Synthesis, immunosuppressive properties, and mechanism of action of a new isoxazole derivative

Article abstract of DOI:10.3390/molecules23071545

This work describes the synthesis of a new series of isoxazole derivatives, their immunosuppressive properties, and the mechanism of action of a representative compound. A new series of N-substituted derivatives of 5-amino-N,3- dimethyl-1,2-oxazole-4-carbohydrazide (MM1–MM10) was synthesized in reaction of 5-amino- N,3-dimethyl-1,2- oxazole-4- carbohydrazide with relevant carbonyl compounds. The isoxazole derivatives were tested in several in vitro models using human cells. The compounds inhibited phytohemagglutinin A (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMCs) to various degrees. The toxicity of the compounds with regard to a reference A549 cell line was also differential. 5-amino-N-(2,4-dihydroxyphenyl) methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM3) compound was selected for further investigation because of its lack of toxicity and because it had the strongest antiproliferative activity. The compound was shown to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF α) production in human whole blood cell cultures. In the model of Jurkat cells, MM3 elicited strong increases in the expression of caspases, Fas, and NF-κB1, indicating that a proapoptotic action may account for its immunosuppressive action in the studied models.

Full text of DOI:10.3390/molecules23071545

molecules
Article
Synthesis, Immunosuppressive Properties,
and Mechanism of Action of a New Isoxazole Derivative
Marcin Ma˛czyn´ski 1,*, Sylwia Borska 2, Katarzyna Mieszała 2, Maja Kocie˛ba 3, Ewa Zaczyn´ska 3,
Iwona Kochanowska 3 and Michał Zimecki 3
1
Faculty of Pharmacy, Department of Organic Chemistry, Wroclaw Medical University, Borowska 211A,
50-556 Wroclaw, Poland
2
Faculty of Medicine, Histology and Embryology Division, Wroclaw Medical University, Chalubinskiego 6a,
50-368 Wroclaw, Poland; sylwia.borska@umed.wroc.pl (S.B.); katarzynamieszala@wp.pl (K.M.)
Institute of Immunology and Experimental Therapy, Laboratory of Immunobiology, Weigla 12,
3
53-114 Wroclaw, Poland; kocieba@iitd.pan.wroc.pl (M.K.); ezacz@iitd.pan.wroc.pl (E.Z.);
kochanie@iitd.pan.wroc.pl (I.K.); zimecki@iitd.pan.wroc.pl (M.Z.)
*
Correspondence: marcin.maczynski@umed.wroc.pl; Tel.: +48-71-7840344
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 June 2018; Accepted: 26 June 2018; Published: 26 June 2018
Abstract: This work describes the synthesis of a new series of isoxazole derivatives,
their immunosuppressive properties, and the mechanism of action of a representative compound.
A new series of N0-substituted derivatives of 5-amino-N,3- dimethyl-1,2-oxazole-4-carbohydrazide
(MM1–MM10) was synthesized in reaction of 5-amino- N,3-dimethyl-1,2- oxazole-4- carbohydrazide
with relevant carbonyl compounds. The isoxazole derivatives were tested in several in vitro models
using human cells. The compounds inhibited phytohemagglutinin A (PHA)-induced proliferation
of peripheral blood mononuclear cells (PBMCs) to various degrees. The toxicity of the compounds
with regard to a reference A549 cell line was also differential. 5-amino-N0-(2,4-dihydroxyphenyl)
methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM3) compound was selected for further
investigation because of its lack of toxicity and because it had the strongest antiproliferative activity.
The compound was shown to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF
production in human whole blood cell cultures. In the model of Jurkat cells, MM3 elicited strong
increases in the expression of caspases, Fas, and NF- B1, indicating that a proapoptotic action may
α)
κ
account for its immunosuppressive action in the studied models.
Keywords: isoxazole; PBMC; proliferation; TNF α; apoptosis; jurkat cells
1. Introduction
Isoxazoles are an important class of heterocyclic compound, displaying a broad spectrum of
biological activities. Modification in their structures has offered a high degree of diversity that has
proved useful for the development of new therapeutic agents with improved potency and lower toxicity.
Isoxazole derivatives display a wide array of pharmacological activities that have been successfully
screened for anticancer [
antiulcer [ ], antiepileptic [
and anxiolytic [ ] activities. The pharmacological profits of employing the isoxazole ring are due
to the fact that this structure acts as a key pharmacophore for the biological activity of such drugs
as Valdecoxib (COX-2 inhibitor) [ ] and Leflunomide (antirheumatic drug) [10]. The compounds
containing the isoxazole ring are also promising therapeutic agents in neurodegenerative diseases
because of their similarity to the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, which is
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)
1], anti-inflammatory [
2
], antimicrobial [
3
], antihistaminic, antitubercular,
4
5
], dual 2-adrenoreceptor and 5-HTreuptekeinhibitors [
α
6
], antiviral [ ],
7
8
9
α
a specific agonist for the
receptor [11].
