (
(dichloromethane/methanol 200:1) yielded a slightly yellow solid (418 mg, 1.38 mmol, 92%);
m.p.: 69–71 ˝C; IR (KBr): [cm´1] = 3291 (m, N-H), 1655 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6):
[ppm] = 0.81 (d, J = 6.7 Hz, 6H, CH3), 1.92 (hept, J = 6.8 Hz, 1H, CH(CH3)2), 2.14 (d, J = 7.2 Hz, 2H,
CH2), 6.79–7.08 (m, 3H, arom. H), 7.08–7.25 (m, 2H, arom. H), 7.28–7.51 (m, 2H, arom. H), 7.88 (dd,
J = 7.6 Hz, 2.2 Hz, 1H, arom. H), 9.38 (s, 1H, NH); 13C-NMR (101 MHz, DMSO-d6)
[ppm] = 22.10 (2C,
3): Purification by column chromatography
ν
δ
δ
CH3), 44.93 (CH2), 25.60, 119.28 (2C), 119.83, 124.34, 124.87, 125.25, 129.56 (2C) (CH), 126.67, 130.18,
147.04, 155.98 (C), 170.89 (C=O); C17H18ClNO2 (303.78): calcd. C 67.21, H 5.97, N 4.61, found C 66.95,
H 6.02, N 4.39; EI-MS: m/z (%): 303.1 [M]+ (16), 219.0 [M
at 280 nm; tR = 6.03 min, tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
95.6%, tR = 13.23 min, tM(DMSO) = 1.28 min.
´
84]+ (100); HPLC: 98.1% at 254 nm, 99.0%
λ
max
[nm] = 249; HPLC-gradient:
N-[2-(4-Chlorophenoxy)phenyl]propanamide (
ether/ethyl acetate 4:1) yielded a colorless solid (265 mg, 0.96 mmol, 63%); m.p.: 99–100 ˝C; IR (KBr):
[cm´1] = 3330 (br, N-H), 1675 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6)
[ppm] = 0.98 (t, J = 7.6 Hz,
3H, CH3), 2.29 (q, J = 7.6 Hz, 2H, CH2), 6.93–7.02 (m, 3H, arom. H), 7.08–7.22 (m, 2H, arom. H), 7.37–7.46
(m, 2H, arom. H), 7.91–7.98 (m, 1H, arom. H), 9.39 (s, 1H, NH); 13C-NMR: (101 MHz, DMSO-d6):
4): Purification by column chromatography (petroleum
ν
δ
δ
[ppm] = 9.65 (CH3), 29.03 (CH2), 119.53 (2C), 124.24, 124.43, 125.02, 129.59 (2C) (CH), 126.78, 130.19,
146.93, 155.85 (C), 172.31 (C=O), one signal missing in 13C-NMR; C15H14ClNO2 (275.73): calcd. C 65.34,
H 5.12, N 5.08, found C 65.33, N 4.98, H 5.02; EI-MS: m/z (%): 275.1 [M]+ (18), 219.0 [M ´ 56]+ (100);
HPLC: 98.9% at 254 nm, 99.4% at 280 nm; tR = 3.95 min, tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
λmax [nm] = 248, 274; HPLC-gradient: 96.0%, tR = 12.23 min, tM(DMSO) = 1.28 min.

Molecules 2016, 21, 223
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N-[2-(4-Chlorophenoxy)phenyl]cyclopropanecarboxyamide (
5
): Crystallization from ethanol (70%) yielded
beige needles (387 mg, 1.34 mmol, 90%); m.p.: 100–102 ˝C; IR (KBr):
ν
[cm´1] = 3371, 3332 (m, N-H),
1663 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6):
δ [ppm] = 0.67–0.77 (m, 4H, CH2), 1.95 (td, J = 7.5 Hz,
3.9 Hz, 1H, CH), 6.94 (dd, J = 7.5 Hz, 2.1 Hz, 1H, arom. H), 6.96–7.03 (m, 2H, arom. H), 7.06–7.26
(m, 2H, arom. H), 7.37–7.41 (m, 2H, arom. H), 7.96 (dd, J = 7.7 Hz, J = 2.1 Hz, 1H, arom. H), 9.71
(s, 1H, NH); 13C NMR (101 MHz, DMSO-d6)
δ [ppm] = 7.16 (2C, CH2), 13.86, 119.14, 119.70 (2C),
124.01, 124.73, 129.55 (2C) (CH), 126.82, 130.09, 146.66, 155.71 (C), 171.99 (C=O); C16H14ClNO2 (287.74):
calcd. C 66.79, H 4.90, N 4.87, found C 66.96, H 4.92, N 4.77; EI-MS: m/z (%): 287.1 [M]+ (21), 219.0
[M ´ 68]+ (100); HPLC: 98.4% at 254 nm, 98.3% at 280 nm; tR = 4.40 min, tM(DMSO) = 1.06 min
(ACN/H2O = 60:40), λmax [nm] = 241; HPLC-gradient: 97.5%, tR = 12.57 min, tM(DMSO) = 1.28 min.
