New acyl derivatives of 3-aminofurazanes and their antiplasmodial activities

Article abstract of DOI:10.3390/ph14050412

An N-acylated furazan-3-amine of a Medicines for Malaria Venture (MMV) project has shown activity against different strains of Plasmodium falciparum. Seventeen new derivatives were prepared and tested in vitro for their activities against blood stages of two strains of Plasmodium falciparum. Several structure–activity relationships were revealed. The activity strongly depended on the nature of the acyl moiety. Only benzamides showed promising activity. The substitution pattern of their phenyl ring affected the activity and the cytotoxicity of compounds. In addition, physicochemical parameters were calculated (log P, log D, ligand efficiency) or determined experimentally (permeability) via a PAMPA. The N-(4-(3,4-diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3(trifluoromethyl)benzamide possessed good physicochemical properties and showed high antiplasmodial activity against a chloroquine-sensitive strain (IC₅₀ (NF54) = 0.019 μM) and even higher antiplasmodial activity against a multiresistant strain (IC₅₀ (K₁ ) = 0.007 μM). Compared to the MMV compound, the permeability and the activity against the multiresistant strain were improved.

Full text of DOI:10.3390/ph14050412

pharmaceuticals
Article
New Acyl Derivatives of 3-Aminofurazanes and Their
Antiplasmodial Activities
Theresa Hermann 1 , Patrick Hochegger 1,
*
, Johanna Dolensky 1, Werner Seebacher 1, Robert Saf 2,
Marcel Kaiser 3 , Pascal Mäser 3 and Robert Weis 1
1
Institute of Pharmaceutical Sciences, Pharmaceutical Chemistry, University of Graz, Schubertstraße 1,
A-8010 Graz, Austria; theresa.hermann@uni-graz.at (T.H.); johanna.faist@uni-graz.at (J.D.);
we.seebacher@uni-graz.at (W.S.); robert.weis@uni-graz.at (R.W.)
Institute for Chemistry and Technology of Materials (ICTM), Graz University of Technology, Stremayrgasse 9,
A-8010 Graz, Austria; robert.saf@tugraz.at
Swiss Tropical and Public Health Institute, Socinstraße 57, CH-4002 Basel, Switzerland;
marcel.kaiser@swisstph.ch (M.K.); pascal.maeser@swisstph.ch (P.M.)
2
3
*
Correspondence: patrick.hochegger@uni-graz.at; Tel.: +43-316-380-5379; Fax: +43-316-380-9846
Abstract: An N-acylated furazan-3-amine of a Medicines for Malaria Venture (MMV) project has
shown activity against different strains of Plasmodium falciparum. Seventeen new derivatives were
prepared and tested in vitro for their activities against blood stages of two strains of Plasmodium
falciparum. Several structure–activity relationships were revealed. The activity strongly depended
on the nature of the acyl moiety. Only benzamides showed promising activity. The substitution
pattern of their phenyl ring affected the activity and the cytotoxicity of compounds. In addition,
physicochemical parameters were calculated (log P, log D, ligand efficiency) or determined ex-
perimentally (permeability) via a PAMPA. The N-(4-(3,4-diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-
(trifluoromethyl)benzamide possessed good physicochemical properties and showed high antiplas-
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
modial activity against a chloroquine-sensitive strain (IC50(NF54) = 0.019
µM) and even higher
Citation: Hermann, T.; Hochegger, P.;
Dolensky, J.; Seebacher, W.; Saf, R.;
Kaiser, M.; Mäser, P.; Weis, R. New
Acyl Derivatives of 3-Aminofurazanes
and Their Antiplasmodial Activities.
Pharmaceuticals 2021, 14, 412. https://
doi.org/10.3390/ph14050412
antiplasmodial activity against a multiresistant strain (IC50(K1) = 0.007 M). Compared to the MMV
µ
compound, the permeability and the activity against the multiresistant strain were improved.
Keywords: antimalarial; furazan derivatives; Plasmodium falciparum; PAMPA
Academic Editor: Marcelo J. Nieto
1. Introduction
Malaria is, to this day, one of the most dangerous infectious diseases worldwide.
It is caused by protozoa of the genus Plasmodium. An estimated number of 229 million
cases occurred in 2019, resulting in more than 400,000 deaths. The burden of this infection
is mostly carried by children under the age of five located in sub-Saharan Africa. Five
Plasmodium species are human pathogenic: P. falciparum, P. vivax, P. malariae, P. ovale and
P. knowlesi of which P. falciparum is the most predominant cause of death [1,2]. The discovery
of chloroquine in the 1960s had an enormous impact on the fight against malaria. Increasing
resistance development in P. falciparum, however, was reported more than fifty years ago,
Received: 29 March 2021
Accepted: 24 April 2021
Published: 27 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
and the cost of it in human lives was severe [3,4]. The first-line treatment for malaria
infections is an artemisinin-based combination therapy (ACT). A rapid-acting artemisinin
derivative is combined with an antimalarial drug with a long half-life period. However,
artemisinin drug resistances are increasingly emerging in P. falciparum, especially within
Copyright:
© 2021 by the authors.
the Southeast Asian region, and are therefore jeopardizing the success of ACTs [
strategy to impede the increasing resistance development temporarily is the application of
triple artemisinin-based combination therapy (TACT) [ ]. Due to the threat of potentially
5,6]. A new
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
7,8
untreatable P. falciparum malaria, the development of drugs with new modes of action is of
utmost importance [9–11].
Phenotypic screening or whole-cell screening represents a significant breakthrough in
the discovery of new antimalarial lead structures. The majority of compounds currently in
Pharmaceuticals 2021, 14, 412. https://doi.org/10.3390/ph14050412
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 412
2 of 23
clinical trials were discovered by means of this screening method. The benefit of phenotypic
screening is that the activity is not determined at isolated targets, but in the physiological
environment. The implementation of this method led to a 100-fold reduction in the cost
for screening compounds against P. falciparum-infected erythrocytes compared to other
methods. Most commonly, the target search starts afterward [12,13].
In 2016, the organization Medicines for Malaria Venture (MMV) published the results
of a major phenotypic screening project. The so-called Malaria Box contains 400 substances
with activity against P. falciparum and serves as a starting point for the development of new
lead structures. Alongside activity assays against different strains and life cycle stages of
P. falciparum, their cytotoxicity against 73 human cell lines was determined as well [14].
The furazan 1 (Scheme 1) from MMV’s Malaria Box project is a promising lead struc-
ture for the development of new antimalarials. It shows antiplasmodial activity against the
multiresistant strains K1 and Dd2 of Plasmodium falciparum [14]. Furthermore, a P. berghei
assay revealed activity against liver schizonts [15,16]. The potential of inhibiting the trans-
mission from humans to mosquitoes by working against early gametocyte stages makes
this compound even more promising [17–21].
◦
Scheme 1. Preparation of compound 1. Reagents and conditions: (
a
) NH2OH x HCl, NaHCO3, MeOH, H2O, 100 C, 2 h;
(
b
) N-chlorosuccinimide, DMF, rt, 20 h; (
c
) NaBH4, MeOH, rt, 1 h; (
d
) thionyl chloride, CH2Cl2, rt, 20 h; (
e
) KCN, DMF, rt,
◦
◦
4 h; (
f
) (1) NaOEt, 0 C, 30 min; (2) 3-methylbutyl nitrite, EtOH, rt, 20 h; (
g
) NH2OH x HCl, NaHCO3, MeOH, H2O, 100 C,
◦
◦
◦
20 h; (h) 2N NaOH, 100 C, 20 h; (i) (1) NaH, DMF, 0 C, 20 min; (2) 3-methylbenzoyl chloride, DMF, 60 C, 48 h.

Pharmaceuticals 2021, 14, 412
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Within two subsequent studies, the possible targets of furazan
1
could be identified.
Firstly, it is likely to inhibit the Na+-efflux pump PfATP4, which is essential in maintaining
the parasite’s ion homeostasis. PfATP4 is localized on the plasma membrane of P. falciparum
and represents an attractive target for novel antimalarials. However, genetic PfATP4 muta-
tions led to an increase in resistance development against several preclinical and clinical
antimalarials. The furazan 1 also interacts with the enzyme deoxyhypusine hydroxylase
(DOHH) which is part of the hypusine biosynthesis [22–25].
The aim of this study was to synthesize new derivatives of compound 1 in order to
increase the antiplasmodial activity and reveal structure–activity relationships (SARs). All
newly synthesized compounds were characterized and tested in vitro for their activities
against the chloroquine-sensitive strain NF54 and the multiresistant K1 strain of P. falci-
parum. The results were compared to those of drugs in use. Furthermore, the passive
diffusion of the compounds was determined by a parallel artificial membrane permeability
assay (PAMPA) to classify compounds according to their permeability. Especially in the
development of new antimalarials, it is important to focus on substances that are orally
bioavailable, because of the potential storage problems with other formulations in countries
where malaria is prevalent and the simpler use. The PAMPA is an early screening assay to
differentiate between compounds that have a good oral absorption potential and those that
do not.
2. Results and Discussion
2.1. Chemistry
The precursor of all derivatives, compound
benzaldehyde in a multistep procedure via the formation of a cyanide-substituted oxime
. Since no synthetic route to generate compound has been published yet, we used a
retrosynthetic approach. Two different methods were applied for the synthesis of the
oxime . Treatment of 3,4-diethoxybenzaldehyde with hydroxylamine hydrochloride
gave the aldoxime in high yields. Conversion of the benzaldoxime to the benzimidoyl
chloride succeeded with N-chloro succinimide. However, subsequent reaction with
potassium cyanide to afford compound failed. Therefore, an alternative method was
1, was synthesized from 3,4-diethoxy-
2
1
2
3
4
2
used, and compound 1 was successfully prepared in a seven-step procedure.
At first, 3,4-diethoxybenzaldehyde was reduced to its corresponding alcohol 5 using
sodium borohydride in methanol. The successful reduction was obvious by the disap-
pearance of the signal of the formyl proton in the 1H NMR spectrum. A resonance at ca.
4.5 ppm appeared for the new methylene grouP. Treatment with thionyl chloride gave the
benzyl chloride
13C resonance of the methylene group was shifted 18 ppm upfield. It was then converted
into the 2-phenylacetonitrile by means of a Kolbe nitrile synthesis [26]. The replacement
of the chlorine atom by a cyano group shifted the proton signal of the methylene group
1 ppm upfield. Its -protons were replaced by a hydroximino grouP. After deprotonation
6. Due to the replacement of the hydroxy group by a chlorine atom the
7
α
with sodium ethylate, 3-methylbutyl nitrite was added, yielding the desired oxime 2 [27].
The oxime carbon gave a new signal at ca. 133 ppm in the 13C NMR spectrum, whereas
the resonance of the methylene group was missing. It was treated with hydroxylamine
hydrochloride giving the amide oxime
doxime shifted the signal of the concerned carbon atom 35 ppm downfield in the 13C NMR
spectrum. Refluxing of with 2N NaOH led to ring closure, affording the 3-aminofurazan 9.
8. The conversion of the cyano group to an ami-
8
Due to the formation of the furazan ring system, the signals of both hydroxy groups disap-
peared in the 1H NMR spectrum. The resonance of the amino protons was shifted 0.5 ppm
downfield. The desired compound
1 was finally obtained by reaction of 9 with sodium
hydride and 3-methylbenzoyl chloride in DMF (Scheme 1) [28]. The successful amide bond
formation was detected by a significant change in the NMR spectrum. The signal of the
aromatic amino protons was replaced by a broadened signal at high frequencies.