α
Molecules 2018, 23, 1545; doi:10.3390/molecules23071545
www.mdpi.com/journal/molecules

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Over the last few decades, non-toxic, low molecular weight, and selectively acting compounds
containing the isoxazole moiety were synthesized by Ryng and Ma˛czyn´ski, and their immunological
activities were reported [12
–
15]. Our previous studies described the immuno-stimulatory [16
,17],
immuno-restituting [18 20], immunosuppressive [13
–
,17,21], and anti-inflammatory [22] activities of
the isoxazole derivatives. Interestingly, isoxazole derivatives differing only in one substituent could
exhibit opposite immunological functions [23]. The best results in designing new immunomodulators
within the isoxazole family are achieved by structural modifications of the previously synthesized
compounds of well-established activity. Interestingly, among all the compounds synthesized by
our team, the modification in position 4 of the 5-amino-3-methyl-4-isoxazolecarboxylic acid led to
unexpected biological activity. The mono-substituted 5-amino-3-methyl-4-isoxazolecarboxylic acid
phenylamides with immune-stimulatory activity equal to or higher than levamisole (a reference drug)
were described [17]. Further characterization of these compounds revealed their very low
toxicity. Compounds substituted in position 4 with a strong electrophilic group were most
active. 4-chlorophenylamide of 5-amino-3-methyl-4-isoxazolecarboxylic acid exerted a strong
stimulating effect on the humoral and cellular immune responses [24–26]. On the other
hand, di- and tri-substituted 5-amino-3-methyl-4-isoxazolecarboxylic acid phenylamides showed
immunosuppressive activity more effectively than cyclosporine A, which was used as a reference drug.
Dichloro- and trimethoxy-derivatives of 5-amino-3-methyl-4-isoxazolecarboxylic acid phenylamides
were inhibitors of the humoral immune response [12,25]. 5-amino-3-methyl-4-isoxazolecarboxylic acid
benzylamides, substituted in the phenyl ring, presented anti-proliferative and anti-inflammatory
properties in vitro and in vivo, in mouse and human models with no apparent toxicity against
these cells. 5-amino-3-methyl-N-(4-methylbenzyl)-4-isoxazolecarboxamide, selected as the most
interesting compound, exhibited strong anti-proliferative and anti-inflammatory properties. On the
other hand, this compound, had the ability to enhance manifestation of the cellular immune
response [27]. 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides
were also synthesized and tested for their immunological activity. The compounds demonstrated
dose-dependent actions ranging from immunosuppressive to immunostimulatory [28
–
30].
0
N -substituted derivatives of 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide exhibited, in turn, other
immunosuppressive activity in mouse and human experimental models [14]. The described results
showed that many of the compounds containing isoxazole moiety exhibited immunosuppressive
effects on phytohemagglutinin-induced peripheral blood mononuclear cell (PBMC) proliferation and
several biological activities and that the isoxazole structure was responsible for the immunological
activity [31].
Differential and interesting activities of described compounds prompted us to continue
modifications of position 4 of the isoxazole moiety. The aim of our study was to synthesize a new series
of N0-substituted derivatives of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide in the reaction
of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide with relevant carbonyl compounds and to
evaluate their immunosuppressive properties using human blood cells in selected models. Further,
we investigated the molecular basis of the immunosuppressive activity of the most active compound
using Jurkat cells.
2. Results
2.1. Chemistry
A series of N0-substituted derivatives of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide-
MM1
–10 (compounds 5 in Scheme 1) was synthesized according to the synthetic pathway presented
in Scheme 1.
The following reagents and substrates were used in the synthesis of the considered compounds.
Starting materials, such as esters of 2-cyano-3-alkoxy-2-butenoate acids and semi-products (i.e., known
ethyl [32] or methyl [33] esters of 5-amino-3-methyl-4-isoxazolecarboxylic acid), were prepared

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according to the highly efficient, environmentally friendly methods precisely described in [34
and [patent priority number PL20110397559, PL 216764 B1 20140530, PL 216770 B1 20140530]. Known
5-amino-3-methyl-4-isoxazolecarboxylic acid (compound , Scheme 1) was obtained by hydrolysis of
its appropriate ethyl or methyl esters with a boiling water solution of sodium hydroxide.
Initially, compounds were obtained using the method described earlier [patent priority
number PL 193939] and were used to obtain compounds and (Scheme 1). Final compound
,35]
1
2–3
4
5
5
(Scheme 1) was synthesized in reaction with the nucleophilic addition of a primary amine group
(terminal group of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide) with appropriate aromatic
aldehyde using indium (III) trifluoromethanesulfonate as a catalyst. The amine group in position
5 in the isoxazole ring is not active in the condition of a realized reaction. The product received
imine derivatives (MM1–10). The applied method provided products in good yields of up to 63–81%.
The structures of the obtained compounds MM1–10 were characterized by a sharp melting point
(m.p.), IR, 1H NMR spectra, and MS.
0
Scheme 1. Synthesis of N -substituted derivatives of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide-
MM1–10 compounds.