N-[2-(4-Chlorophenoxy)phenyl]cyclobutanecarboxyamide (
(toluene/petroleum ether 10:1 20:1) yielded a slightly brown solid (246, 0.82 mmol, 53%):
m.p.: 88–89 ˝C; IR (KBr): [cm´1] = 3433 (br, N-H), 1657 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6)
[ppm] = 1.66–2.14 (m, 6H, CH2), 3.19–3.33 (m, 1H, CH), 6.90–7.03 (m, 3H, arom. H), 7.12–7.20 (m,
2H, arom. H), 7.36–7.45 (m, 2H, arom. H), 7.91 (dd, J = 7.4 Hz, 2.1 Hz, 1H, arom. H), 9.22 (s, 1H,
NH); 13C-NMR: (101 MHz, DMSO-d6)
[ppm] = 17.65, 24.47 (2C) (CH2), 38.88, 119.28 (2C), 119.78,
6): Purification by column chromatography
Ñ
ν
δ
δ
124.36, 124.74, 125.18, 129.55 (2C) (CH), 126.68, 130.18, 147.00, 155.87 (C), 173.09 (C=O); C17H16ClNO2
(301.77): calcd. C 67.66, H 5.34, N 4.64, found C 67.81, H 5.43, N 4.55; EI-MS: m/z (%): 301.1 [M]+ (20),
219.0 [M
´
82]+ (100); HPLC: 99.4% at 254 nm, 99.8% at 280 nm; tR = 5.86 min; tM(DMSO) = 1.06 min
(ACN/H2O = 60:40), λmax [nm] = 229; HPLC-gradient: 98.4%, tR = 13.15 min; tM(DMSO) = 1.28 min.
Methyl 4-{[2-(4-chlorophenoxy)phenyl]amino}-4-oxobutanoate ( ): Crystallization from methanol yielded
a beige solid (278 mg, 0.83 mmol, 56%); m.p.: 109–110 ˝C; IR (KBr): [cm´1] = 3341 (m, N-H),1721
(s, C=O, ester) 1687 (s, C=O, amide); 1H-NMR: (400 MHz, CDCl3):
[ppm] = 2.35–2.97 (m, 4H, CH2),
7
ν
δ
3.67 (s, 3H, OCH3), 6.83 (dd, J = 8.1 Hz, 1.4 Hz, 1H, arom. H), 6.93–7.05 (m, 3H, arom. H), 7.12 (td,
J = 7.8 Hz, 1.5 Hz, 1H, arom. H), 7.28–7.38 (m, 2H, arom. H), 7.91 (s, 1H, NH), 8.41 (d, J = 7.8 Hz, 1H,
arom. H); 13C-NMR (101 MHz, CDCl3)
δ [ppm] = 51.93 (CH3) 29.15, 32.27 (CH2), 117.77, 119.84 (2C),
121.14, 124.10, 124.44, 129.95 (2C) (CH), 128.98, 129.78, 145.22, 155.13 (C), 169.69 (C=O), 173.23 (C=O);
C17H16ClNO4 (333.77): calcd. C 61.18, H 4.83, N 4.20, found: C 61.15, H 4.83, N 4.21; EI-MS: m/z
(%): 333.1 [M]+ (12), 174.1 [M
tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
tM(DMSO) = 1.28 min.
´
159]+ (100); HPLC: 99.3% at 254 nm, 99.7% at 280 nm; tR = 3.44 min,
λmax [nm] = 228; HPLC-gradient: 98.5%, tR = 11.99 min,
N-(2-(4-Chlorophenoxy)phenyl)-4-methoxybenzamide (
needles (346mg,0.98 mol, 65%); m.p.: 101–102 ˝C; IR (KBr):
1H-NMR: (400 MHz, DMSO-d6):
[ppm] = 3.81 (s, 3H, OCH3), 6.89–7.13 (m, 5H, arom. H), 7.17–7.30
(m, 2H, arom. H), 7.30–7.48 (m, 2H, arom. H), 7.65–7.76 (m, 1H, arom. H), 7.75–7.99 (m, 2H, arom.