Pharmaceuticals 2021, 14, 412
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To obtain some insight considering structure–activity relationship, the amino furazan
was subsequently coupled with different carboxylic acids. Furthermore, the importance
9
9
of the meta-methyl group was investigated. Compounds 10
–
17 and 26
29 were obtained by coupling the amino furazan
with the respective benzoyl chlorides that were commercially available or generated
29]. Reaction of the
yielded 15 30].
–29 were synthesized.
The amides 1, 10–14, 16, 17 and 26–
by the reaction of benzoic acids and oxalyl dichloride (Scheme 2) [28
,
N-hydroxy succinimide ester of the 3-(trifluoromethoxy)benzoic acid with
9
[
◦
Scheme 2. Preparation of compounds
chloride, DMF, 60 ◦C, 48 h (compounds
DMF, 0 ◦C, 20 min; (3) acid chloride, DMF, 60 ◦C, 48 h (compounds 16
1
,
10
1
–
17 and 26
–
29. Reagents and conditions: (
a
) (1) NaH, DMF, 0 C, 20 min; (2) acyl
and 10
–
14); or (1) carboxylic acid, oxalyl dichloride, CH2Cl2, rt, 20 h; (2) NaH,
29); or (1) carboxylic acid, N-hydroxy
succinimide, DCC, THF, rt, 24 h; (2) DMF, rt, 15 min; (3) NaH, DMF, 0 C, 20 min; (4) acid chloride, DMF, 60 C, 48 h
,
17 and 26
–
◦
◦
(compound 15).
The carboxylic acids used for the synthesis of compounds 26–29 had to be synthesized
from methyl 3-(bromomethyl)benzoate and methyl 4-(bromomethyl)benzoate (Scheme 3).
The benzyl bromides reacted with the respective heterocyclic amines, potassium carbonate
and catalytic amounts of sodium iodide in acetonitrile to form the tertiary amines 18, 19,
22 and 23. Treatment of the methyl esters with 2N NaOH in methanol gave the desired
carboxylic acids 20, 21, 24 and 25 [31].
Within another series of derivatives, the planar aromatic system was replaced by
different aliphatic heterocycles. Furthermore, we also modified the chain length between
the amide carbonyl groups and the
linker, the influence of an ethyl linker was investigated.
To obtain compounds 31 32 34 and 35, the amino furazan
the corresponding -chloroacyl chloride, yielding the -chloroalkanamides 30 and 33 [
ω-(dialkylamino) groups. Aside from a methylene
,
,
9
was treated at first with
32].
ω
ω
These were treated with the corresponding amines, giving the pyrrolidine derivatives 31
and 34, as well as the morpholine derivatives 32 and 35 (Scheme 4).

Pharmaceuticals 2021, 14, 412
5 of 23
◦
, 21, 24 and 25. Reagents and conditions: (a) amine, NaI, K2CO3, acetonitrile, 80 C,
Scheme 3. Preparation of compounds 20
20 h; (b) 2N NaOH, MeOH, rt, 20 h.
Scheme 4. Preparation of compounds 31
,
32
,
34 and 35. Reagents and conditions: (
a
) chloroacetyl chloride, pyridine, diethyl
ether, benzene, 80 ◦C, 20 h; (
b
) 3-chloropropanoyl chloride, pyridine, diethyl ether, benzene, 80 ◦C, 20 h; (
c) amine, NaI,
◦
K2CO3, acetonitrile, 80 C, 20 h.
To gain further insight regarding SARs, we also synthesized compound 39. This
compound possesses a 4-phenyl substituent instead of a 4-(3,4-diethoxyphenyl) substituent
when compared to compound 1.
The synthesis was similar to the synthesis of compound 1 (Scheme 1). Starting from
benzyl cyanide, the oxime 36 was obtained by treatment of the nitrile with 3-methylbutyl
nitrite after deprotonation with sodium ethylate. The cyano group was further converted

Pharmaceuticals 2021, 14, 412
6 of 23
to the amidoxime 37 and further on to the aminofurazan 38 after a cyclization reaction.
The final compound 39 was obtained by an amide reaction of the aminofurazan 38 with
3-methylbenzoyl chloride (Scheme 5).
◦
Scheme 5. Preparation of compound 39. Reagents and conditions: (
a
) (1) NaOEt, 0 C, 30 min;
(2) 3-methylbutyl◦nitrite, EtOH, rt, 20 h; (
b
) N◦H2OH x HCl, NaHCO3, MeOH, H2O, 100 ◦C, 20 h;
◦
(c) 2N NaOH, 100 C, 20 h; (
d
) (1) NaH, DMF, 0 C, 20 min; (2) 3-methylbenzoyl chloride, DMF, 60 C,
48 h.
2.2. Antiplasmodial Activity and Cytotoxicity
All newly synthesized compounds were at first tested in vitro for their antiplasmodial
activity against the chloroquine-sensitive strain NF54 of P. falciparum. The most active
compounds were further tested against the K1 strain of P. falciparum, which is resistant to
chloroquine and pyrimethamine. Cytotoxicity was determined using rat skeletal myoblasts
(L-6 cells). As standards served chloroquine, artemisinin and podophyllotoxin.
In order to evaluate the influence of a series of inserted acyl moieties, the 4-(3,4-
diethoxyphenyl) substituent of the 3-aminofurazan was left unaltered. The substitution
pattern of the aromatic system was diversified to evaluate the importance of the meta-
methyl group of furazan
including (dialkylamino)methyl substitution in meta- and para-position. Furthermore, the
benzoyl moiety was exchanged with -(dialkylamino)acyl residues in order to observe
1. The methyl group was replaced by several other substituents,
ω
the importance of the aromatic system for activity. The variation of the linker length
between the amide and the aliphatic heterocyclic amines might increase the flexibility of
the substituents, resulting in a positive effect on cytotoxicity.
Compound
It shows no difference in activity against the chloroquine-sensitive strain NF54 (PfNF54
IC50 = 0.011 M) and the multiresistant K1 strain (PfK1 IC50 = 0.011 M) of Plasmod-
ium falciparum. The low cytotoxicity (L-6 cells IC50 = 159.3 M) combined with the
high antiplasmodial activity results in an excellent selectivity index for both strains
(SIPf = SIPf = 14,483). Compound 10 is the 3-demethyl analog of . It shows still
M) and PfK1 (IC50 = 0.091 M)
. The 2-substituted compounds
M) and the 4-substituted compounds 26 and 27
M) possess only moderate or very weak antiplasmodial activ-
ity. With the exception of the only moderately active 3-(morpholin-4-yl)methyl derivative
1 served as comparison for all newly synthesized compounds (Table 1).
µ
µ
µ
1
NF54
K1
high antiplasmodial activity against PfNF54 (IC50 = 0.076
but a 7–8-fold decrease in activity when compared to
11 and 12 (PfNF54 IC50 = 0.343–0.831
(PfNF54 IC50 = 0.674–4.055
µ
µ
1
µ
µ

Pharmaceuticals 2021, 14, 412
7 of 23
29 (PfNF54 IC50 = 0.712
µM), the series of 3-substituted analogs exhibited good to excellent
activity (PfNF54 IC50 = 0.014–0.192
4-fold increase in activity (PfNF54 IC50 = 0.192
were among the most active of the new compounds (PfNF54 IC50 = 0.019–0.098
pared to its activity against PfNF54 (IC50 = 0.049 M), the 3-fluoro derivative 14 showed the
expected decrease in activity against the multiresistant strain PfK1 (IC50 = 0.108 M). How-
ever, its 3-(trifluoromethyl) analog 13 was even more active against the multiresistant strain
PfK1 (IC50 = 0.007 M) than against PfNF54 (IC50 = 0.019 M). Against this strain, it even
surpassed the activity of compound (PfK1 IC50 = 0.011 M) and showed similar activity
to artemisinin (PfK1 IC50 = 0.0064 M). Likewise, the 3-substituted 4-methoxy compounds
16 and 17 exhibited promising activity against PfNF54 (IC50 = 0.014–0.167 M). Against
the sensitive strain, the 3-chloro-4-methoxybenzamide 16 possessed the highest activity
(PfNF54 IC50 = 0.014 M), which is similar to (PfNF54 IC50 = 0.011 M). Replacement
of the benzamide by a -(dialkylamino)acetamide or a -(dialkylamino)propanamide
moiety led to inactive compounds 31 32 34 and 35 (PfNF54 IC50 = 7.24–56.01 M). In
comparison to (L-6 cells IC50 = 159.3 M), all of the more active compounds showed
increased cytotoxicity (L-6 cells IC50 = 9.24–111.2 M). However, the selectivity indexes of
the most promising compounds 10, 13, 14 and 16 were still very high (S.I. = 380–1463).
µ
M). Its 3-(pyrrolidin-4-yl)methyl analog 28 showed a
M). The fluorinated analogs 13 14 and 15
M). Com-
µ
,
µ
µ
µ
µ
µ
1
µ
µ
µ
µ
1
µ
ω
ω
,
,
µ
1
µ
µ
Table 1. Activities of compounds
cells, expressed as IC50 (µM) a.
1, 10–17, 26–29, 31, 32, 34, 35 and 39 against P. falciparum NF54, P. falciparum K1 and L-6
S.I.=
IC50 (Cyt.)/IC50
(P.f. NF54)
S.I. =
IC50 (Cyt.)/IC50
(P.f. K1)
Cytotoxicity
L-6 Cells
IC50 (µM)
c
P.f. NF54 b
Compound
P.f. K1
IC50 (µM)
IC50 (µM)
1
10
11
12
13
14
15
16
17
26
27
28
29
31
32
34
35
0.011
0.076
0.343
0.831
0.019
0.049
0.098
0.014
0.167
4.055
0.674
0.192
0.712
7.24
0.011
0.091
14483
1463
43.49
17.13
524.6
838.6
142.8
1431
209.8
25.07
172.7
402.2
146.0
33.46
10.95
2.50
14483
1222
159.3
111.2
14.92
14.24
9.97
0.007
0.108
1424
380.5
41.09
13.99
20.03
35.03
101.7
116.4
77.23
104.0
242.3
265.7
139.8
147.3
65.95
450.5
188.5
0.012
1.87
0.586
54.25
199.7
5.02
19.53
2.41
18.99
39.92
0.0064
0.15
48.25
13.61
58.09
7.76
1.65
70390
1570
24.25
56.01
30.15
21.52
4.88
3.07
39
ART
CQ
POD
a
ART = artemisinin; CQ = chloroquine; POD = podophyllotoxin. Values represent the average of four determinations (two determinations
of two independent experiments); b sensitive to chloroquine; c resistant to chloroquine and pyrimethamine.
The removal of both ethoxy groups of the left-hand side ring, like in compound
39, resulted in a massive loss in activity against the chloroquine-sensitive strain PfNF54
(IC50 = 21.52 µM) and the multiresistant strain PfK1 (IC50 = 39.92 µM). This affirms the
positive impact of the 3,4-diethoxyphenyl substitution pattern.
2.3. Physicochemical Properties and Permeability
In addition to antiplasmodial activity tests of compounds 1, 10–17, 26–29, 31, 32,
34, 35 and 39, physicochemical parameters like log P and log D7.4 were calculated (us-