2.2. Biology
In pilot experiments (Supplementary Materials, Figure S3), we found that compounds MM2
MM3 MM5 MM6, and MM7 inhibited to various degrees concanavalin A-induced proliferation of
,
,
,
mouse splenocytes. The best inhibitory effects were observed with MM3, but MM2 and MM7 were
also strongly suppressive. The determination of the cytotoxicity in relation to the L929 cell line did
not reveal toxic effects of the compounds (Supplementary Materials, Figure S1). However, cytotoxic
actions of some compounds (MM4, MM6, and MM9) could be demonstrated at a higher concentration
by using the A549 reference cell line (Figure 1). MM3, in turn, appeared to be devoid of cell toxicity
until a tested concentration of 250 µM (Supplementary Materials, Figure S2).

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Figure 1. The toxicity of the compounds against the A549 cell line.
The cytotoxicity of the compounds was determined by measuring the growth of the human tumor
epithelial lung A549 cell line. The results are presented as optical density (OD) values. Statistics are
presented as p < 0.05 versus appropriate dilutions of the solvent (DMSO).
The results showed that the compounds exhibited differential abilities to suppress
phytohemagglutinin A (PHA)-induced lymphocyte proliferation (Figure 2). The most suppressive was
MM3, as in the case of concanavalin-induced splenocyte proliferation (Supplementary Materials,
Figure S3). Its inhibitory action, in terms of kinetics, resembled that of teriflunomide but was
somewhat weaker.
Figure 2. Effects of the MM compounds on phytohemagglutinin A (PHA)-induced peripheral blood
mononuclear cell (PBMC) proliferation.

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MM3 inhibited, in a dose-dependent manner, inducible tumor necrosis factor (TNF
(Figure 3). The inhibition was still significant at a concentration of 6.25
The interdependence between activities, toxicity, and selected biological activities are presented in Table 1.
α) production
µM (about 40% inhibition).
Figure 3. Effect of MM3 compound on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF
production in whole blood culture. * p < 0.05 versus appropriate dilutions of the solvent (DMSO).
α)
Table 1. Interdependence between structure, toxicity, and biological activities of the studied compounds.
Modification/R
Toxicity against
Compound
Biological Activity
A549 Cells
MM1
MM2
ethylidene
absent
absent
not suppressive in the proliferation test
4-hydroxyphenyl
moderately suppressive in the proliferation test
suppression of PBMC proliferation, TNF α
activity and induction of a proapoptotic pathway
MM3
2,4-dihydroxyphenyl
absent
MM4
MM5
MM6
MM7
MM8
MM9
MM10
5-nitrothiophen-2-yl
phenyl
high
absent
strongly suppressive in the proliferation test
not suppressive in the proliferation test
4-chlorophenyl
2-chlorophenyl
2-methoxyphenyl
4-nitrophenyl
3-nitrophenyl
moderate
absent
moderately suppressive in the proliferation test
weakly suppressive in the proliferation test
weakly suppressive in the proliferation test
moderately suppressive in the proliferation test
moderately suppressive in the proliferation test
absent
moderate
absent
MM3 compound was incubated for 24 h at a concentration of 50
changes in the expression of signaling molecules were measured as described in Section 4. The results
(Table 2) demonstrated strong upregulation of caspase expression, as well as NF- B1 and Fas signaling
proteins. No increase of p53 and a negligible change in Bcl-2 expression was noted.
µM with Jurkat cells, and the
κ

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Table 2. The changes in expression of selected signaling molecules induced by MM3 in cultures of
Jurkat cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene
to normalize values obtained for the target genes. The values presented in the table reflect relative
fold changes in response of the cells to the MM3 compound, when the GAPDH gene expression was a
control and assumed as equal to one.
Signaling Molecule
MM3 (50 µM)
Bcl-2
Casp-3
Casp-7
Casp-8
Casp-9
Fas
0.5
20.6
16.0
50.7
2.7
7.8
NF-κB1
p53
50.1
0
3. Discussion
In this investigation, we evaluated the ability of the MM1
that of MM3, to suppress proliferation of mitogen-induced human blood lymphocytes and TNF
–
10 compounds and subsequently,
α
production by human blood cultures. Teriflunomide, an isoxazole drug [36], served as a reference
compound in the proliferation test. MM3 was not toxic with regard to the reference cell line A549 and,
up to the studied concentration of 250 µM, showed a moderate inhibition of the cell proliferation but a
strong, dose-dependent suppression of TNF α production.
The design of a compound’s structure with a more beneficial therapeutic property and less toxicity
than a leading structure is a key task in the search for new potential drugs. In the described MM1–10
series, 5-amino-N,3- dimethyl-N0-phenylmethylidene-1,2-oxazole-4-carbohydrazide (MM5 derivative),
containing an unsubstituted phenyl ring, has been considered a leading structure. Its immunosuppressive
activity was moderate. Modifications of a leading structure may be achieved by exchanging substituents,
increasing or decreasing the size of the ring, and simplifying or stiffening a molecule. The exchange of
a substituent presents the simplest way to adjust a modified structure to an action (target) site. In the
described MM1–10 compound series, the location of a substituent in the phenyl ring was subject to
modification. In these cases, the hydrogen atom was exchanged for a chlorine atom or a hydroxyl,
metoxyl, or nitryl group. In addition, in the MM4 compound, the phenyl ring (aromatic one) was
exchanged for a heteroaromatic, 5-nitrothiophen-2-yl ring.