H), 9.69 (s, 1H, NH); 13C-NMR (101 MHz, DMSO-d6)
[ppm] = 55.29 (CH3), 113.44 (2C), 119.36 (2C),
8
): Crystallization from methanol yielded colorless
ν
[cm´1] = 3420 (m, N-H), 1660 (s, C=O);
δ
δ
119.69, 124.15, 126.32, 126.83, 129.38 (2C), 129.48 (2C) (CH), 126.25, 126.64, 129.79, 149.02, 155.71, 161.80
(C), 164.69 (C=O); C20H16ClNO3 (353.80): calcd. C 67.90, H 4.56, N 3.96, found: C 67.78, H 4.43, N
3.95; EI-MS: m/z (%): 353.1 [M]+ (8), 135.0 [M
tR = 6.37 min, tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
tR = 13.37 min, tM(DMSO) = 1.28 min.
´
218]+ (100); HPLC: 99.7% at 254 nm, 99.8% at 280 nm;
λ
max
[nm] = 266; HPLC-gradient: 99.0%,
General Procedure for the Synthesis of Compounds 11–18
To a stirred and cooled solution of 2-(4-methoxyphenoxy)aniline hydrochloride (1.50 mmol) and
triethylamine (4.00 mmol) in toluene (5–8 mL), the appropriate acid chloride (pivaloyl chloride,
2,2-dimethylbutyryl chloride, 3-methylbutyryl chloride, propyl chloride, cyclopropanecarbonyl
chloride, cyclobutanecarbonyl chloride, methyl succinyl chloride, 4-methoxybenzoyl chloride)
(1.65 mmol) was added. Thereafter the reaction mixture was allowed to warm to room temperature.
The progress of the reaction was monitored by TLC. After 4–24 h an aqueous work-up was performed

Molecules 2016, 21, 223
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similar to that of compounds
1–
8. The resulting residue was further purified by recrystallization or
column chromatography over silica gel.
N-[2-(4-Methoxyphenoxy)phenyl]-2,2-dimethylpropanamide (11):˝ Crystallization from ethanol (70%)
yielded a colorless solid (401 mg, 1.34 mmol, 89%); m.p.: 70–71 C; IR (KBr):
ν
[cm´1] = 3317 (m, N-H),
1661 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6):
δ
[ppm] = 1.11 (s, 9H, C(CH3)3), 3.72 (s, 3H, OCH3),
6.85–6.90 (m, 1H, arom. H), 6.92 (s, 4H, arom. H), 7.06–7.16 (m, 2H, arom. H), 7.69–7.78 (m, 1H, arom.
H), 8.62 (s, 1H, NH); 13C-NMR (101 MHz, DMSO-d6)
[ppm] = 27.06 (3C), 55.41 (CH3), 114.86 (2C),
δ
118.35, 119.09 (2C), 123.25, 125.06, 125.40 (CH), 38.90, 129.48, 148.96, 149.97, 155.27 (C), 176.10 (C=O);
C18H21NO3 (299.37): calcd. C 72.22, H 7.07, N 4.68, found C 72.21, H 7.25, N 4.73; EI-MS: m/z (%):
299.1 [M]+ (48), 176.1 [M
tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
tM(DMSO) = 1.28 min.
´
123]+(100); HPLC: 99.4% at 254 nm, 99.4% at 280 nm; tR = 5.21 min,
λmax [nm] = 248; HPLC-Gradient: 99.3%, tR = 12.88 min,
N-[2-(4-Methoxyphenoxy)phenyl]-2,2-dimethylbutanamide (12): Crystallization from ethanol (70%) yielded
˝
a slightly yellow solid (415 mg, 1.32 mmol, 88%): m.p.: 71–72 C; IR (KBr):
ν
[cm´1] = 3433 (m, N-H),
1674 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6):
δ
[ppm] = 0.72 (t, J = 7.4 Hz, 3H, CH3), 1.07 (s, 6H,
C(CH3)2), 1.51 (q, J = 7.4 Hz, 2H, CH2), 3.72 (s, 3H, OCH3), 6.83–6.92 (m, 1H, arom. H), 6.92 (s, 4H,
arom. H), 7.05–7.18 (m, 2H, arom. H), 7.66–7.74 (m, 1H, arom. H), 8.60 (s, 1H, NH); 13C-NMR (101
MHz, DMSO-d6) δ [ppm] = 8.90, 32.96 (2C), 55.42 (CH3), 24.61 (CH2), 114.90 (2C), 118.31, 119.17 (2C),
123.21, 125.34, 125.50 (CH), 42.56, 129.43, 149.21, 150.00, 155.28 (C), 175.42 (C=O); C19H23NO3 (313.40):
calcd. C 72.84, H 7.40, N 4.47, found C 72.72, H 7.45, N 4.52; EI-MS: m/z (%): 313.1 [M]+ (51), 190.1
[M ´ 123]+ (100); HPLC: 98.0% at 254 nm, 98.3% at 280 nm; tR = 6.69 min, tM(DMSO) = 1.06 min
(ACN/H2O = 60:40), λmax [nm] = 228; HPLC-gradient: 98.1%, tR = 13.43 min, tM(DMSO) = 1.28 min.