Pharmaceuticals 2021, 14, 412
8 of 23
ing the ChemAxon software JChem for Excel). Furthermore, test compounds had to
meet conditions of sufficient ligand efficiency (LE) [33] and effective permeability (Pe).
The latter was determined by a PAMPA (Table 2) [34]. The log P values of the com-
pounds range between 1.50 and 5.11. The inactive
ω-(dialkylamino)alkanamides 31, 32,
34 and 35 have by far the lowest log P values (log P = 1.50–2.36). The most active com-
pounds have good log p values (log P = 3.66–4.56). All compounds with considerable
antiplasmodial activity have log D7.4 values ranging from 3.09 to 5.11. The permeability of
compounds was only detectable for selected compounds due to insufficient solubility or ex-
cessive mass retention in the PAMPA. All new compounds showed increased permeability
(Pe = 3.90–10.80
ing compound 13 has only slightly higher permeability than
whereas the inactive -aminoacetamides 31 and 32 show by far the best permeabilities
×
10−6 cm/s) in comparison to
1
(Pe = 2.77
×
10−6 cm/s). The most promis-
(Pe = 4.26
10−6 cm/s),
1
×
ω
(Pe = 10.25–10.80
×
10−6 cm/s). However, in general, substances with a permeability above
1.5 × 10−6 cm/s are considered as having good permeability.
Table 2. Key physicochemical parameters and PAMPA values of compounds
34, 35 and 39.
1, 10–17, 26–29, 31, 32,
LE
a
log P a
log D7.4
Pe b (10 cm/s)
−6
Compound
(kcal/mol/HA)
1
4.19
3.68
4.19
4.56
4.56
3.82
5.11
4.13
3.66
3.90
3.40
4.03
3.40
2.12
1.50
2.36
1.74
3.79
4.19
3.68
4.19
4.56
4.56
3.82
5.11
4.13
3.66
2.47
3.34
3.09
3.39
1.83
1.38
0.57
1.53
3.79
0.404
0.375
0.328
0.278
0.353
0.371
0.310
0.371
0.320
0.231
0.256
0.288
0.255
0.271
0.234
0.216
0.221
0.287
2.77
n.d.
n.d.
n.d.
4.26
n.d.
n.d.
n.d.
3.90
8.13
n.d.
7.26
5.65
10.80
10.25
6.93
6.08
8.47
10
11
12
13
14
15
16
17
26
27
28
29
31
32
34
35
39
a
Log P and log D were calculated using the ChemAxon software JChem for Excel 14.9.1500.912 (2014); b deter-
mined by PAMPA; n.d., could not be determined.
Ligand efficiency has become an important concept in drug development, partly
due to the realization that large ligands have a disadvantage in terms of the molecular
properties necessary for bioavailability. It is defined as the binding free energy for a
ligand divided by its number of heavy atoms (HA). Proposed acceptable values for drug
candidates are ~0.3 kcal/mol/HA and higher. Out of all tested compounds,
1
has the
highest ligand efficiency (LE = 0.404 kcal/mol/HA). The compounds 10, 13, 14 and 16 also
exhibit promising ligand efficiencies (LE = 0.353–0.375 kcal/mol/HA).
3. Materials and Methods
3.1. Instrumentation and Chemicals
Melting points were obtained on an Electrothermal IA 9200 melting point apparatus.
IR spectra: Bruker Alpha Platinum ATR FTIR spectrometer (KBr discs); frequencies are
reported in cm−1. The structures of all newly synthesized compounds were determined
by one- and two-dimensional NMR spectroscopy. NMR spectra: Varian UnityInova 400
1
(298 K) 5 mm tubes, TMS as internal standard. Shifts in H NMR (400 MHz) and 13C
NMR (100 MHz) spectra are reported in ppm; 1H- and 13C-resonances were assigned using

Pharmaceuticals 2021, 14, 412
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1
1H,1H- and H,13C-correlation spectra and are numbered as given in Scheme 1. Signal
multiplicities are abbreviated as follows: br, broad; d, doublet; dd, doublet of doublets;
ddd, doublet of doublet of doublets; m, multiplet; s, singlet; t, triplet; td, triplet of doublets;
q, quartet. HRMS: Micromass Tofspec 3E spectrometer (MALDI) and GCT-Premier, Waters
(EI, 70 eV). Materials: column chromatography (CC): silica gel 60 (Merck 70–230 mesh, pore
diameter 60 Å), aluminium oxide (pH: 9.5, Fluka), thin-layer chromatography (TLC): TLC
plates silica gel 60 F254 (Merck), aluminium oxide 60 F254 (neutral, Merck). Experiments
to assess the purity of final compounds were carried out on an Agilent 1110 HPLC device
(Agilent Technologies, Palo Alto, CA, USA) equipped with an auto sampler and a VWL
detector. Ultraviolet detection was performed at 214 nm. The measurements were carried
out under isocratic conditions at ambient temperature with a flowrate of 2 mL/min and
an injection volume of 20 µL. Data were collected with Chemstation Rev. B. 0903 (Agilent
Technologies, Waldbronn, Germany) software. A LiChrospher 100 RP-18e, 125 mm
×
4 mm,
3
µm from Merck KGaA (Darmstadt, Germany) served as stationary phase. Mobile phase
was prepared by mixing a 20 mM ammonium phosphate buffer adjusted to pH 2.4 and
acetonitrile (2:1). Samples were prepared by dissolving 1 mg each in 1 mL methanol. Unless
otherwise noted, all compounds were found to be >95% pure by this method. PAMPA:
96-well precoated Corning Gentest PAMPA plate (Corning, Glendale, AZ, USA), 96-well
UV-Star Microplates (Greiner Bio-One, Kremsmünster, Austria), SpectraMax M3 UV plate
reader (Molecular Devices, San Jose, CA, USA). 1H-NMR and 13C-NMR spectra of new
compounds are available in Supplementary Materials Section (Figures S1–S23).
3.2. Syntheses
(3,4-Diethoxyphenyl)methanol (5): NaBH4 (0.57 g (15.00 mmol)) was added in portions to
an ice-cooled solution of 3,4-diethoxybenzaldehyde (2.91 g (15.00 mmol)) in dry methanol
◦
(16 mL). After that, the ice bath was removed and the reaction mixture was stirred at 25 C
for 1 h. Then, the solvent was evaporated in vacuo and the residue was mixed with water
and extracted with CH2Cl2. The combined organic phases were washed with water, dried
over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo giving
compound 5 as colorless oil (2.80 g (95%)), which was used without further purification.
NMR data were in accordance with literature data [35].
4-(Chloromethyl)-1,2-diethoxybenzene (
added dropwise via a dropping funnel to an ice-cooled solution of benzyl alcohol
(12.00 mmol)) in dry CH2Cl2 (50 mL). The ice bath was removed and the reaction mixture
6): Thionyl chloride (4.14 g (34.80 mmol)) was
5
(2.36 g
◦
◦
was stirred at 25 C for 20 h. Then, the reaction was quenched with 2N NaOH at 0 C
and the mixture was basified to a pH of 10–11. The aqueous and organic phases were
separated and the aqueous phase was extracted with CH2Cl2. The combined organic phases
were dried over anhydrous sodium sulfate and filtered and the solvent was removed in
vacuo, giving compound
purification. IR = 2977, 1604, 1510, 1476, 1428, 1392, 1256, 1232, 1135, 1038, 985, 805; 1H
NMR (CDCl3, 400 MHz) = 1.42–1.47 (m, 6H, 2 CH3), 4.06–4.13 (m, 4H, 2 CH2), 4.55 (s,
2H,CH2Cl), 6.82 (d, J = 8.1 Hz, 1H, 5-H), 6.90 (dd, J = 8.1, 2.0 Hz, 1H, 6-H), 6.91 (d, J = 2.0 Hz,
1H, 2-H); 13C NMR (CDCl3, 100 MHz)
= 14.76 (2 CH3), 46.71 (CH2Cl), 64.56 (2 CH2),
6 as brown oil (2.47 g (96%)), which was used without further
δ
δ
113.08 (C-5), 113.86 (C-2), 121.21 (C-6), 129.96 (C-1), 148.81 (C-3), 148.93 (C-4); HRMS (EI+)
calcd for C11H15ClO2: 214.0761; found: 214.0755.
2-(3,4-Diethoxyphenyl)acetonitrile (7): Benzyl chloride 6 (2.15 g (10.00 mmol)) was
dissolved in dry DMF (20 mL). KCN (1.30 g (20.00 mmol)) was added and the suspension
◦
was stirred at 100 C for 4 h. After that, the solvent was evaporated in vacuo and the
residue was mixed with water and extracted with ethyl acetate. The organic phases were
combined and washed with water and brine, dried over anhydrous sodium sulfate and
filtered. The solvent was evaporated in vacuo, giving compound
(95%)), which was used without further purification.
7 as brown oil (1.95 g
NMR data were in accordance with literature data [36].

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(3,4-Diethoxyphenyl)(hydroxyimino)acetonitrile (
2
): Sodium (0.37 g (12.00 mmol)) was
dissolved in dry ethanol (15 mL) and then cooled to 0 ◦C with an ice bath. A solution of
compound
7 (1.64 g (8.00 mmol)) in dry ethanol (10 mL) was added dropwise. Finally,
isopentyl nitrite (1.41 g (12.00 mmol)) was added dropwise with a syringe through a
septum. The ice bath was removed and the reaction mixture stirred at 25 ◦C for 20 h. The
solution was diluted with ethyl acetate (80 mL) and washed with 2N HCl, 8% aq NaHCO3
and brine. The organic phase was dried over anhydrous sodium sulfate and filtered and
the solvent was removed in vacuo, giving compound
(99%)), which was used without further purification. IR = 3364, 1601, 1512, 1438, 1337, 1273,
1210, 1146, 1076, 1006, 845, 806; 1H NMR (CDCl3, 400 MHz)
= 1.42 (t, J = 6.9 Hz, 3H, CH3),
2 as white amorphous solid (1.86 g
δ
1.43 (t, J = 6.9 Hz, 3H, CH3), 4.08 (q, J = 6.9 Hz, 2H, OCH2), 4.10 (q, J = 6.9 Hz, 2H, OCH2),
7.00 (d, J = 8.5 Hz, 1H, 5-H), 7.26 (dd, J = 8.5, 1.9 Hz, 1H, 6-H), 7.33 (d, J = 1.9 Hz, 1H, 2-H);
13C NMR (CDCl3, 100 MHz)
δ = 15.16 (CH3), 15.19 (CH3), 65.75 (OCH2), 65.89 (OCH2),
110.68 (C-2), 111.04 (CN), 114.03 (C-5), 121.22 (C-6), 124.25 (C-1), 132.87 (C=NOH), 150.49
(C-3), 152.73 (C-4); HRMS (EI+) calcd for C12H14N2O3: 234.1004; found: 234.0996.
2-(3,4-Diethoxyphenyl)-N’-hydroxy-2-(hydroxyimino)ethanimidamide (
8): Oxime
2 (1.41 g
(6.00 mmol)) was dissolved in methanol (18 mL) and a solution of NH2OH
×
HCl (0.50 g
(7.20 mmol)) and NaHCO3 (0.61 g (7.20)) in water (9 mL) was added. The mixture was
refluxed for 20 h and then the solvent was evaporated in vacuo. The residue was mixed
with water (20 mL) and extracted with ethyl acetate. The organic phases were combined
dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo,
giving the raw amidoxime which was r◦ecrystallized in CH2Cl2 affording compound
8 as
beige crystals (0.55 g (34%)). m.P. 148 C. IR = 3370, 2981, 1661, 1598, 1516, 1444, 1393,
1270, 1201, 1144, 1040, 978, 810; 1H NMR (DMSO-d6, 400 MHz)
δ = 1.33 (t, J = 7.0 Hz, 6H,
2 CH3), 3.97–4.07 (m, 4H, 2 OCH2), 5.67 (s, 2H, NH2), 6.95 (d, J = 8.4 hz, 1H, 5-H), 7.06
(dd, J = 8.4, 1.9 Hz, 1H, 6-H), 7.22 (d, J = 1.9 Hz, 1H, 2-H), 9.39 (s, 1H, NOH), 11.34 (s, 1H,
NOH); 13C NMR (DMSO-d6, 100 MHz)
δ = 14.66 (CH3), 14.72 (CH3), 63.75 (OCH2), 63.81
(OCH2), 110.59 (C-2), 112.59 (C-5), 120.24 (C-6), 127.24 (C-1), 146.64 (C(=NOH)NH2), 147.66
(C-3), 149.20 (C-4), 149.22 (C=NOH); HRMS (EI+) calcd for C12H17N3O4: 267.1219; found:
267.1230.
4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-amine (
9): Amidoxime 8 (0.80 g (3.00 mmol))
was dissolved in 2N NaOH (30 mL) and refluxed for 20 h. The formed precipitate was
filtered, washed with water and dried. The beige precipitate was pure product 9 (0.67 g
(83%)) and was used without further purification. m.P. 155 ◦C. IR = 3374, 3322, 3244, 2984,
1593, 1536, 1501, 1396, 1327, 1305, 1253, 1215, 1141, 1111, 1041, 941, 862, 848, 815; 1H NMR
(DMSO-d6, 400 MHz)
6.12 (s, 2H, NH2), 7.09 (d, J = 8.4 hz, 1H, 5’-H), 7.25 (d, J = 1.8 Hz, 1H, 2’-H), 7.29 (dd, J = 8.4,
1.8 Hz, 1H, 6’-H); 13C NMR (DMSO-d6, 100 MHz)
= 14.62 (CH3), 14.66 (CH3), 63.85
δ = 1.35 (t, J = 6.9 Hz, 6H, 2 CH3), 4.10 (q, J = 6.9 Hz, 4H, 2 OCH2),
δ
(2 OCH2), 112.20 (C-2’), 113.25 (C-5’), 117.65 (C-1’), 120.60 (C-6’), 146.69 (C-4), 148.35 (C-3’),
149.87 (C-4’), 155.21 (C-3); HRMS (EI+) calcd for C12H15N3O3: 249.1113; found: 249.1099.
The general procedure for the synthesis of tertiary amines (18, 19, 22 and 23) is as
follows: The respective heterocyclic amine (8.00 mmol), K2CO3 (8.00 mmol) and a catalytical
amount of NaI (3.20 mmol) were added to a solution of the corresponding benzyl bromide
(4.00 mmol) in dry acetonitrile (40 mL). The mixture was refluxed for 20 h. The suspension
was cooled and filtered. The filtrate was evaporated in vacuo to dryness. The residue
was mixed with water and the aqueous phase was extracted with CH2Cl2. The combined
organic phases were washed with 8% aq NaHCO3 and brine. The organic phase was dried
over anhydrous sodium sulfate and filtered and the solvent was removed in vacuo, giving
pure amines 18, 19, 22 and 23, which were used without further purification.
Methyl 3-((pyrrolidin-1-yl)methyl)benzoate (18): The reaction of methyl 3-(bromomethyl)-
benzoate (0.92 g (4.00 mmol)), pyrrolidine (0.57 g (8.00 mmol)), K2CO3 (1.11 g (8.00 mmol))
and NaI (0.48 g (3.20 mmol)) in dry acetonitrile (40 mL) gave compound 18 as pale brown
oil (0.87 g (99%)).
NMR data were in accordance with literature data [37].