The assumption of these modifications was to obtain derivatives of a higher immunosuppressive
activity in relation to lymphocyte proliferation and/or proinflammatory cytokine production and
lower toxicity than the leading structure. The MM5 derivative, containing an unsubstituted phenyl
ring was characterized by a moderate suppression of lymphocyte proliferation. Literature data [37
]
indicate that the phenyl ring belongs to groups preferably binding to flat, hydrophobic sites
by means of van der Waals bonds. The introduction of the hydroxyl group, which potentially
belongs to groups interacting with an acting site by hydrogen bonds, increased the strength of
the action of the MM2 derivative in relation to the unsubstituted phenyl ring in the MM5 leading
structure. MM2, containing a 4-hydroxyphenyl group, exhibited higher suppressive potency
than MM5 in the described proliferation test. The most active in this series of compounds was
0
5-amino-N -(2,4-dihydroxyphenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (the MM3
compound) containing a 2,4-dihydroxy phenyl substituent. A higher number of hydroxyl groups in
MM3 should in theory cause an increase of the molecule’s polarity and a possibility to form more
hydrogen bonds in comparison with MM2, which contains only one hydroxyl group. An increased
number of groups capable of forming hydrogen bonds lowers the susceptibility of a molecule to
absorption, with the exception of compounds, such as methotrexate or erythromycin, which are
transferred by transporting proteins [37].

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It is known that derivatives containing nitroaromatic groups are metabolized to toxic
compounds [37]. Our compounds, containing 4-nitrophenyl (MM9) or 3-nitrophenyl (MM10
)
fragments did not show toxicity in our test, but their suppressive actions were lower than that of MM5
.
Such a modification, based on the introduction of the nitryl group, worsened the biological activity but
did not significantly influence toxicity. In turn, the exchange of the aromatic ring for the heteroaromatic
one with the nitryl substituent led to a high increase in toxicity in the MM4 compound. Literature
reports [37] prove that derivatives containing 5-nitrophen-2-yl group display a higher pharmacological
activity than derivatives bearing an unsubstituted aromatic ring. Bearing in mind that our compounds
are expected to be potential immunosuppressive drugs in such immunological disorders, such as
autoimmunity or inflammation, compound MM4 was not attractive enough because of its toxicity
with regard to the A549 reference cell line.
The exchange of the hydrogen atoms in the phenyl ring for electro-acceptor chlorine atoms in
MM6 and MM7 compounds did not significantly increase their immunosuppressive potencies in
comparison to MM5. These effects were observed in every concentration used. The replacement of the
hydrogen atom by a big methoxyl group in the MM8 derivative did not positively affect its activity in
comparison with MM5 leading structure. Thus, it may be concluded that both the lack of ability to
form hydrogen bonds in MM8, in contrast with the MM3 compound, as well as the introduction of the
substituent to position 2 of the phenyl ring, may hinder adjustment of this fragment to a binding site.
The presented consequences of the structural modifications of the MM1–10 compound series allow
us to draw a conclusion that the possibility of forming hydrogenous bonds may have a major impact
on the biological activity of the MM3 compound displaying the highest activity. The interdependence
between the structure, toxicity, and activity of the compounds is depicted in Table 1.
The mechanisms of the immunosuppressive actions of the isoxazole derivatives may differ.
For example, leflunomide interferences with pyrimidine synthesis [36], and some isoxazoles may
act as p38 mitogen-activated protein kinase (MAP kinase) inhibitors [38], or, as in the case of
oxazolones, inhibit tyrosinase activity [39]. An isoxazole derivative, synthesized in our laboratory,
MZO-2 (ethyl N-{4-[(2,4-dimethoxybenzyl)carbamoyl]-3-methylisoxazol-5-yl}acetimidate) [40], had no
effect on the induction phase of the humoral immune response to sheep red blood cells (SRBC)
in vitro and in vivo and moderately suppressed the induction phase of delayed-type hypersensitivity
(DTH) to ovalbumin (OVA). Its inhibitory effect on carrageenan-induced paw inflammation was
potent. Likewise, MZO-2 applied in ointment was very effective in reducing contact sensitivity to
oxazolone compared with tacrolimus, the reference drug. Its mechanism of action we associated
with inhibition of caspase 3, 8, and 9 expression in Jurkat cells, because caspases are essential
for interleukin-2 (IL-2) release upon T cell activation [41]. In the case of 01 K compounds
(4-phenyl-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide), suppressive in relation to
interleukin-1β (IL-1β) and tumor necrosis factor α (TNF α) production by lipopolysaccharide
(LPS)stimulated splenocytes and concanavalin A (ConA)–induced thymocyte proliferation [42],
the compound upregulated in Jurkat cells’ expression of caspases 3 and 9, Fas and Bcl2, indicating
induction of cell apoptosis as its major mechanism of action. The investigations on mechanism of action
of MM3 (Table 2) revealed some resemblance to the action of 01 K compounds indicating elicitation
of cell apoptosis as a major cause of the immunosuppressive property of the compound. The results
strongly suggest initiation by MM3 of an apoptotic pathway associated with activation by caspase 8 of
NFκB [43] and Fas [44]. On the other hand, based on the obtained data, the involvement of Bcl2 [45]
and p53 [46] in this process seems to be excluded.