N-[2-(4-Methoxyphenoxy)phenyl]-3-methylbutanamide (13): Purification by column chromatography
(petroleum ether/ethyl acetate 4:1) yielded a colorless oil (303 mg, 1.01 mmol, 67%); IR (NaCl):
ν
[cm´1] = 3426 (m, N-H), 3317 (m, br, N-H), 1679 (s, C=O); 1H-NMR: (600 MHz, DMSO-d6):
δ [ppm]
= 0.87 (d, J = 6.7 Hz, 6H, CH2), 1.99 (hept, J = 6.8 Hz, 1H, CH), 2.21 (d, J = 7.2 Hz, 2H, CH2), 3.73 (s, 3H,
OCH3), 6.72–6.83 (m, 1H, arom. H), 6.89–7.01 (m, 4H, arom. H), 7.01–7.10 (m, 2H, arom. H), 7.77–8.09
(m, 1H, arom. H), 9.35 (s, 1H, NH); 13C-NMR (151 MHz, DMSO-d6)
δ [ppm] = 22.12 (2C), 55.32 (CH3),
44.94 (CH2) 25.60, 114.81 (2C), 117.42, 120.01 (2C), 122.71, 123.95, 124.65 (CH), 129.16, 148.63, 149.70,
155.35 (C), 170.87 (C=O); C18H21NO3 (299.37): calcd. C 72.22, H 7.07, N 4.68, found C 71.88, H 7.33, N
4.52; EI-MS: m/z (%): 299.1 [M]+ (48), 176.1 [M
tR = 4.37 min, tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
tR = 12.47 min, tM(DMSO) = 1.28 min.
´
123]+ (100); HPLC: 98.5% at 254 nm, 98.4% at 280 nm;
λ
max
[nm] = 248; HPLC-gradient: 98.1%,
N-[2-(4-Methoxyphenoxy)phenyl]propanamide (14): Crystallization from ethanol (70%) yielded colorless
crystals (379 mg, 1.40 mmol, 93%); m.p.: 107–109 ˝C; IR (KBr): [cm´1] = 3299 (m, br, N-H), 1651 (s,
C=O); 1H-NMR: (400 MHz, DMSO-d6):
[ppm] = 1.02 (t, J = 7.6 Hz, 3H, CH3), 2.35 (q, J = 7.5 Hz, 2H,
CH2), 3.74 (s, 3H, OCH3), 6.70–6.79 (m, 1H, arom. H), 6.90–7.09 (m, 6H, arom. H), 7.87–8.10 (m, 1H,
arom. H), 9.33 (s, 1H, NH); 13C-NMR (101 MHz, DMSO-d6)
[ppm] = 9.68, 55.39 (CH3), 29.09 (CH2),
ν
δ
δ
114.89 (2C), 117.23, 120.24 (2C), 122.70, 123.53, 124.45 (CH), 129.26, 148.49, 149.64, 155.48 (C), 172.28
(C=O); C16H17NO3 (271.32): calcd. C 70.83, H 6.32, N 5.16, found C 70.70, H 6.37, N 5.12; EI-MS: m/z
(%): 271.1 [M]+ (54), 215.1 [M
tM(DMSO) = 1.06 min (ACN/H2O = 50:50),
tM(DMSO) = 1.28 min.
´
59]+ (100); HPLC: 98.6% at 254 nm, 97.9% at 280 nm; tR = 4.89 min,
λmax [nm] = 239; HPLC-gradient: 97.5%, tR = 11.41 min,
N-[2-(4-Methoxyphenoxy)phenyl]cyclopropanecarboxyamide (15): Crystallization from ethanol (70%)
yielded a colorless solid (360 mg, 1.27 mmol, 85%); m.p.: 138–139 ˝C; IR (KBr): [cm´1] = 3312 (m,
br, N-H), 1660 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6):
[ppm] = 0.65–0.87 (m, 4H, CH2), 1.95–2.14
(m, 1H, CH), 3.75 (s, 3H, OCH3), 6.64–6.81 (m, 1H, arom. H), 6.88–7.06 (m, 6H, arom. H), 7.88–8.05
(m, 1H, arom. H), 9.69 (s, 1H, NH); 13C-NMR (101 MHz, DMSO-d6)
[ppm] = 55.39 (CH3), 7.25 (2C,
ν
δ
δ

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CH2), 14.02, 114.93 (2C), 116.93, 120.47 (2C), 122.57, 123.38, 124.32 (CH), 129.21, 148.38, 149.54, 155.56
(C), 172.04 (C=O); C17H17NO3 (283.33): calcd. C 72.07, H 6.05, N 4.94, found C 71.86, H 6.20, N 4.93;
EI-MS: m/z (%): 283.1 [M]+ (43), 215.1 [M
tR = 5.79 min, tM(DMSO) = 1.06 min (ACN/H2O = 50:50),
tR = 11.73 min, tM(DMSO) = 1.28 min.