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Methyl 3-((morpholin-4-yl)methyl)benzoate (19): The reaction of methyl 3-(bromomethyl)-
benzoate (0.92 g (4.00 mmol)), morpholine (0.70 g (8.00 mmol)), K2CO3 (1.11 g (8.00 mmol))
and NaI (0.48 g (3.20 mmol)) in dry acetonitrile (40 mL) gave compound 19 as yellow oil
(0.93 g (99%)).
NMR data were in accordance with literature data [38].
Methyl 4-((pyrrolidin-1-yl)methyl)benzoate (22): The reaction of methyl 4-(bromomethyl)-
benzoate (0.92 g (4.00 mmol)), pyrrolidine (0.57 g (8.00 mmol)), K2CO3 (1.11 g (8.00 mmol))
and NaI (0.48 g (3.20 mmol)) in dry acetonitrile (40 mL) gave compound 22 as pale brown
oil (0.82 g (94%)).
NMR data were in accordance with literature data [39].
Methyl 4-((morpholin-4-yl)methyl)benzoate (23): The reaction of methyl 4-(bromomethyl)-
benzoate (0.92 g (4.00 mmol)), morpholine (0.70 g (8.00 mmol)), K2CO3 (1.11 g (8.00 mmol))
and NaI (0.48 g (3.20 mmol)) in dry acetonitrile (40 mL) gave compound 23 as yellow oil
(0.90 g (96%)).
NMR data were in accordance with literature data [40].
2-(Hydroxyimino)-2-phenylacetonitrile (36): Sodium (0.37 g (12.00 mmol)) was dissolved
◦
in dry ethanol (15 mL) and then cooled to 0 C with an ice bath. A solution of benzyl
cyanide (0.94 g (8.00 mmol)) in dry ethanol (10 mL) was added dropwise. Finally, isopentyl
nitrite (1.41 g (12.00 mmol)) was added dropwise with a syringe through a septum. The
ice bath was removed and the reaction mixture stirred at 25 ◦C for 20 h. The solution was
diluted with ethyl acetate (80 mL) and washed with 2N HCl, 8% aq NaHCO3 and brine.
The organic phase was dried over anhydrous sodium sulfate and filtered and the solvent
was removed in vacuo, giving compound 36 as yellow amorphous solid (1.15 g (98%)),
which was used without further purification.
NMR data were in accordance with literature data [41].
N’-Hydroxy-2-(hydroxyimino)-2-phenylethanimidamide (37): Oxime 36 (0.88 g (6.00 mmol))
was dissolved in methanol (18 mL) and a solution of NH2OH
×
HCl (0.50 g (7.20 mmol))
and NaHCO3 (0.61 g (7.20)) in water (9 mL) was added. The mixture was refluxed for
20 h and then the solvent was evaporated in vacuo. The residue was mixed with water
(20 mL) and extracted with ethyl acetate. The organic phases were combined, dried over
anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo, giving the
raw amidoxime which was recrystallized in CH2Cl2 affording compound 37 as white
crystals (0.25 g (23%)).
NMR data were in accordance with literature data [42].
4-Phenyl-1,2,5-oxadiazol-3-amine (38): Amidoxime 37 (0.54 g (3.00 mmol)) was dissolved
in 2N NaOH (30 mL) and refluxed for 20 h. The formed precipitate was filtered, washed
with water and dried. The white precipitate was pure product 38 (0.17 g (36%)) and was
used without further purification.
NMR data were in accordance with literature data [43].
The general procedure for the synthesis of carboxylic acids (20, 21, 24 and 25) is as
follows: To a solution of the respective methyl ester (3.00◦mmol) in methanol (10 mL), 2N
NaOH (9.0 mL) was added. The mixture was stirred at 25 C for 20 h and then the reaction
mixture was acidified with conc HCl to a pH of 6. The solvent was evaporated in vacuo
and the residue was mixed with CH2Cl2 (20 mL) and sonicated for 5 min. The suspension
was filtered and the filtrate was evaporated in vacuo to dryness, giving pure carboxylic
acids 20, 21, 24 and 25, which were used without further purification.
3-((Pyrrolidin-1-yl)methyl)benzoic acid (20): The reaction of methyl ester 18 (0.66 g
(3.00 mmol)) and 2N NaOH (9 mL) in methanol (10 mL) gave compound 20 as white foam
(0.17 g (27%)). IR = 2927, 2597, 1613, 1567, 1455, 1381, 1263; 1H NMR (CDCl3, 400 MHz)
δ
= 2.12 (br, s, 4H, (CH2)2), 3.26 (br, 4H, N(CH2)2), 4.20 (br, s, 2H, ArCH2), 7.40 (t, J = 7.7 Hz,
1H, 5-H), 7.55 (br, 1H, 4-H), 8.07 (d, J = 7.7 Hz, 1H, 6-H), 8.50 (br, s, 1H, 2-H); 13C NMR
(CDCl3, 100 MHz) = 23.19 ((CH2)2), 52.71 (N(CH2)2), 58.49 (ArCH2), 128.33 (C-5), 130.39
δ
(C-6), 130.75 (C-1), 132.47 (C-2), 132.55 (C-4), 136.70 (C-3), 170.88 (COOH); HRMS (EI+)
calcd for C12H15NO2: 205.1103; found: 205.1088.

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3-((Morpholin-4-yl)methyl)benzoic acid (21): The reaction of methyl ester 19 (0.71 g
(3.00 mmol)) and 2N NaOH (9 mL) in methanol (10 mL) gave compound 21 as white
amorphous solid (0.64 g (97%)).
NMR data were in accordance with literature data [44].
4-((Pyrrolidin-1-yl)methyl)benzoic acid (24): The reaction of methyl ester 22 (0.66 g
(3.00 mmol)) and 2N NaOH (9 mL) in methanol (10 mL) gave compound 24 as white foam
(0.55 g (90%)).
NMR data were in accordance with literature data [39].
4-((Morpholin-4-yl)methyl)benzoic acid (25): The reaction of methyl ester 23 (0.71 g
(3.00 mmol)) and 2N NaOH (9 mL) in methanol (10 mL) gave compound 25 as yellow oil
(0.62 g (94%)).
NMR data were in accordance with literature data [40].
The general procedure for the synthesis of carboxamides (1, 10–17, 26–29) is detailed
in the following subsections.
3.2.1. Method A
An ice-cooled suspension of NaH (60% dispersion in mineral oil; 2.00 mmol) in dry
DMF (14 mL) was mixed with aminofurazan 9 (1.00 mmol) and stirred for 20 min. Then,
a solution of acid chloride (1.30 mmol) in dry DMF (2 mL) was added dropwise and the
reaction mixture was stirred at 60 ◦C for 20 h. Afterward, the mixture was quenched with
water at 0 ◦C and the aqueous phase was extracted with CH2Cl2. The organic layer was
washed with 8% aq NaHCO3 and brine, dried over anhydrous sodium sulfate and filtered,
and the solvent was evaporated in vacuo, yielding the raw carboxamides
which were further purified by crystallization.
1 and 10–14,
3.2.2. Method B
To an ice-cooled solution of carboxylic acid (1.50 mmol) in dry CH2Cl2 (14 mL), oxalyl
dichloride 2 M in CH2Cl2 (1.90 mmol) was added dropwise under stirring. After 1 h, the
ice bath was removed and the reaction batch was stirred 20 h at 25 ◦C in an atmosphere of
Ar. Subsequently, the solvent was evaporated in vacuo and the crude acyl chloride was
dissolved in dry DMF (9 mL). An ice-cooled suspension of NaH (60% dispersion in mineral
oil; 2.00 mmol) in dry DMF (14 mL) was mixed with aminofurazan
9 (1.00 mmol) and
stirred for 20 min. Then, the solution of acyl chloride in dry DMF was added dropwise and
the reaction mixture was stirred at 60 ◦C for 48 h. Afterward, the mixture was quenched
with water at 0 ◦C and the aqueous phase was extracted with CH2Cl2. The organic layer
was washed with 8% aq NaHCO3 and brine, dried over anhydrous sodium sulfate and
filtered, and the solvent was evaporated in vacuo, yielding the raw carboxamides 16
and 26–29, which were further purified by column chromatography or crystallization.
,
17
3.2.3. Method C
0
A solution of benzoic acid (1.50 mmol), N-hydroxysuccinimide (1.58 mmol) and N,N -
dicyclohexylcarbodiimide (1.50 mmol) was dissolved in dry THF (10 mL) and stirred at
◦
25 C for 20 h. The formed precipitate was filtered and the filtrate was evaporated in vacuo
to dryness to obtain the crude NHS ester which was used without further purification.
An ice-cooled suspension of NaH (60% dispersion in mineral oil; 2.00 mmol) in dry DMF
(14 mL) was mixed with aminofurazan
9 (1.00 mmol) and stirred for 20 min. Then, the
solution of NHS◦ester in dry DMF (4 mL) was added dropwise and the reaction mixture
◦
was stirred at 60 C for 20 h. Afterward, the mixture was quenched with water at 0 C and
the aqueous phase was extracted with CH2Cl2. The organic layer was washed with 8% aq
NaHCO3 and brine, dried over anhydrous sodium sulfate and filtered, and the solvent
was evaporated in vacuo, yielding the raw carboxamide 15, which was further purified
by crystallization.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-methylbenzamide (1): Method A: The re-
action of compound (249 mg (1.00 mmol)), 3-methylbenzoyl chloride (201 mg (1.30 mmol))
9