In conclusion, among the synthesized group of isoxazole derivatives, we selected the
representative, nontoxic MM3 compound with strong immunosuppressive properties and suggested
its molecular mechanism of action. The compound is a good candidate for further studies in in vivo
models to evaluate its potential therapeutic utility.

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4. Materials and Methods
4.1. Chemistry
Melting points were determined by Büchi apparatus (Laboratoriums-Technik AG,
Flawil, Switzerland) and Kofler system (Wagner & Munz) and were uncorrected. The progress of the
reaction was monitored by thin layer chromatography (TLC) on silica gel Polygram SIL G/UV 254 nm
coated TLC plates (Macherey-Nagel) and visualized by ultraviolet (UV) light at 254 nm (Fisher Bioblock
Scientific 254 nm lamps). Infrared (IR) spectra were collected on a Thermo Scientific Nicolet iS50
FT-IR spectrometer with built-in iS50 ATR single reflection crystal. Frequencies are reported in cm−1
.
The samples were applied as solids. The proton nuclear magnetic resonance (1H NMR) spectra were
obtained using a Bruker ARX 300 MHz NMR spectrometer in d6-dimethylsulfoxide (DMSO-d6).
Chemical shifts are given in ppm units. Signal multiplicities are represented by the following
abbreviations: s (singlet), d (doublet), t (triplet), and m (multiplet). Values of coupling constant
are reported as J in Hz. Mass spectrometry (MS) was performed on a Bruker Daltonic Electrospray
ionisation-Quadrupole-Time of Flight (ESI-Q-TOF) apparatus. Monoisotopic mass was calculated
(calc.) by Compass Data Analysis 4.2. All chemicals were purchased from commercial suppliers. Dry
solvents were obtained according to the standard procedure.
General Procedure for the Synthesis of N0-Substituted Derivatives of 5-Amino-N,3-dimethyl-
1,2-oxazole-4-Carbohydrazide Compounds MM1
–
MM10
.
To 1 mmol of 5-amino-N,3-dimethyl-1,2-
oxazole-4-carbohydrazide (obtained according to an analogously described method, patent PL 193939)
dissolved in 10 mL of 2-propanol, 5 mL of relevant aldehyde and indium (III) trifluoromethanesulfonate
◦
were added. The mixture was stirred and heated at a boiling temperature (82 C) for 4 h. At the end of
the reaction (controlled in a TLC), the mixture was cooled. The solution was evaporated in vacuum
from the mixture. The crude product, which separated out, was collected on a filter. The unrefined
compound was purified by recrystallization in methanol. As a result, a pure product was obtained.
5-Amino-N0-(ethylidene)-N,3-dimethy◦l-1,2-oxazole-4-carbohydrazide (MM1).
Anal.
C8H12N4O2
(m.w. 196.206 g/mol); m.p. 211–212 C; yield 68%; FTIR (ATR, selected lines) vmax/cm−1: 1605 (C=O),
1
3441 (NH2). H NMR (DMSO-d6)
δ (ppm): 1.25 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.93 (s, 3H, CH3), 7.22
(s, 2H, NH2), 8.62 (s, 1H, CH). MS (ESI) [M + H]+ m/z 197.1054, calc. m/z 197.1033, [M + Na]+ m/z
219.0876, calc. m/z 219.0852.
5-Amino-N0-(4-hydroxyphenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM2).
Anal.
◦
−1
C13H14N4O3 (m.w. 274.275 g/mol); m.p. 149–151 C; yield 71%; FTIR (ATR, selected lines) vmax/cm
:
1
1615 (C=O), 3446 (NH2). H NMR (DMSO-d6)
δ (ppm): 2.09 (s, 3H, CH3), 3.33 (s, 3H, CH3), 6.79–6.82
(d, J = 8.7 Hz, 2H, CH-aromat), 7.07 (s, 2H, NH2), 7.50–7.53 (d, J = 8.7 Hz, 2H, CH-aromat), 7.88 (s, 1H,
CH), 9.807 (s, 1H, OH). MS (ESI) m/z [M + H]+ 275.1157, calc. m/z 275.1139, m/z [M + Na]+ 297.0976,
calc. m/z 297.0958.
5-amino-N0-(2,4-dihydroxyphenyl)methylidene-N,3-◦dimethyl-1,2-oxazole-4-carbohydrazide (MM3). Anal.