´
68]+ (100); HPLC: 99.2% at 254 nm, 99.4% at 280 nm;
λ
max
[nm] = 247; HPLC-gradient: 98.6%,
N-[2-(4-Methoxyphenoxy)phenyl]cyclobutanecarboxyamide (16): Crystallization from ethanol (70%) yielded
a colorless solid (378 mg, 1.27 mmol, 85%); m.p.: 72–74 ˝C; IR (KBr): [cm´1] = 3294 (m, br, N-H),
1665 (s, C=O); 1H-NMR: (400 MHz, CDCl3):
[ppm] = 1.82–2.07 (m, 2H, CH2), 2.13–2.27 (m, 2H, CH2),
ν
δ
2.28–2.45 (m, 2H, CH2), 3.08–3.29 (m, 1H, CH), 3.81 (s, 3H, OCH3), 6.74 (dd, J = 8.1 Hz, 1.4 Hz, 1H,
arom. H), 6.84–7.01 (m, 5H, arom. H), 7.06 (td, J = 7.8 Hz, 1.4 Hz, 1H, arom. H), 7.71 (s, 1H, NH), 8.47
(dd, J = 8.1 Hz, 1.6 Hz, 1H, arom. H); 13C-NMR (101 MHz, CDCl3) δ [ppm] = 55.67 (CH3) 18.02, 25.32
(2C) (CH2), 41.10, 114.99 (2C), 116.31, 120.29 (2C), 120.51, 123.35, 123.54 (CH) 129.29, 146.55, 149.48,
156.21 (C), 173.22 (C=O); C17H17NO3 (283.33): calcd. C 72.07, H 6.05, N 4.94, found C 71.86, H 6.20, N
4.93; EI-MS: m/z (%): 297.1 [M]+ (38) 215.1 [M ´ 82]+ (100); HPLC: 99.3% at 254 nm, 99.2% at 280 nm;
tR = 4.15 min, tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
tR = 12.93 min, tM(DMSO) = 1.28 min.
λ
max
[nm] = 248; HPLC-Gradient: 98.5%,
Methyl 4-{[2-(4-methoxyphenoxy)phenyl]amino}-4-oxobutanoate (17): Crystallization from methanol
yielded slightly yellow crystals (347 mg, 1.05 mmol, 70%); m.p.: 82–83 ˝C; IR (KBr): ν [cm´1] = 3335
(m, br, N-H), 1721 (s, C=O, ester), 1682 (s, C=O, amide); 1H-NMR: (400 MHz, DMSO-d6):
δ [ppm] =
2.55 (t, J = 6.8 Hz, 2H, CH2), 2.66 (t, J = 6.8 Hz, 2H, CH2), 3.58 (s, 3H, COOCH3), 3.75 (s, 3H, OCH3),
6.67–6.80 (m, 1H, arom. H), 6.86–7.12 (m, 6H, arom. H), 7.84–8.05 (m, 1H, arom. H), 9.52 (s, 1H,
NH); 13C-NMR (101 MHz, DMSO-d6)
δ [ppm] = 51.27, 55.39 (CH3), 28.61, 30.62 (CH2), 114.90 (2C),
117.00, 120.43 (2C), 122.60, 123.19, 124.41 (CH), 129.06, 148.39, 149.54, 155.54 (C), 170.19, 172.77 (C=O);
C18H19NO5 (329.35): calcd. C 65.64, H 5.82, N 4.25, found C 65.52, H 5.91, N 4.12; EI-MS: m/z (%): 329.1
[M]+ (32), 297.1 [M
1.06 min (ACN/H2O = 50:50),
1.28 min.