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and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in dry DMF (16 mL) gave the
raw carboxamide which was purified by recrystallization in CH2Cl2 to yield compound
◦
1
as white crystals (63 mg (17%)). m.P. 162 C. IR = 3198, 2981, 1665, 1591, 1531, 1500,
1394, 1377, 1325, 1277, 1260, 1218, 1142, 1042, 940, 862, 810; 1H NMR (DMSO-d6, 400 MHz)
δ
= 1.20 (t, J = 6.9 Hz, 3H, CH3), 1.32 (t, J = 6.9 Hz, 3H, CH3), 2.40 (s, 3H, ArCH3), 3.90
(q, J = 6.9 Hz, 2H, OCH2), 4.05 (q, J = 6.9 Hz, 2H, OCH2), 7.07 (d, J = 8.4 hz, 1H, 5”-H),
7.32-7.37 (m, 2H, 2”-H, 6”-H), 7.45 (t, J = 7.6, 1H, 5-H), 7.49 (d, J = 7.6 Hz, 1H, 4-H), 7.79
(br, d, J = 7.4 hz, 1H, 6-H), 7.82 (br, s, 1H, 2-H), 11.17 (br, 1H, NH); 13C NMR (DMSO-d6,
100 MHz) δ = 14.47 (CH3), 14.56 (CH3), 20.86 (ArCH3), 63.79 (2 OCH2), 111.68 (C-2”), 113.21
(C-5”), 116.92 (C-1”), 120.44 (C-6”), 125.17 (C-6), 128.52 (C-2), 128.61 (C-5), 132.22 (C-1),
133.41 (C-4), 138.13 (C-3), 148.10 (C-3”), 150.06 (C-3’), 150.33 (C-4”), 151.23 (C-4’), 166.78
(C=O); HRMS (EI+) calcd for C20H21N3O4: 367.1532; found: 367.1526.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)benzamide (10): Method A: The reaction
of compound
9 (249 mg (1.00 mmol)), benzoyl chloride (183 mg (1.30 mmol)) and NaH
(60% dispersion in mineral oil) (80 mg (2.00 mmol)) in dry DMF (16 mL) gave the raw
carboxamide which was purified by recrystallization in CH2Cl2 to yield compound 10 as
◦
white crystals (81 mg (23%)). m.P. 199 C. IR = 3212, 2980, 1660, 1591, 1530, 1501, 1468,
1395, 1378, 1324, 1275, 1258, 1216, 1141, 1040, 916, 852, 811; 1H NMR (DMSO-d6, 400 MHz)
δ
= 1.19 (t, J = 6.9 Hz, 3H, CH3), 1.31 (t, J = 6.9 Hz, 3H, CH3), 3.88 (q, J = 7.0 Hz, 2H, OCH2),
4.05 (q, J = 6.9 Hz, 2H, OCH2), 7.07 (d, J = 8.4 hz, 1H, 5”-H), 7.31 (d, J = 1.7 Hz, 1H, 2”-H),
7.33 (dd, J = 8.4, 1.7 Hz, 1H, 6”-H), 7.58 (t, J = 7.4 hz, 2H, 3-H, 5-H), 7.68 (t, J = 7.4 hz, 1H,
4-H), 8.00 (d, J = 7.2 Hz, 2H, 2-H, 6-H), 11.24 (br s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz)
δ
= 14.45 (CH3), 14.57 (CH3), 63.78 (2 OCH2), 111.65 (C-2”), 113.19 (C-5”), 116.87 (C-1”),
120.43 (C-6”), 128.05 (C-2, C-6), 128.74 (C-3, C-5), 132.10 (C-1), 132.91 (C-4), 148.11 (C-3”),
149.95 (C-3’), 150.36 (C-4”), 151.24 (C-4’), 166.66 (C=O); HRMS (EI+) calcd for C19H19N3O4:
353.1375; found: 353.1373.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-2-methylbenzamide (11): Method A:
The reaction of compound
9 (249 mg (1.00 mmol)), 2-methylbenzoyl chloride (201 mg
(1.30 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in dry DMF
(16 mL) gave the raw carboxamide which was purified by recrystallization in CH2Cl2
◦
to yield compound 11 as white crystals (103 mg (28%)). m.P. 170 C. IR = 3214, 2983,
1663, 1591, 1503, 1395, 1377, 1324, 1259, 1216, 1142, 1042, 918, 854, 803, 741, 687, 657,
618; 1H NMR (DMSO-d6, 400 MHz)
δ = 1.28 (t, J = 7.0 Hz, 3H, CH3), 1.34 (t, J = 7.0 Hz,
3H, CH3), 2.34 (s, 3H, ArCH3), 3.99 (q, J = 7.0 Hz, 2H, OCH2), 4.08 (q, J = 7.0 Hz, 2H,
OCH2), 7.11 (d, J = 8.4 hz, 1H, 5”-H), 7.33–7.37 (m, 4H, 2”-H, 3-H, 5-H, 6”-H), 7.45 (t,
J = 7.3 Hz, 1H, 4-H), 7.65 (d, J = 7.3 Hz, 1H, 6H), 11.12 (br, 1H, NH); 13C NMR (DMSO-d6,
100 MHz)
113.13 (C-5”), 116.96 (C-1”), 120.67 (C-6”), 125.79 (C-5), 127.89 (C-6), 130.92 (C-4), 131.10
(C-3), 133.97 (C-1), 136.63 (C-2), 148.21 (C-3”), 149.83 (C-3 ), 150.34 (C-4”), 151.22 (C-4 ),
168.70 (C=O); HRMS (EI+) calcd for C19H21N3O4: 367.1532; found: 367.1526.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-2-(trifluoromethyl)benzamide (12): Method
A: The reaction of compound (249 mg (1.00 mmol)), 2-(trifluoromethyl)benzoyl chloride
δ = 14.61 (2 CH3), 19.56 (ArCH3), 63.84 (OCH2), 63.97 (OCH2), 112.06 (C-2”),
´
´
9
(271 mg (1.30 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in
dry DMF (16 mL) gave the raw carboxamide which was purified by recry◦stallization in
methanol to yield compound 12 as white crystals (38 mg (9%)). m.P. 171 C. IR = 3209,
2983, 1676, 1604, 1531, 1494, 1397, 1377, 1316, 1277, 1217, 1177, 1129, 1043, 921, 854, 811,
1
772, 651, 617; H NMR (DMSO-d6, 400 MHz)
δ = 1.32 (t, J = 7.0 Hz, 3H, CH3), 1.34 (t,
J = 7.0 Hz, 3H, CH3), 4.05 (q, J = 7.0 Hz, 2H, OCH2), 4.09 (q, J = 7.0 Hz, 2H, OCH2), 7.13
(d, J = 8.2 Hz, 1H, 5”-H), 7.34 (d, J = 1.8 Hz, 1H, 2”-H), 7.36 (dd, J = 8.2, 1.8 Hz, 1H, 6”-H),
7.76-7.84 (m, 3H, 4-H, 5-H, 6-H), 7.89 (d, J = 8.2 Hz, 1H, 3-H), 11.46 (br, 1H, NH); 13C NMR
(DMSO-d6, 100 MHz)
δ = 14.61 (CH3), 14.64 (CH3), 63.84 (OCH2), 63.96 (OCH2), 112.37
(C-2”), 113.09 (C-5”), 116.52 (C-1”), 120.94 (C-6”), 123.52 (q, J = 274 hz, CF3), 126.25 (q,
J = 31.7 Hz, C-2), 126.71 (q, J = 4.9 Hz, C-3), 128.69 (C-6), 131.05 (C-4), 132.69 (C-5), 134.03

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(m, C-1), 148.23 (C-3”), 149.00 (C-3’), 150.41 (C-4”), 150.72 (C-4’), 166.47 (C=O); HRMS (EI+)
calcd for C20H18F3N3O4: 421.1249; found: 421.1248.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-(trifluoromethyl)benzamide (13): Method
A: The reaction of compound
9 (249 mg (1.00 mmol)), 3-(trifluoromethyl)benzoyl chloride
(271 mg (1.30 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in dry
DMF (16 mL) gave the raw carboxamide which was purified by recrystallization in CH2Cl2
to yield compound 13 as white crystals (131 mg (31%)). m.P. 153 ◦C. IR = 3203, 1675, 1498,
1
1378, 1336, 1260, 1217, 1172, 1121, 1042, 815; H NMR (DMSO-d6, 400 MHz)
δ = 1.19 (t,
J = 7.0 Hz, 3H, CH3), 1.32 (t, J = 7.0 Hz, 3H, CH3), 3.90 (q, J = 7.0 Hz, 2H, OCH2), 4.06 (q,
J = 7.0 Hz, 2H, OCH2), 7.08 (d, J =8.3 Hz, 1H, 5”-H), 7.33 (d, J = 1.9 Hz, 1H, 2”-H), 7.34
(dd, J = 8.3, 1.9 Hz, 1H, 6”-H), 7.84 (t, J = 7.7 Hz, 1H, 5-H), 8.06 (d, J = 7.7 Hz, 1H, 4-H),
8.29 (d, J = 7.7 Hz, 1H, 6-H), 8.36 (s, 1H, 2-H), 11.53 (br, 1H, NH); 13C NMR (DMSO-d6,
100 MHz) δ = 14.40 (CH3), 14.56 (CH3), 63.77 (OCH2), 63.81 (OCH2), 111.72 (C-2”), 113.24
(C-5”), 116.83 (C-1”), 120.59 (C-6”), 123.82 (q, J = 272 Hz, CF3), 124.66 (q, J = 3.9 Hz, C-2),
129.33 (q, J = 4.4 hz, C-4), 129.44 (q, J = 32.2 Hz, C-3), 130.18 (C-5), 132.27 (C-6), 133.21 (C-1),
148.11 (C-3”), 149.80 (C-3’), 150.36 (C-4”), 151.08 (C-4’), 165.25 (C=O); HRMS (EI+) calcd for
C20H18F3N3O4: 421.1249; found: 421.1242.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-fluorobenzamide (14): Method A: The re-
action of compound
9 (249 mg (1.00 mmol)), 3-fluorobenzoyl chloride (206 mg (1.30 mmol))
and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in dry DMF (16 mL) gave the
raw carboxamide which was purified by recrystallization in CH2Cl2 to yield compound 14
as beige crystals (186 mg (50%)). m.P. 250 ◦C. IR = 3216, 2983, 1663, 1591, 1505, 1364, 1275,
1215, 1142, 1039, 880, 857, 809; 1H NMR (DMSO-d6, 400 MHz)
δ = 1.27 (t, J = 7.0 Hz, 3H,
CH3), 1.33 (t, J = 7.0 Hz, 3H, CH3), 3.99 (q, J = 7.0 Hz, 2H, OCH2), 4.07 (q, J = 7.0 Hz, 2H,
OCH2), 7.06 (d, J =8.4 hz, 1H, 5”-H), 7.32 (td, J = 8.4, 2.1 Hz, 1H, 4-H), 7.48 (ddd, J = 8.4, 7.6,
5.7 Hz, 1H, 5-H), 7.63 (dd, J = 8.3, 1.4 hz, 1H, 6”-H), 7.81-7.87 (m, 2H, 2-H, 2”-H), 7.93 (d,
J = 7.6 Hz, 1H, 6-H); 13C NMR (DMSO-d6, 100 MHz)
δ = 14.60 (CH3), 14.66 (CH3), 63.76
(2 OCH2), 112.34 (C-2”), 113.08 (C-5”), 114.69 (d, J = 22.1 Hz, C-2), 117.25 (d, J = 21.2 Hz,
C-4), 119.08 (C-1”), 120.60 (C-6”), 124.25 (d, J = 2.7 Hz, C-6), 129.87 (d, J = 7.9 Hz, C-5),
140.35 (C-1), 147.84 (C-3”), 149.66 (C-4”), 150.19 (C-4’), 155.35 (C-3’), 161.94 (d, J = 242 Hz,
C-3), 166.84 (C=O); HRMS (EI+) calcd for C19H18FN3O4: 371.1261; found: 371.1276.
3-Chloro-N-(4-(3,4-diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-4-methoxybenzamide (16): Method
B: The reaction of 3-chloro-4-methoxybenzoic acid (280 mg (1.50 mmol)) and oxalyl dichlo-
ride 2 M in CH2Cl2 (0.95 mL (1.90 mmol)) in dry CH2Cl2 (14 mL) gave the raw acid
chloride which was suspended in dry DMF (9 mL) and added to a suspension of com-
pound 9 (249 mg (1.00 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol))
in dry DMF (14 mL), giving the raw carboxamide which was purified by recrystallization
in toluene to yield compound 16 as white crystals (255 mg (61%)). m.P. 260 ◦C (decomp.).
IR = 2978, 1663, 1600, 1501, 1470, 1395, 1360, 1271, 1215, 1142, 1057, 1040, 937, 855, 811; 1H
NMR (DMSO-d6, 400 MHz) δ = 1.30 (t, J = 7.0 Hz, 3H, CH3), 1.34 (t, J = 7.0 Hz, 3H, CH3),
3.91 (s, 3H, OCH3), 4.02 (q, J = 7.0 Hz, 2H, OCH2), 4.07 (q, J = 7.0 Hz, 2H, OCH2), 7.06
(d, J = 8.4 hz, 1H, 5”-H), 7.18 (d, J = 8.7 Hz, 1H, 5-H), 7.65 (dd, J = 8.4, 1.6 Hz, 1H, 6”-H),
7.92 (d, J = 1.6 Hz, 1H, 2”-H), 8.05 (dd, J = 8.7, 1.9 Hz, 1H, 6-H), 8.17 (d, J = 1.6 Hz, 1H,
2-H), 11.27 (br, 1H, NH); 13C NMR (DMSO-d6, 100 MHz)
δ = 14.69 (2 CH3), 56.27 (OCH3),
63.72 (2 OCH2), 111.80 (C-5), 112.33 (C-2”), 112.99 (C-5”), 119.43 (C-1”), 120.18 (C-3), 120.55
(C-6”), 128.55 (C-6), 129.79 (C-2), 131.72 (C-1), 147.76 (C-3”), 149.48 (C-4”), 150.09 (C-4’),
153.07 (C-3’), 156.03 (C-4), 166.89 (C=O); HRMS (EI+) calcd for C20H20ClN3O5: 417.1092;
found: 417.1081.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-fluoro-4-methoxybenzamide (17): Method
B: The reaction of 3-fluoro-4-methoxybenzoic acid (255 mg (1.50 mmol)) and oxalyl dichlo-
ride 2 M in CH2Cl2 (0.95 mL (1.90 mmol)) in dry CH2Cl2 (14 mL) gave the raw acid
chloride which was suspended in dry DMF (9 mL) and added to a suspension of com-
pound
9 (249 mg (1.00 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol))
in dry DMF (14 mL), giving the raw carboxamide which was purified by recrystallization