−1
C13H14N4O4 (m.w. 290.275 g/mol); m.p. 211–212 C; yield 73%; FTIR (ATR, selected lines) vmax/cm
1620 (C=O), 3442 (NH2). H NMR (DMSO-d6)
:
1
δ (ppm): 2.08 (s, 3H, CH3), 3.33 (s, 3H, CH3), 6.28–6.32
(d, J = 8.6 Hz, 2H, CH-aromat), 7.11 (s, 2H, NH2), 7.36–7.39 (d, J = 8.6 Hz, 1H, CH-aromat), 8.05 (s, 1H,
CH), 9.78 (s, 1H, OH), 10.25 (s, 1H, OH). MS (ESI) m/z [M + H]+ 291.1114, calc. m/z 291.1087, m/z
[M + Na]+ 313.0930, calc. m/z 313.0907.
5-Amino-N,3-dimethyl-N0-(5-nitrothiophen-2-yl)met◦hylidene-1,2-oxazole-4-carbohydrazide (MM4). Anal.
−1
C11H11N5O4S (m.w. 309.301 g/mol); m.p. 230–231 C; yield 81%; FTIR (ATR, selected lines) vmax/cm
1628 (C=O), 3445 (NH2). H NMR (DMSO-d6)
:
1
δ (ppm): 2.11 (s, 3H, CH3), 3.34 (s, 3H, CH3), 7.28 (s, 2H,
NH2), 7.44–7.46 (d, J = 4.4 Hz, 1H, CH-aromat), 8.10–8.11 (d, J = 4.4 Hz, 1H, CH-aromat), 8.17 (s, 1H,
CH). MS (ESI) m/z [M + H]+ 310.0630, calc. m/z 310.0604, m/z [M + Na]+ 332.0449, calc. m/z 332.0424.

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5-Amino-N,3-dimethyl-N0-phenylmethylidene-1,2-oxazole-4-carbohydrazide (MM5). Anal. C13H14N4O2
◦
(m.w. 258.276 g/mol); m.p. 134–135 C; yield 75%; FTIR (ATR, selected lines) vmax/cm−1: 1635 (C=O),
3443 (NH2). H NMR (DMSO-d6)
1
δ (ppm): 2.09 (s, 3H, CH3), 3.37 (s, 3H, CH3), 7.13 (s, 2H, NH2),
7.40–7.43 (m, 3H, CH-aromat), 7.67–7.70 (d, J = 6.4 Hz, 2H, CH-aromat), 7.97 (s, 1H, CH). MS (ESI) m/z
[M + H]+ 259.1215, calc. m/z 259.1189, m/z [M + Na]+ 281.1035, calc. m/z 281.1009.
5-Amino-N0-(4-chlorophenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide
(
MM6).
Anal.
◦
C13H13N4O2Cl (m.w. 292.721 g/mol); m.p. 210–211 C; yield 69%; FTIR (ATR, selected lines)
1
v
max/cm−1: 1628 (C=O), 3447 (NH2). H NMR (DMSO-d6)
δ
(ppm): 2.08 (s, 3H, CH3), 3.32 (s, 3H,
CH3), 7.13 (s, 2H, NH2), 7.48–7.51 (d, J = 8.6 Hz, 2H, CH-aromat), 7.68–7.71 (d, J = 8.6 Hz, 2H,
CH-aromat), 7.97 (s, 1H, CH). MS (ESI) m/z [M + H]+ 293.0811, calc. m/z 293.0800, m/z [M + Na]+
315.0631, calc. m/z 315.0613.
5-Amino-N0-(2-chlorophenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide
(
MM7).
Anal.
◦
C13H13N4O2Cl (m.w. 292.721 g/mol); m.p. 208–209 C; yield 65%; FTIR (ATR, selected lines)
1
v
max/cm−1: 1630 (C=O), 3438 (NH2). H NMR (DMSO-d6)
δ
(ppm): 2.09 (s, 3H, CH3), 3.39 (s, 3H,
CH3), 7.19 (s, 2H, NH2), 7.367–7.41 (m, 2H, CH-aromat), 7.51–7.52 (m, 1H, CH-aromat), 7.85–7.88
(m, 1H, CH-aromat), 8.06 (s, 1H, CH). MS (ESI) m/z [M + H]+ 293.0811, calc. m/z 293.0800, m/z
[M + Na]+ 315.0631, calc. m/z 315.0619.
5-Amino-N0-(2-methoxyphenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM8).
Anal.
◦
−1
C14H16N4O3 (m.w. 288.302 g/mol); m.p. 224–225 C; yield 63%; FTIR (ATR, selected lines) vmax/cm
:
1625 (C=O), 3445 (NH2). 1H NMR (DMSO-d6)
δ (ppm): 2.08 (s, 3H, CH3), 3.35 (s, 3H, CH3), 3.87
(s, 3H, OCH3), 6.95–7.09 (t, J = 8.9 Hz, 2H, CH-aromat), 7.12 (s, 1H, CH-aromat), 7.13 (s, 2H, NH2),
7.35–7.40 (m, 1H, CH-aromat), 8.08 (s, 1H, CH). MS (ESI) m/z [M + H]+ 289.1313, calc. m/z 289.1295,
m/z [M + Na]+ 311.1132, calc. m/z 311.1115.