´
32]+ (100); HPLC: 98.0% at 254 nm, 98.2% at 280 nm; tR = 4.39 min, tM(DMSO) =
λmax [nm] = 245; HPLC-gradient: 97.4%, tR = 11.20 min, tM(DMSO) =
N-(2-(4-Methoxyphenoxy)phenyl)-4-methoxybenzamide (18): Crystallization from methanol yielded
˝
colorless needles (344 mg, 0.98 mmol, 66%); m.p.: 88–89 C; IR (KBr):
ν
[cm´1] = 3442 (m, N-H),
1674 (s, C=O); 1H-NMR: (400 MHz, DMSO-d6):
δ
[ppm] = 3.72 (s, 3H, OCH3), 3.82 (s, 3H, OCH33),
6.80–6.88 (m, 1H, arom. H), 6.90–6.96 (m, 2H, arom. H), 6.96–7.04 (m, 4H, arom. H), 7.06–7.20 (m, 2H,
arom. H), 7.76 (dd, J = 7.5 Hz, 2.1 Hz, 1H, arom. H), 7.84–7.93 (m, 2H, arom. H), 9.61 (s, 1H, NH);
13C-NMR (101 MHz, DMSO-d6)
δ [ppm] = 55.37 (2C, CH3), 113.57 (2C), 114.88 (2C), 117.63, 120.08
(2C), 122.78, 125.89, 126.05, 129.45 (2C) (CH), 126.48, 128.93, 149.69, 150.56, 155.44, 161.87 (C), 164.68
(C=O); C21H19NO4 (349.39): calcd. C 72.19, H 5.48, N 4.01, found C 72.01, H 5.46, N 3.90; EI-MS: m/z
(%): 349.1 [M]+ (20), 135.0 [M
tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
tM(DMSO) = 1.28 min.
´
214]+ (100); HPLC: 99.4% at 254 nm, 99.4% at 280 nm; tR = 4.71 min,
λmax [nm] = 268; HPLC-gradient: 98.8%, tR = 12.78 min,
tert-Butyl 2-{[2-(4-chlorophenoxy)phenyl]amino}-2-oxoethylcarbamate (9)
A stirred solution of Boc-glycine (1.50 mmol), PyBOP (1.60 mmol) and DIPEA (3.50 mmol)
in dichloromethane (10 mL) was cooled to 0 ˝C with an ice bath. To this solution
2-(4-chlorophenoxy)aniline (1.50 mmol), dissolved in a minimum amount of dichloromethane,
was added dropwise. The reaction mixture was allowed to warm to room temperature and
was then stirred for 24 h. Thereafter, an aqueous work-up was performed similar to that of
compounds
(dichloromethane/methanol 200:1) to give a slightly orange solid (358 mg, 0.95 mmol, 63%); m.p.:
50–51 ˝C; IR (KBr): [cm´1] = 3409 (m, N-H), 3342 (s, N-H), 1691 (br, s, C=O); 1H-NMR: (600 MHz,
DMSO-d6): [ppm] = 1.34 (s, 9H, CH3), 3.71 (d, J = 6.1 Hz, 2H, CH2), 6.87–7.06 (m, 3H, arom. H),
1–8. The resulting oil was further purified by column chromatography over silica gel
ν
δ

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7.05–7.30 (m, 3H, arom. H), 7.35–7.53 (m, 2H, arom. H), 8.12 (d, J = 7.7 Hz, 1H, NH), 9.33 (s, 1H,
NH); 13C-NMR (151 MHz, DMSO-d6)
[ppm] = 28.00 (3C, CH3), 43.92 (CH2), 118.82, 119.90 (2C),
122.35, 124.15, 124.63, 129.70 (2C) (CH), 78.17, 127.17, 129.64, 146.09, 155.47, 155.86, 168.51 (C); EI-MS:
m/z (%): 376.1 [M]+ (11), 219.1 [M 157]+ (100); HREI-MS: calcd. for C19H21ClN2O4 376.11844,
δ
´
found 376.11803; HPLC: 96.3% at 254 nm, 96.3% at 280 nm; tR = 4.84 min, tM(DMSO) = 1.06 min
(ACN/H2O = 60:40), λmax [nm] = 230; HPLC-gradient: 95.8%, tR = 12.80 min, tM(DMSO) = 1.28 min.
tert-Butyl 2-{[2-(4-methoxyphenoxy)phenyl]amino}-2-oxoethylcarbamate (19)
A stirred solution of Boc-glycine (2.00 mmol), PyBOP (2.10 mmol) and DIPEA (5.00 mmol)
in dichloromethane (10 mL) was cooled to 0 ˝C with an ice bath. To this solution,
2-(4-methoxyphenoxy)aniline hydrochloride (2.00 mmol), dissolved in dichloromethane (5 mL)
together with DIPEA (2.00 mmol), was added dropwise. The reaction mixture was allowed to warm to
room temperature and was then stirred for 20 h. Subsequently, an aqueous work-up was performed
similar to that of compounds 1–8. The resulting oil was taken up in cold diethyl ether (10 mL) leading
to precipitation of a beige solid, which was filtered off and discarded. The ether was evaporated under
reduced pressure and the resulting residue was recrystallized from methanol/water (5:1) to give a
slightly beige solid(495 mg, 1.33 mmol, 66%); m.p.: 92–93 ˝C; IR (KBr):
ν
[cm´1] = 3393 (w, N-H),
3313 (s, br, N-H), 1696 (s, C=O, carbamate), 1666 (s, C=O, amide); 1H-NMR: (600 MHz, DMSO-d6):
δ
[ppm] = 1.35 (s, 9H, C(CH3)3), 3.73–3.75 (m, 5H, CH2, OCH3), 6.73 (dd, J = 8.0 Hz, 1.7 Hz, 1H, arom.