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in toluene to yield compound 17 as beige crystals (124 mg (31%)). m.P. 232 ◦C (decomp.).
IR = 3199, 2982, 1663, 1615, 1500, 1469, 1396, 1377, 1327, 1286, 1217, 1140, 1040, 853, 802; 1H
NMR (DMSO-d6, 400 MHz) δ = 1.25 (t, J = 6.9 Hz, 3H, CH3), 1.33 (t, J = 6.9 Hz, 3H, CH3),
3.92 (s, 3H, OCH3), 3.96 (q, J = 6.9 Hz, 2H, OCH2), 4.07 (q, J = 6.9 Hz, 2H, OCH2), 7.06 (d,
J = 8.6 Hz, 1H, 5”-H), 7.27 (t, J = 8.7 Hz, 1H, 5-H), 7.51 (dd, J = 8.4, 2.0 Hz, 1H, 6”-H), 7.62
(d, J = 2.0 Hz, 1H, 2”-H), 7.89 (d, J = 8.3 Hz, 1H, 6-H), 7.90 (s, 1H, 2-H), 11.29 (br, 1H, NH);
13C NMR (DMSO-d6, 100 MHz)
δ = 14.55 (CH3), 14.63 (CH3), 56.23 (d, J = 2.3 Hz, OCH3),
63.74 (2 OCH2), 112.02 (C-2”), 113.06 (C-5, C-5”), 115.54 (d, J = 19.0 Hz, C-2), 118.23 (C-1”),
120.50 (C-6”), 125.30 (d, J = 3.1 Hz, C-6), 128.22 (C-1), 147.90 (C-3”), 149.86 (d, J = 23.6 Hz,
C-4), 149.89 (C-4”), 150.61 (C-4’), 150.76 (d, J = 244 hz, C-3), 153.19 (C-3’), 166.11 (C=O);
HRMS (EI+) calcd for C20H20FN3O5: 401.1387; found: 401.1380.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-4-((pyrrolidin-1-yl)methyl)benzamide (26):
Method B: The reaction of carboxylic acid 20 (308 mg (1.50 mmol)) and oxalyl dichloride
2 M in CH2Cl2 (0.95 mL (1.90 mmol)) in dry CH2Cl2 (14 mL) gave the raw acid chloride
which was suspended in dry DMF (9 mL) and added to a suspension of compound
9
(249 mg (1.00 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in
dry DMF (14 mL), giving the raw carboxamide which was purified by recrystallizati◦on
in toluene to yield compound 26 as light yellow crystals (140 mg (32%)). m.P. 251 C
(decomp.). IR = 2973, 2788, 1584, 1471, 1365, 1271, 1246, 1201, 1141, 1091, 1038, 997, 937, 880,
854, 809; 1H NMR (DMSO-d6, 400 MHz)
δ = 1.32 (t, J = 7.0 Hz, 3H, CH3), 1.35 (t, J = 7.0 Hz,
3H, CH3), 1.69 (br, s, 4H, (CH2)2), 2.43 (br, s, 4H, N(CH2)2), 3.59 (s, 2H, ArCH2), 4.05 (q,
J = 7.0 Hz, 2H, OCH2), 4.08 (q, J = 7.0 Hz, 2H, OCH2), 7.07 (d, J = 8.5 Hz, 1H, 5”-H), 7.27
(d, J = 7.8 Hz, 2H, 3-H, 5-H), 7.92 (dd, J = 7.8, 1.9 Hz, 1H, 6”-H), 8.11 (d, J = 7.8 Hz, 2H,
2-H, 6-H), 8.24 (d, J = 1.9 Hz, 1H, 2”-H); 13C NMR (DMSO-d6, 100 MHz)
δ = 14.74 (CH3),
14.79 (CH3), 23.14 ((CH2)2), 53.54 (N(CH2)2), 59.54 (ArCH2), 63.68 (2 OCH2), 112.67 (C-2”),
112.86 (C-5”), 120.64 (C-1”), 120.72 (C-6”), 127.37 (C-3, C-5), 128.24 (C-2, C-6), 139.78 (C-1),
140.34 (C-4), 147.60 (C-3”), 149.12 (C-4”), 149.56 (C-4’), 158.93 (C-3’), 169.56 (C=O); HRMS
(EI+) calcd for C24H28N4O4: 436.2111; found: 436.2102.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-4-((morpholin-4-yl)methyl)benzamide (27):
Method B: The reaction of carboxylic acid 25 (332 mg (1.50 mmol)) and oxalyl dichloride
2 M in CH2Cl2 (0.95 mL (1.90 mmol)) in dry CH2Cl2 (14 mL) gave the raw acyl chloride
which was suspended in dry DMF (9 mL) and added to a suspension of compound
9
(249 mg (1.00 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in
dry DMF (14 mL), giving the raw carboxamide which was purified by recrystallization in
◦
toluene to yield compound 27 as pale yellow crystals (258 mg (57%)). m.P. 269 C (decomp.).
IR = 2977, 1585, 1499, 1472, 1360, 1271, 1202, 1141, 1109, 1035, 867; 1H NMR (DMSO-d6,
400 MHz)
δ = 1.32 (t, J = 7.0 Hz, 3H, CH3), 1.35 (t, J = 7.0 Hz, 3H, CH3), 2.36 (br, s, 4H,
N(CH2)2), 3.49 (s, 2H, ArCH2), 3.58 (t, J = 4.5 Hz, 4H, O(CH2)2), 4.05 (q, J = 7.0 Hz, 2H,
OCH2), 4.08 (q, J = 7.0 Hz, 2H, OCH2), 7.07 (d, J = 8.6 Hz, 1H, 5”-H), 7.28 (d, J = 8.2 Hz,
2H, 3-H, 5-H), 7.92 (dd, J = 8.6, 1.6 Hz, 1H, 6”-H), 8.12 (d, J = 7.8 Hz, 2H, 2-H, 6-H), 8.23 (d,
J = 1.6 Hz, 1H, 2”-H); 13C NMR (DMSO-d6, 100 MHz)
δ = 14.75 (CH3), 14.79 (CH3), 53.22
(N(CH2)2), 62.36 (ArCH2), 63.69 (2 OCH2), 66.24 (O(CH2)2), 112.66 (C-2”), 112.86 (C-5”),
120.64 (C-1”), 120.73 (C-6”), 127.86 (C-3, C-5), 128.28 (C-2, C-6), 138.60 (C-1), 140.08 (C-4),
147.61 (C-3”), 149.12 (C-4”), 149.56 (C-4’), 158.97 (C-3’), 169.47 (C=O); HRMS (EI+) calcd for
C24H28N4O5: 452.2060; found: 452.2043.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-((pyrrolidin-1-yl)methyl)benzamide (28):
Method B: The reaction of carboxylic acid 20 (308 mg (1.50 mmol)) and oxalyl dichloride
2 M in CH2Cl2 (0.95 mL (1.90 mmol)) in dry CH2Cl2 (14 mL) gave the raw acid chloride
which was suspended in dry DMF (9 mL) and added to a suspension of compound
9
(249 mg (1.00 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in dry
DMF (14 mL), giving the raw carboxamide which was purified by column chromatography
(silica gel, CH2Cl2/toluene/MeOH, 20:1:1) to yield compound 28 as yellow oil (262 mg
(60%)). IR = 3212, 2974, 2799, 1661, 1591, 1500, 1473, 1395, 1377, 1259, 1217, 1140, 1041,
849, 800; 1H NMR (CDCl3, 400 MHz)
δ = 1.40 (t, J = 7.0 Hz, 3H, CH3), 1.46 (t, J = 7.0 Hz,