5-Amino-N,3-dimethyl-N0-(4-nitrophenyl)methylidene-1,2-oxazole-4-carbohydrazide
(
MM9).
Anal.
◦
−1
C13H13N5O4 (m.w. 303.273 g/mol); m.p. 214–216 C; yield 73%; FTIR (ATR, selected lines) vmax/cm
:
1
1635 (C=O), 3446 (NH2). H NMR (DMSO-d6)
δ (ppm): 2.09 (s, 3H, CH3), 3.39 (s, 3H, CH3), 7.20 (s,
2H, NH2), 7.89–7.92 (d, J = 8.9 Hz, 2H, CH-aromat), 8.08 (s, 1H, CH), 8.27–8.31 (d, J = 8.9 Hz, 2H,
CH-aromat). MS (ESI) m/z [M + H]+ 304,1055, calc. m/z 304.1040, m/z [M + Na]+ 326.0875, calc. m/z
326.0860.
5-Amino-N,3-dimethyl-N0-(3-nitrophenyl)methylidene-1,2-oxazole-4-carbohydrazide
(
MM10).
Anal.
◦
−1
C13H13N5O4 (m.w. 303.273 g/mol); m.p. 209–210 C; yield 77%; FTIR (ATR, selected lines) vmax/cm
:
1
1638 (C=O), 3438 (NH2). H NMR (DMSO-d6)
δ (ppm): 2.10 (s, 3H, CH3), 3.34 (s, 3H, CH3), 7.19 (s, 2H,
NH2), 7.69–7.75 (t, J = 8.1 Hz, 1H, CH-aromat), 8.08–8.12 (d, J = 8.1 Hz, 2H, CH-aromat), 8.19–8.23 (m,
1H, CH-aromat), 8.53 (s, 1H, CH). MS (ESI) m/z [M + H]+ 304.1059, calc. m/z 304.1040, m/z [M + Na]+
326.0876, calc. m/z 326.0860.
Additional spectroscopic data is available in the Supplementary Materials (Figures S4–S21).
4.2. Biology
4.2.1. Reagents
Fetal calf serum (FCS), RPMI-1640, and Hanks’ medium were purchased from Biowest
(Nuaillé, France). Lipopolysaccharide from Escherichia coli 0111:B4 (LPS), phytohemagglutinin (PHA),
dimethyl sulfoxide (DMSO), and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
and all other reagents were from Sigma-Aldrich (St. Louis, MO, USA).

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4.2.2. Preparation of the Compounds for Biological Assays
◦
The compounds were dissolved in DMSO to a concentration of 10 mM and kept at 4 C.
◦
Then, the compound solutions were incubated at 37 C for 10 min with vigorous shaking and
subsequently diluted in the culture medium to the respective concentrations used in the cell cultures.
4.2.3. Determination of the Toxicity of the MM Compounds against the A549 Cell Line
The cytotoxicity of the compounds was determined by measuring growth of human lung epithelial
A549 cells (ATCC CCL 185). The cells were suspended in a density of 2
medium, referred to below as “the culture medium”, consisting of RPMI-1640 with the addition of
10% fetal calf serum (FCS), 100 U/mL of penicillin, 100 g/mL streptomycin, and 2 mM L-glutamine.
The compounds were tested at a concentration range of 100–12.5 M and the MM3 compound at
250–1.9 M. The test was performed in 96-flat bottom plates containing 2
105 cells incubated
×
106 mL in a culture
µ
µ
µ
×
initially for 24 h in a cell culture incubator. Then, the supernatants were removed and to the cells,
appropriate dilutions of the compounds (0.2 mL) were added in a cell culture medium containing
2% FCS. The cultures were incubated for 72 h followed by the determination of cell viability by a
colorimetric MTT method. In parallel, control cultures containing appropriate dilutions of the solvent
(DMSO) were also incubated. The results were presented as mean optical density (OD) values from
four wells ± standard deviation.
4.2.4. Isolation of the Peripheral Blood Mononuclear Cells (PBMCs)
Venous blood from a single donor was withdrawn into heparinized syringes and diluted twice
with phosphate buffered saline (PBS). PBMCs were is◦olated by centrifugation on a Ficoll-Uropoline
gradient (density 1.077 g/mL) at 800
times with Hanks’ medium and re-suspended in the culture medium at a density of 2
×
g for 20 min at 4 C. The interphase cells were then washed three
×
106 cells/mL.
4.2.5. PHA-Induced Proliferation of Human PBMC
PBMC were distributed into 96-well flat-bottom plates in 100
µ
L aliquots (2
×
105 cells/well).