H), 6.95–7.07 (m, 6H, arom. H), 7.24 (t, J = 5.2 Hz, 1H, arom. H), 8.13 (d, J = 7.0 Hz, 1H, NH), 9.30
(s, 1H, NH); 13C-NMR (151 MHz, DMSO-d6)
δ [ppm] = 28.02 (3C), 55.33 (CH3), 44.06 (CH2), 114.92
(2C), 116.55, 120.52 (2C), 121.58, 122.67, 124.19 (CH), 78.22 128.63, 147.69, 149.16, 155.65, 155.90, 168.43
(C); C20H24N2O5 (372.42): calcd. C 64.50, H 6.50, N 7.52, found C 64.10, H 6.68, N 7.61; EI-MS: m/z
(%): 372.2 [M]+ (18), 215.1 [M
tM(DMSO) = 1.06 min (ACN/H2O = 60:40),
tM(DMSO) = 1.28 min.
´
157]+ (100); HPLC: 96.5% at 254 nm, 95.5% at 280 nm; tR = 3.41 min,
λmax [nm] = 241; HPLC-gradient: 95.1%, tR = 11.99 min,
General Procedure for the Synthesis of Compounds 10 and 20
The reaction was performed under nitrogen atmosphere. The Boc-protected amine
9 (490 mg,
1.31 mmol) or 19 (250 mg, 0.67 mmol) was dissolved in dichloromethane (10 mL). The solution was
cooled with an ice bath. To the well stirred solution 10 equivalents of trifluoroacetic acid (1.01 mL,
13.1 mmol or 0.516 mL, 6.70 mmol) were added dropwise. The reaction mixture was allowed to
warm to room temperature and was stirred for 24h. Afterwards, the solvent was evaporated under
reduced pressure. The resulting residue was taken up with propan-2-ol (4–5 mL). Then a mixture of
hydrochloric acid (37%) and propan-2-ol (1:1) was added dropwise until a precipitate was formed.
Thereafter, diethyl ether (20–30 mL) was added and the mixture was refluxed for 2 h. After cooling to
˝
room temperature the formed precipitate was filtered off and dried under reduced pressure at 60 C.
2-{[2-(4-Chlorophenoxy)phenyl]amino}-2-oxoethan-1-aminium chloride (10): Crystallization from diethyl
ether/propan-2-ol yielded a beige solid (220 mg, 0.70 mmol, 54%); m.p.: 255–257 ˝C (decomp.); IR
(KBr):
[ppm] = 3.78 (s, 2H, CH2), 6.95 (dd, J = 8.0 Hz, 1.6 Hz, 1H, arom. H), 7.01–7.05 (m, 2H, arom. H),
7.13–7.21 (m, 2H, arom. H), 7.42–7.48 (m, 2H, arom. H), 8.03 (dd, J = 8.0 Hz, 1.9 Hz, 1H, arom. H),
8.14 (s, 3H, NH3`), 10.12 (s, 1H, NH); 13C-NMR (151 MHz, DMSO-d6)
[ppm] = 40.91 (CH2), 118.99,
120.12 (2C), 123.27, 124.12, 125.52, 129.72 (2C) (CH), 127.22, 128.90, 146.92, 155.45 (C), 165.48 (C=O);
ν
[cm´1] = 3434 (m, br, N-H), 3124 (m, N-H), 1673 (s, C=O); 1H-NMR: (600 MHz, DMSO-d6):
δ
δ
C14H13ClN2O2
m/z (%): 276.0 [Mfree base
tR = 3.41 min, tM(DMSO) = 1.06 min (ACN/Buffer = 30:70), λmax [nm] = 231.
¨
HCl (313.18): calcd. C 53.69, H 4.51, N 8.95, found C 53.89, H 4.51, N 8.57; EI-MS:
+.