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3H, CH3), 1.78–1.81 (m, 4H, (CH2)2), 2.52–2.56 (m, 4H, N(CH2)2), 3.69 (s, 2H, ArCH2), 4.04
(q, J = 7.0 Hz, 2H, OCH2), 4.11 (q, J = 7.0 Hz, 2H, OCH2), 6.91 (d, J = 8.2 Hz, 1H, 5”-H),
7.23 (dd, J = 8.2, 1.9 Hz, 1H, 6”-H), 7.25 (d, J = 1.9 Hz, 1H, 2”-H), 7.42 (t, J = 7.7 Hz, 1H,
5-H), 7.57 (d, J = 7.6 Hz, 1H, 4-H), 7.75 (d, J = 7.8 Hz, 1H, 6-H), 7.87 (s, 1H, 2-H); 13C
NMR (CDCl3, 100 MHz)
δ = 14.64 (CH3), 14.65 (CH3), 23.42 ((CH2)2), 54.20 (N(CH2)2),
60.09 (ArCH2), 64.44 (OCH2), 64.58 (OCH2), 112.36 (C-2”), 112.94 (C-5”), 117.28 (C-1”),
120.33 (C-6”), 126.40 (C-6), 127.82 (C-2), 128.92 (C-5), 132.39 (C-1), 133.45 (C-4), 140.34 (C-3),
148.88 (C-3’), 149.08 (C-3”), 149.83 (C-4’), 150.82 (C-4”), 165.69 (C=O); HRMS (EI+) calcd for
C24H28N4O4: 436.2111; found: 436.2086.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-((morpholin-4-yl)methyl)benzamide (29):
Method B: The reaction of carboxylic acid 21 (332 mg (1.50 mmol)) and oxalyl dichloride
2 M in CH2Cl2 (0.95 mL (1.90 mmol)) in dry CH2Cl2 (14 mL) gave the raw acid chloride
which was suspended in dry DMF (9 mL) and added to a suspension of compound
9
(249 mg (1.00 mmol)) and NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in
dry DMF (14 mL), giving the raw carboxamide which was purified by recrystallization in
◦
toluene to yield compound 29 as white crystals (113 mg (25%)). m.P. 199 C. IR = 2978, 1605,
1502, 1468, 1361, 1271, 1209, 1141, 1114, 1036, 854; 1H NMR (CD3OD, 400 MHz)
δ
= 1.28
(t, J = 7.0 Hz, 3H, CH3), 1.40 (t, J = 7.0 Hz, 3H, CH3), 2.48 (br, s, 4H, N(CH2)2), 3.58 (s, 2H,
ArCH2), 3.70 (t, J = 4.5 Hz, 4H, O(CH2)2), 3.94 (q, J = 7.0 Hz, 2H, OCH2), 4.09 (q, J = 7.0 Hz,
2H, OCH2), 6.98 (d, J = 8.3 Hz, 1H, 5”-H), 7.36 (t, J = 7.7 Hz, 1H, 5-H), 7.43 (d, J = 7.7 Hz, 1H,
4-H), 7.64 (dd, J = 8.3, 1.6 Hz, 1H, 6”-H), 7.72 (d, J = 1.6 Hz, 1H, 2”-H), 8.00 (d, J = 7.7 Hz,
1H, 6-H), 8.03 (s, 1H, 2-H); 13C NMR (CD3OD, 100 MHz)
δ = 15.23 (2 CH3), 54.77 (N(CH2)2),
64.49 (ArCH2), 65.72 (OCH2), 65.76 (OCH2), 67.92 (O(CH2)2), 114.03 (C-2”), 114.47 (C-5”),
121.81 (C-1”), 122.20 (C-6”), 128.81 (C-6), 128.95 (C-5), 131.01 (C-2), 132.31 (C-4), 137.99
(C-3), 140.85 (C-1), 149.92 (C-3”), 151.48 (C-4”), 151.79 (C-4’), 160.84 (C-3’), 173.71 (C=O);
HRMS (EI+) calcd for C24H28N4O5: 452.2060; found: 452.2042.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-(trifluoromethoxy)benzamide (15): Method
C: The reaction of 3-(trifluoromethoxy)benzoic acid (309 mg (1.50 mmol)), N-hydroxysuccini-
mide (182 mg (1.58 mmol)) and N,N0-dicyclohexylcarbodiimide (310 mg (1.50 mmol)) in
dry THF (10 mL) gave the raw NHS ester. It was suspended in dry DMF (4 mL) and added
to a suspension of compound
9 (249 mg (1.00 mmol)) and NaH (60% dispersion in mineral
oil) (80 mg (2.00 mmol)) in dry DMF (14 mL), giving the raw carboxamide which was
purified by recrystallization in CH2Cl2 to yield compound 15 as white crystals (136 mg
(31%)). m.P. 145–147 ◦C. IR = 3209, 1675, 1590, 1501, 1378, 1260, 1216, 1170, 1042, 809, 633;
1H NMR (DMSO-d6, 400 MHz)
δ = 1.19 (t, J = 7.0 Hz, 3H, CH3), 1.32 (t, J = 7.0 Hz, 3H, CH3),
3.90 (q, J = 7.0 Hz, 2H, OCH2), 4.05 (q, J = 7.0 Hz, 2H, OCH2), 7.06 (d, J = 8.3 Hz, 1H, 5”-H),
7.39–7.44 (m, 2H, 2”-H, 6”-H), 7.63 (d, J = 8.3 Hz, 1H, 4-H), 7.68 (t, J = 7.8 Hz, 1H, 5-H),
7.96 (s, 1H, 2-H), 8.06 (d, J = 7.5 Hz, 1H, 6-H), 11.32 (br, 1H, NH); 13C NMR (DMSO-d6,
100 MHz) δ = 14.45, 14.62 (2 CH3), 63.72, 63.78 (2 OCH2), 111.79 (C-2”), 113.13 (C-5”), 117.50
(C-1”), 120.08 (q, J = 257 Hz, CF3), 120.50 (C-2), 120.56 (C-6”), 124.68 (C-4), 127.27 (C-6),
130.75 (C-5), 136.13 (C-1), 148.01 (C-3”), 148.34 (C-3’), 150.14 (C-4”), 150.84 (C-4’), 151.53
(C-3), 165.46 (C=O); HRMS (EI+) calcd for C20H18F3N3O5: 437.1198; found: 437.1171.
3.2.4. General Procedure for the Synthesis of Compounds 31, 32, 34 and 35
Amino furazan 9 (1.00 mmol) was dissolved in a mixture of dry diethyl ether (4 mL)
and dry benzene (16 mL). Dry pyridine (1.13 mmol) was added and then a solution of
chloroacyl chloride (1.10 mmol) in dry diethyl ether (4 mL) was added dropwise. The
mixture was refluxed for 20 h and then the solvent was evaporated in vacuo. The residue
was mixed with water (35 mL) and the aqueous phase was extracted with CH2Cl2. The
combined organic layers were dried over anhydrous sodium sulfate and filtered, and the
solvent was evaporated in vacuo, yielding the raw carboxamide which was used without
further purification. It was dissolved in dry acetonitrile (20 mL), and the respective amine
(2.00 mmol), K2CO3 (2.00 mmol) and a catalytical amount of NaI (0.65 mmol) were added.
◦
The mixture was stirred at 70 C for 20 h and then cooled to ambient temperature. The sus-

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pension was filtered and the filtrate was evaporated in vacuo to dryness. The residue was
mixed with water (20 mL) and the aqueous phase was extracted with CH2Cl2. The organic
layer was washed with 8% aqueous NaHCO3 and brine, dried over anhydrous sodium
sulfate and filtered, and the solvent was evaporated in vacuo, yielding the carboxamide,
which was purified by column chromatography.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-2-(pyrrolidin-1-yl)acetamide (31): The reac-
tion of amino furazan 9 (249 mg (1.00 mmol)), chloroacetyl chloride (124 mg (1.10 mmol))
and pyridine (89 g (1.13 mmol)) in a mixture of dry diethyl ether (8 mL) and dry ben-
zene (16 mL) gave the raw carboxamide. This was mixed with pyrrolidine (142 mg
(2.00 mmol)), K2CO3 (276 mg (2.00 mmol)) and NaI (97 mg (0.65 mmol)) in dry acetonitrile
(20 mL), giving the tertiary amine which was purified by column chromatography (silica
gel, CH2Cl2/MeOH, 100:1) to yield compound 31 as pale yellow amorphous solid (87 mg
(24%)). IR = 2983, 1726, 1593, 1545, 1519, 1461, 1256, 1216, 1149, 1042, 844, 811; 1H NMR
(CDCl3, 400 MHz) δ = 1.48 (t, J = 7.0 Hz, 3H, CH3), 1.50 (t, J = 7.0 Hz, 3H, CH3), 1.79–1.85
(m, 4H, (CH2)2), 2.68–2.73 (m, 4H, N(CH2)2), 3.38 (s, 2H, CH2CO), 4.13 (q, J = 7.0 Hz, 2H,
OCH2), 4.17 (q, J = 7.0 Hz, 2H, OCH2), 6.97 (d, J = 8.4 hz, 1H, 5”-H), 7.12 (dd, J = 8.4,
2.1 Hz, 1H, 6”-H), 7.22 (d, J = 2.1 Hz, 1H, 2”-H), 9.73 (br, 1H, NH); 13C NMR (CDCl3,
100 MHz) δ = 14.63 (CH3), 14.70 (CH3), 24.04 ((CH2)2), 54.62 (N(CH2)2), 58.76 (CH2), 64.63,
64.88 (2 OCH2), 113.07 (C-5”), 113.13 (C-2”), 117.07 (C-1”), 120.06 (C-6”), 147.54 (C-3’),
148.36 (C-4’), 149.53 (C-3”), 151.07 (C-4”), 168.87 (C=O); HRMS (EI+) calcd for C18H24N4O4:
360.1797; found: 360.1782.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-2-(morpholin-4-yl)acetamide (32): The reac-
tion of amino furazan 9 (249 mg (1.00 mmol)), chloroacetyl chloride (124 mg (1.10 mmol))
and pyridine (89 g (1.13 mmol)) in a mixture of dry diethyl ether (8 mL) and dry benzene
(16 mL) gave the raw carboxamide. It was mixed with morpholine (174 mg (2.00 mmol)),
K2CO3 (276 mg (2.00 mmol)) and NaI (97 mg (0.65 mmol)) in dry acetonitrile (20 mL),
giving the tertiary amine which was purified by column chromatography (silica gel,
CH2Cl2/MeOH, 40:1) to yield compound 32 as white amorphous solid (233 mg (62%)).
IR = 3326, 2982, 1731, 1595, 1542, 1520, 1462, 1328, 1254, 1216, 1146, 1112, 1041, 1009, 867,
1
809; H NMR (CDCl3, 400 MHz)
δ = 1.46–1.53 (m, 6H, 2 CH3), 2.64 (t, J = 4.6 Hz, 4H,
N(CH2)2), 3.24 (s, 2H, 2-H), 3.68 (br, t, J = 4.6 Hz, 4H, O(CH2)2), 4.12–4.20 (m, 4H, 2 OCH2),
7.00 (d, J = 8.4 hz, 1H, 5”-H), 7.11 (dd, J = 8.4, 2.0 Hz, 1H, 6”-H), 7.23 (d, J = 2.0 Hz, 1H,
2”-H), 9.75 (br, s, 1H, NH); 13C NMR (CDCl3, 100 MHz)
δ = 14.62 (CH3), 14.71 (CH3), 53.79
(N(CH2)2), 61.76 (C-2), 64.69, 65.01 (2 OCH2), 66.85 (O(CH2)2), 113.05 (C-5”), 113.57 (C-2”),
116.84 (C-1”), 120.00 (C-6”), 147.42 (C-3’), 148.30 (C-4’), 149.65 (C-3”), 151.29 (C-4”), 167.80
(C=O); HRMS (EI+) calcd for C18H24N4O5: 376.1747; found: 376.1743.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-(pyrrolidin-1-yl)propanamide (34): The
reaction of amino furazan
9 (249 mg (1.00 mmol)), 3-chloropropanoyl chloride (140 mg
(1.10 mmol)) and pyridine (89 g (1.13 mmol)) in a mixture of dry diethyl ether (8 mL) and
dry benzene (16 mL) gave the raw carboxamide. It was mixed with pyrrolidine (142 mg
(2.00 mmol)), K2CO3 (276 mg (2.00 mmol)) and NaI (97 mg (0.65 mmol)) in dry acetonitrile
(20 mL), giving the tertiary amine which was purified by column chromatography (silica
gel, CH2Cl2/MeOH, 15:1) to yield compound 34 as yellow amorphous solid (300 mg (80%)).
1
IR = 2977, 1719, 1551, 1476, 1434, 1396, 1323, 1274, 1250, 1217, 1149, 1043, 916, 845; H
NMR (CDCl3, 400 MHz) δ = 1.46–1.51 (m, 6H, 2 CH3), 1.53–1.57 (m, 4H, (CH2)2), 2.51–2.56
(m, 4H, N(CH2)2), 2.63 (t, J = 5.9 Hz, 2H, 2-H), 2.82 (t, J = 5.9 Hz, 2H, 3-H), 4.10–4.18 (m,
4H, 2 OCH2), 6.96 (d, J = 8.3 Hz, 1H, 5”-H), 7.11 (dd, J = 8.3, 1.8 Hz, 1H, 6”-H), 7.19 (d,
J = 1.8 Hz, 1H, 2”-H), 12.02 (br, 1H, NH); 13C NMR (CDCl3, 100 MHz)
δ = 14.68 (CH3),
14.72 (CH3), 23.35 ((CH2)2), 34.06 (C-2), 51.07 (C-3), 53.08 (N(CH2)2), 64.89, 64.65 (2 OCH2),
112.95 (C-5”), 113.50 (C-2”), 117.36 (C-1”), 120.46 (C-6”), 148.42 (C-3’), 148.71 (C-4’), 149.27
(C-3”), 150.88 (C-4”), 170.64 (C=O); HRMS (EI+) calcd for C19H26N4O4: 374.1954; found:
374.1973.
N-(4-(3,4-Diethoxyphenyl)-1,2,5-oxadiazol-3-yl)-3-(morpholin-4-yl)propanamide (35): The
reaction of amino furazan
9 (249 mg (1.00 mmol)), 3-chloropropanoyl chloride (140 mg