PHA was added at a concentration of 5 g/mL. The compounds were tested at the following doses:
µ
100, 50, and 25 uM. DMSO at appropriate dilutions served as the control. After three days of incubation
in a cell culture incubator, the proliferative response of the cells was determined by colorimetric
MTT [47]. The results were presented as optical density (OD) values, and appropriate DMSO dilutions
served as the control.
4.2.6. Lipopolysaccharide-Induced TNF-a Production in Whole Blood Cell Culture
Human whole blood was diluted 10
culture plates in 1 mL aliquots. The cultures were stimulated with LPS (100 ng/mL), and the studied
compound was added at concentrations of 50, 25, 12.5, and 6.15 M. The control cultures contained
DMSO in appropriate concentrations. After an overnight incubation, the supernatants were harvested
and frozen at concentrations were determined in the
80 ◦C until cytokine determination. TNF
×
with RPMI-1640 medium and distributed to 24-well
µ
−
α
supernatants by using an ELISA kit from eBioscience in a presence of TNF
expressed in pg/mL.
α standard and originally
4.2.7. Colorimetric MTT Assay for Cell Growth and Kill
The assay was performed according to [47]. Briefly, 25
added per well at the end of cell incubation period, and the plates were incubated for an additional 3 h
in a cell culture incubator. Then, 100 L of the extraction buffer (20% SDS with 50% dimethylformamide
µL of MTT (5 mg/mL) stock solution was
µ
(DMF), pH 4.7) was added. After an overnight incubation, the OD was measured at 550 nm with the
reference wavelength of 630 nm in a Dynatech 5000 spectrophotometer.

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4.2.8. Cultures of Jurkat Cells and Total RNA Isolation
Jurkat cells (105/mL) in the culture medium were cultured overnight with the MM3 compound
M) Total RNA isolation was carried with TRIzol Reagent (Ambion) according to the
manufacturer’s recommendations. The cell pellet (2
106 cells) was suspended in 1 mL of TRIzol
reagent, shaken, incubated for 10 min at room temperature (RT), supplemented with 0.2 mL of
(50
µ
×
chloroform, shaken vigorously for 15 s, incubated for 3 min at RT, and centrifuged at 12,000
for 15 min at 4 C. The water phase was collected, transferred to a new tube, supplemented with
×
g
◦
0.5 mL of isopropanol, incubated at RT for 10 min, and centrifuged at 12,000
×
g for 10 min at 4 ◦C.
The RNA pellet was washed with 1 mL of 75% ethanol, dried in air, and dissolved in 20–30 µL of sterile
diethylpyrocarbonate-treated Mili-Q water. RNA samples were stored at −20 ◦C.
4.2.9. Reverse Transcription
Single stranded complementary DNA (cDNA) was synthesized with oligo (dT) 12–18 primers
from 5 µg of total RNA using Novazym VerteKit, according to the manufacturer’s instruction.
4.2.10. Quantitation of Gene Expression by Real Time PCR
The expression of the genes (i.e., caspase 3, 7, 8, and 9, Bcl-2, Fas, NFκB1, and p53) were measured
using an APA SYBR FAST qPCR Kit. The sequences of primers are enclosed in the Supplementary
Materials. The reaction was performed in an Applied Biosystems ViiA7 thermocycler starting with
5 min of preincubation at 95 ◦C, followed by 35 amplification cycles as follows: 95 ◦C for 30 s and
simultaneous annealing-extension-data acquisition for 45 s and 60 ◦C. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as a housekeeping gene for arbitrary unit calculation for every
tested gene.
4.2.11. Statistics
The results are presented as mean values
±
standard deviation (SD). The Brown–Forsythe test
was used to determine the homogeneity of the variance between the groups. When the variance
was homogenous, analysis of the variance (one-way ANOVA) was applied, followed by post hoc
comparisons with the Tukey test to estimate the significance of the difference between the groups.
Nonparametric data were evaluated with the Kruskal–Wallis analysis of variance. The significance
was determined at p < 0.05. Statistical analysis was performed using STATISTICA 6.1 for Windows.
Supplementary Materials: Supplementary materials includes detailed experimental procedures, characterizations
of all new compounds, as well as additional figures and tables.
Author Contributions: M.M. performed the synthetic experiments, analyzed the chemical data, created the
concept, designed the study, coordinated the work, and wrote the manuscript. S.B. planned the cytotoxicity tests
and performed data analysis. K.M. performed the MTT tests. M.K. performed tests and measured the production
of cytokines. E.Z. determined the toxicity of the compounds. I.K. performed molecular studies. M.Z. created the
concept, designed the study, coordinated the work, and wrote the manuscript. All the authors read and approved
the final manuscript.
Funding: This research was funded by Wroclaw Medical University, grant number STD090.17.012.
Acknowledgments: The authors thank Magdalena Korab for the excellent technical assistance and Aleksandra
´
Sochacka-Cwikła for the processing of ESI-MS data. MS measurements were carried out in the Laboratory
of Elemental Analysis and Structural Research, Faculty of Pharmacy with Division of Laboratory Diagnostics,
Wroclaw Medical University.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 1545
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©
2018 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).