]
(36), 219.0 [M
´
54]+ (100); HPLC: 99.9% 254 nm, 99.8% at 280 nm;
2-{[2-(4-Methoxyphenoxy)phenyl]amino}-2-oxoethan-1-aminium chloride (20): Crystallization from diethyl
ether/propan-2-ol yielded a beige solid (189 mg, 0.61 mmol, 91%); m.p.: 249–250 ˝C (decomp.); IR

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(KBr):
[ppm] = 3.76 (s, 2H, OCH3), 3.84 (s, 2H, CH2), 6.72–6.77 (m, 1H, arom. H), 6.95–7.04 (m, 4H, arom.
H), 7.05–7.13 (m, 2H, arom. H), 7.88–8.14 (m, 1H, arom. H), 8.26 (s, 3H, NH3`), 10.11 (s, 1H, NH);
13C-NMR (151 MHz, DMSO-d6)
[ppm] = 55.35 (CH3), 40.85 (CH2), 114.95 (2C), 116.75, 120.73 (2C),
122.61, 122.82, 125.16 (CH), 127.88, 148.66, 149.11, 155.69 (C), 165.28 (C=O); C15H16N2O3 HCl (308.76):
calcd. C 58.35, H 5.55, N 9.07, found C 58.28, H 5.52, N 8.82; EI-MS: m/z (%): 272.1 [Mfree base
+. (55),
200.1 [M
72]+ (100); HPLC: 99.2% at 254 nm, 99.3% at 280 nm; tR = 7.89 min, tM(DMSO) = 1.06 min
ν
[cm´1] = 3434 (m, br, N-H), 3114 (m, N-H), 1680 (s, C=O); 1H-NMR: (600 MHz, DMSO-d6):
δ
δ
¨
]
´
(ACN/Buffer = 20:80), λmax [nm] = 229.
3.2.1. Calculations of Lipinski Properties
Calculations of the octanol/water partition coefficients (logP) were performed using
chemicalize.org [15]. The calculator plugins are based on Viswanadhan’s fragmentation methods [22],
the PHYSPROP© database [23] and an additional data set [24]. All three approaches were weighted
equally for the calculations.
3.2.2. In Vitro Antimalarial Activity Assay
Erythrocytic stages of transgenic NF54-luc P. falciparum were used for the luciferase-based viability
assay. These parasites constitutively express high levels of luciferase. The parasites were cultured as
described earlier [19
,25]. Firstly, the culture was dispensed in triplicate into white 96-well flat bottom
plates (each well contains 250
µL) (NUNC, Roskilde, Denmark) with parasitemia of 0.5%–1%. Then
the˝cultures were incubated in the presence of a 3
µ
M concentration of the test compounds for 48 h
(
37 C, 90% N2, 5% CO2, and 5% O2). Subsequently, 100 µL RPMI1640 media was removed from each
well and a 100
µ
L volume of the Bright-Glo® substrate solution was added to each well. One of the
cleavage products of the reaction is light, which was measured by a FLUOROSKAN FL luminometer
(Thermo) machine, thereby detecting the amount of living parasites. The experiments were repeated
with an incubation of 96 h. Untreated cultures were used as negative controls and to calculate the
inhibition rate (0% inhibition of parasite growth). Blasticidin (Sigma-Aldrich, St. Louis, MO, USA), a
drug used for selection of transfected parasites, was included as a positive control on each plate and
gave >90% inhibition of parasite growth at 2 µg/mL.
4. Conclusions
In conclusion, the previously published N-[2-(4-chlorophenoxy)phenyl]-2,2-dimethylpropanamide
(TCMDC-137332,
fully characterized regarding identity and purity. The predicted antimalarial activity of the herein
examined hit structure of the TCAMS ( ) was not confirmed in biological evaluation experiments.
1) [7] and 19 derivatives were synthesized in satisfactory to excellent yields and were
1
The growth of Plasmodium falciparum NF54 strains was not diminished in the expected nanomolar
concentration by the 2-phenoxyanilide congeners. However, in a prolonged in vitro assay, in which
cultures were incubated for 96 h, N-[2-(4-methoxyphenoxy)phenyl]cyclobutanecarboxyamide (16) was
identified to be a moderate inhibitor of the proliferation of Plasmodium falciparum NF54 strains.
Acknowledgments: The project was funded by the German Federal Ministry of Education and Research (BMBF
BioDisc 7; 13GW0024). Support by the COST action CM1307 “Targeted chemotherapy towards diseases caused by
endoparasites” (to Thomas Weidner and Conrad Kunick) is gratefully acknowledged.
Author Contributions: Thomas Weidner: experimental synthetic work, synthesis planning, chemoinformatics,
instrumental analysis, writing of manuscript; Abed Nasereddin and Ron Dzikowski: biological evaluation;
Lutz Preu: NMR interpretation, chemoinformatics; Johann Grünefeld: synthesis planning; Conrad Kunick:
synthesis planning, literature search, writing of manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016, 21, 223
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).