Pharmaceuticals 2021, 14, 412
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(1.10 mmol)) and pyridine (89 g (1.13 mmol)) in a mixture of dry diethyl ether (8 mL) and
dry benzene (16 mL) gave the raw carboxamide. It was mixed with morpholine (174 mg
(2.00 mmol)), K2CO3 (276 mg (2.00 mmol)) and NaI (97 mg (0.65 mmol)) in dry acetonitrile
(20 mL), giving the tertiary amine which was purified by column chromatography (silica
gel, CH2Cl2/MeOH, 40:1) to yield compound 35 as white amorphous solid (223 mg (57%)).
1
IR = 3205, 2978, 1677, 1537, 1472, 1375, 1274, 1215, 1142, 1119, 1042, 870, 814; H NMR
(CDCl3, 400 MHz) δ = 1.48 (q, J = 6.9 Hz, 6H, 2 CH3), 2.49 (br, t, J = 4.6 Hz, 4H, N(CH2)2),
2.64 (t, J = 5.7 Hz, 2H, 2-H), 2.72 (t, J = 5.7 Hz, 2H, 3-H), 3.36–3.41 (m, 4H, O(CH2)2),
4.10–4.17 (m, 4H, 2 OCH2), 6.96 (d, J = 8.2 Hz, 1H, 5”-H), 7.15 (dd, J = 8.2, 2.1 Hz, 1H, 6”-H),
7.19 (d, J = 2.1 Hz, 1H, 2”-H), 11.28 (br, 1H, NH); 13C NMR (CDCl3, 100 MHz)
δ = 14.62
(CH3), 14.70 (CH3), 31.56 (C-2), 52.70 (N(CH2)2), 53.56 (C-3), 64.63 (OCH2), 64.98 (OCH2),
66.17 (O(CH2)2), 113.09 (C-5”), 113.56 (C-2”), 117.09 (C-1”), 120.83 (C-6”), 148.22 (C-3’),
148.95 (C-4’), 149.28 (C-3”), 151.14 (C-4”), 170.19 (C=O); HRMS (EI+) calcd for C19H26N4O5:
390.1903; found: 390.1892.
3-Methyl-N-(4-phenyl-1,2,5-oxadiazol-3-yl)benzamide (39): Method A: The reaction of
compound 38 (161 mg (1.00 mmol)), 3-methylbenzoyl chloride (201 mg (1.30 mmol)) and
NaH (60% dispersion in mineral oil) (80 mg (2.00 mmol)) in dry DMF (16 mL) gave the
raw carboxamide which was purified by column chromatography (aluminium oxide basic,
cyclohexane/ethyl acetate, 3:1) to yield compound 39 as white amorphous solid (75 mg
1
(27%)). IR = 3288, 1663, 1588, 1566, 1521, 1481, 1383, 1284, 885, 807; H NMR (CDCl3,
400 MHz)
7.45–7.53 (m, 3H, 3”-H, 4”-H, 5”-H), 7.63 (d, J = 7.5 Hz, 1H, 6-H), 7.69 (s, 1H, 2-H), 7.70 (d,
J = 7.5 Hz, 2H, 2”-H, 6”-H), 8.29 (s, 1H, NH); 13C NMR (CDCl3, 100 MHz)
= 21.32 (CH3),
δ = 2.41 (s, 3H, CH3), 7.38 (t, J = 7.5 Hz, 1H, 5-H), 7.42 (d, J = 7.5 Hz, 1H, 4-H),
δ
124.45 (C-6), 125.29 (C-1”), 127.57 (C-2”, C-6”), 128.39 (C-2), 128.88 (C-5), 129.32 (C-3”, C-5”),
130.81 (C-4”), 131.99 (C-1), 133.95 (C-4), 139.13 (C-3), 148.73 (C-3’), 149.94 (C-4’), 165.38
(CO); HRMS (EI+) calcd for C16H13N3O2: 279.1008; found: 279.0997.
3.3. Biological Tests
3.3.1. In Vitro Microplate Assay against P. falciparum
In vitro activity against erythrocytic stages of P. falciparum was determined using a
3H-hypoxanthine incorporation assay [45
,46], using the drug-sensitive NF54 strain [47] or
the chloroquine- and pyrimethamine-resistant K1 strain [48]. Chloroquine (Sigma C6628)
was used as standard. Compounds were dissolved in DMSO at 10 mg/mL and added to
parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented
with HEPES (5.94 g/L), NaHCO3 (2.1 g/L), neomycin (100 U/mL), Albumax (5 g/L) and
washed human red cells A+ at 2.5% hematocrit (0.3% parasitemia). Serial drug dilutions
of 11 3-fold dilution steps covering a range from 100 to 0.002 µg/mL were prepared. The
96-well plates were incubated in a humidified atmosphere at 37 ◦C; 4% CO2, 3% O2, 93%
3
N2. After 48 h, 0.05 mL of H-hypoxanthine (=0.5
µCi) was added to each well of the
plate. The plates were incubated for a further 24 h under the same conditions. The plates
were then harvested with a Betaplate cell harvester (Wallac, Zurich, Switzerland). The red
blood cells were transferred onto a glass fiber filter and washed with distilled water. The
dried filters were inserted into a plastic foil with 10 mL of scintillation fluid and counted
in a Betaplate liquid scintillation counter (Wallac, Zurich, Switzerland). IC50 values were
calculated from sigmoidal inhibition curves by linear regression [49] using Microsoft Excel.
Artemisinin and chloroquine were used as control. Dose-response curves of measured
compounds and activities with standard deviation values are available in Supplementary
Materials Section (Figures S24–S41 and Table S1).
3.3.2. In Vitro Cytotoxicity with L-6 Cells
Assays were performed in 96-well microtiter plates, each well containing 0.1 mL of
RPMI 1640 medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine
serum and 4000 L-6 cells (a primary cell line derived from rat skeletal myoblasts) [50
,51].
Serial drug dilutions of 11 3-fold dilution steps covering a range from 100 to 0.002 g/mL
µ

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were prepared. After 70 h of incubation, the plates were inspected under an inverted
microscope to assure the growth of the controls and sterile conditions. Then, 0.01 mL of
Alamar Blue was then added to each well and the plates incubated for another 2 h. Then,
the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular
Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and
an emission wavelength of 588 nm. The IC50 values were calculated by linear regression [44
]
from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices
Cooperation, Sunnyvale, CA, USA). Podophyllotoxin (Sigma P4405) was used as control.
3.3.3. Parallel Artificial Membrane Permeability Assay (PAMPA)
The PAMPA was performed using a 96-well precoated Corning Gentest PAMPA plate
at a pH of 7.4. The PAMPA plate system consists of a donor plate and an acceptor plate.
When both plates are coupled, each well is divided into two chambers that are separated
by a lipid–oil–lipid trilayer constructed in a porous filter. Stock solutions (10 mM) of each
compound were prepared in DMSO and then further diluted to a concentration of 200 µM
in phosphate-buffered saline (PBS) at pH 7.4. The compound dissolved in PBS was then
added to the donor plate and pure PBS was added to the acceptor plate. Four replicates
of each compound and negative control (PBS) were transferred into different wells of the
donor plate. Both plates were coupled and left at room temperature for 5 h. Then, the plates
were separated, and the solutions of each donor well and acceptor well were transferred
to 96-well UV-Star Microplates (Greiner Bio-One). The UV absorbance of compounds
in donor wells and acceptor wells were analyzed by a SpectraMax M3 UV plate reader
(Molecular Devices). The concentrations were received from a calibration curve for each
substance. The plates were analyzed at a wavelength where the R2 value of the calibration
curve was higher than 0.99 [34]. The effective permeability (Pe) was calculated as shown in
the following Equations (1)–(3):
h
i
c
A(t)
− ln 1 −
cequ
Pe(nm/s) = 107 ∗
(1)
ꢀ
ꢁ
1
1
S ∗
+
∗ t
VD
VA
where:
Pe—effective permeability;
S—filter area (0.3 cm2);
VD—donor well volume;
VA—acceptor well volume;
t—incubation time (14,400 s);
cA(t)—concentration of compound in acceptor well at time t;
c
equ—equilibrium concentration.
[cD(t) ∗ VD + cA(t) ∗ VA]
(VD + VA)
cequ
=
(2)
where:
cD(t) —concentration of compound in donor well at time t.
Recovery of compounds from donor and acceptor wells (mass retention) was calculated
as shown in the equation below. Data were only accepted when recovery exceeded 70%.
[cD(t) ∗ VD + cA(t) ∗ VA]
R = 1 −
(3)
(c0 ∗ VD)
where:
R—mass retention (%);
cD(t)—concentration of compound in donor well at time t;
cA(t)—concentration of compound in acceptor well at time t;
c0—initial concentration of compound in donor well;

Pharmaceuticals 2021, 14, 412
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VD—donor well volume;
VA—acceptor well volume.
3.3.4. Ligand Efficiency (LE)
Ligand efficiency was calculated as shown in the following Equation (4) [33]:
1.37
HA
LE =
∗ pIC50
(4)
where:
LE—ligand efficiency;
HA—number of heavy atoms;
pIC50—negative logarithm of IC50.
4. Conclusions
This paper deals with the synthesis and antiplasmodial activities of a series of new
4-substituted N-acyl derivatives of 1,2,5-oxadiazol-3-amines. The substitution of the phenyl
ring in ring position 4 of the furazan ring has a remarkable impact on the antiplasmodial
activity. A compound with an unsubstituted ring was practically ineffective against both
strains of P. falciparum, whereas its 3,4-diethoxy analog was active in low nanomolar
concentration. Moreover, the activity strongly depended on the nature of the acyl moiety.
Benzoyl derivatives were much more active than their alkanoyl analogs. Substitution of
the phenyl ring strongly influenced the activity depending on the substituent and its ring
position (Scheme 6). The most promising derivative 13 with a 3-(trifluoromethyl) group was
highly active against the chloroquine-sensitive strain NF54 but even more active against
the multiresistant K1 strain of P. falciparum. Like artemisinin, it possessed activity against
this strain in low nanomolar concentration. Compared to the parent compound, it showed
improved permeability. Further investigations should reveal if the (3,4-dimethoxyphenyl)
substitution is already the optimum in ring position 4 of the furazan ring.
Scheme 6. Summary of SARs of benzoyl derivatives.

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Supplementary Materials: The following materials are available online at https://www.mdpi.com/
article/10.3390/ph14050412/s1. Figures S1–S23. 1H- and 13C-NMR spectra for compounds
6
,
2
,
8
1
,
,
9
,
20
,
1
,
10
–
17
,
26
–
29
,
31
,
32
35 and 39 against P.f. NF54, P.f. K1 and L-6 cells. Table S1
10 17, 26 29 31 32 34 35 and 39 against P. falciparum
NF54, P. falciparum K1 and L-6 cells, expressed as IC50 (µM).
, 34, 35 and 39. Figures S24–S41. Dose-response curves of compounds
10
–
17,
26 29
–
,
31
,
32,
34
,
. Activities with
standard deviation values of compounds
1,
–
–
,
,
,
,
Author Contributions: Conceptualization, P.H. and R.W.; investigation, T.H., P.H., J.D., W.S., R.S.,
M.K., P.M. and R.W.; methodology T.H. and P.H; data curation, T.H., P.H., J.D., W.S., R.S., M.K., P.M.
and R.W.; writing—original draft preparation, T.H., P.H. and R.W.; writing—review and editing, T.H.,
P.H. and R.W.; supervision, R.W.; project administration, P.H. and R.W. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this article.
Acknowledgments: The authors acknowledge Open Access Funding by the University of Graz.
Conflicts of Interest: The authors declare no conflict of interest.
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