A family of substituted hydrazonoisoxazolones with potential biological properties

Article abstract of DOI:10.1039/c5nj02604k

The synthesis, characterization and biological study of a new 3,4,5-trisubstituted isoxazolones have been reported, whereby a series of (Z)-3-methyl-4-(2-(R-phenyl)hydrazinylidene)isoxazol-5(4H)-ones were prepared by the reaction of a β-diketohydrazone with hydroxylammonium chloride. All the products were characterized using EA, UV-Vis, FT-IR, ¹H-NMR, ¹³C-NMR spectroscopy and HMBC. The crystalline and molecular structures of three compounds were solved by X-ray diffraction methods. Density functional theory (DFT) and time-dependent DFT (TDDFT) calculations were performed to obtain a better explanation of the observed experimental behaviour of these newly synthetized compounds. Furthermore, the reports of cytotoxicity and an antiproliferative effect in human promyelocytic leukaemia cells, HL-60, was tested by the MTT reduction method, showing that most of the newly synthetized compounds had important antineoplastic activity. The most active isoxazolones were used in reverse transcription polymerase chain reaction (RT-PCR) experiments to determine the effect on the expression levels on mRNA encoding using the anti-apoptotic, Bcl 2, pro-apoptotic, Bax, and the proliferation inhibition, p21^WAF-1, proteins. Therefore, it was possible to fully characterize the complete library of 15 isoxazolones and to show that most of them are antineoplastic trough an apoptotic pathway.

Full text of DOI:10.1039/c5nj02604k

View Article Online
View Journal
NJC
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: C. Bustos, E.
Molins, J. Carcamo, M. Aguilar, C.O. Sánchez, I. Moreno-Villoslada, H. Nishide, X. Zarate and E. Schott,
New J. Chem., 2015, DOI: 10.1039/C5NJ02604K.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/njc

Page 1 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
A family of substituted hydrazonoisoxazolones with
potential biological properties
Carlos Bustosa,*, Elies Molinsb, JuanꢀGuillermo Cárcamoc, Marcelo N. Aguilarc, Christian Sáncheza,
Ignacio MorenoꢀVillosladaa, Hiroyuki Nishided, Ximena Zaratee and Eduardo Schottf,*
aInstituto de Ciencias Químicas, Universidad Austral de Chile, Las Encinas 220, Campus Isla Teja, Valdivia, Chile.
bInstitut de Ciència de Materials de Barcelona (ICMABꢀCSIC), Campus de la Universitat Autònoma de Barcelona,
08193 Bellaterra, Spain.
cInstituto de Bioquímica y Microbiología, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile.
dDepartment of Applied Chemistry, School of Science and Engineering, Waseda University, Tokyo 169ꢀ8555, Japan.
eInstituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, Av. Pedro de
Valdivia 641, Santiago, Chile.
fDepartamento de Química Inorgánica, Facultad de Química, Pontificia Universidad Católica de Chile, Avda. Vicuña
Mackenna 4860, Santiago, Chile
*email: maschotte@gmail.com, Phone/Fax: (562)ꢀ2354ꢀ1422
.
Keywords: 3,4ꢀRꢀIsoxazolones, Organic synthesis, Antiproliferative, Cytitoxic effect, XꢀRay
Abstract
The synthesis, characterization and biological study of a new 3,4,5ꢀtrisubstituted isoxazolones have
been reported. In this sense a series of (Z)ꢀ3ꢀmethylꢀ4ꢀ(2ꢀ(Rꢀphenyl)hydrazinylidene)isoxazolꢀ
5(4H)ꢀones were prepared by reaction of a βꢀdiketohydrazone with hydroxilammonium chloride. All
1
products were characterized using EA, UVꢀVis, FTꢀIR, HꢀNMR, 13CꢀNMR spectroscopy and
HMBC. The crystalline and molecular structure of three compounds were solved by XꢀRay
diffraction methods. Density functional theory (DFT) and Timeꢀdependent DFT (TDDFT)
calculations were performed to give a better explanation of the observed experimental behaviour of

New Journal of Chemistry
Page 2 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
these newly synthetized compounds. Furthermore, the report of cytotoxicity and the antiproliferative
effect in human promielocytic leukemia cells, HLꢀ60 was tested by the MTT reduction method,
showing most of the newly synthetized compounds important antineoplastic activity. The most
active isoxazolones were used in experiments of reverse transcription polymerase chain reaction
(RTꢀPCR) to determine the effect on the expression levels on mRNA encoding using the antiꢀ
apoptotic, Bcl 2, proꢀapoptotic, Bax, and the proliferation inhibition p21WAFꢀ1 proteins. Therefore, it
was possible to fully characterize the complete library of 15 isoxazolones and to show that most of
them are antineoplastic trough an apoptotic pathway.
INTRODUCTION
The biological activity of substituted isoxazoles has made them an interesting focus for medicinal
chemistry in the last years1. In fact, isoxazoles are potent, selective agonists at human cloned
dopamine D4 receptor2 and exhibit GABAA antagonist3, analgesic4 antiinflamatory4, ulcerogenic4,
,
antimicrobial5, antifungal5, COXꢀ2 inhibitory6,7, antinociceptive8 and anticancer9 activity.
Many synthetic methods have been employed to obtain substituted isoxazoles including reactions of
hydroxilamine with 1,3ꢀdicarbonyl compounds10, α,βꢀunsaturated carbonyl compounds11 and
α,β−unsaturated nitriles12. The reaction of an oximeꢀderivative dianion with esters13 or amides14,15
also yields isoxazoles. On the other hand, [3+2] cycloaddition reactions of alkynes and nitrile oxides
have also been developed16. However, these methods often require strong bases, strong mineral
acids, high temperatures or they provide poor regioselectivity. Isoxazoles can be also halogenated to
give substituted haloisoxazoles17, however under the reaction conditions high temperatures are
required.In this work we report a oneꢀpot synthesis of isoxazolones, of the type (Z)ꢀ3ꢀmethylꢀ4ꢀ(2ꢀ
(Rꢀphenyl)hydrazinylidene)isoxazolꢀ5(4H)ꢀones. These compounds were obtained by cyclization of
an in situ generated oxime, formed by reaction of (E)ꢀethyl 2ꢀ(2ꢀ(Rꢀphenyl)hydrazinylidene)ꢀ3ꢀ

Page 3 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
oxobutanoates with hydroxilammonium chloride in acid media (R= 4ꢀOH(1), 4ꢀCH3(2), 4ꢀOCH3(3),
4ꢀH(4), 4ꢀCl(5), 4ꢀBr(6), 4ꢀCO2H(7), 4ꢀCOCH3(8), 4ꢀCN(9), 4ꢀNO2(10), 2ꢀOH (11), 2ꢀCl(12), 2ꢀ
CO2H(13), 2ꢀNO2(14), 3ꢀCl(15)). Besides, we present the spectroscopic characterization and
structures of three representative compounds. Finally, considering that numerous isoxazole
derivatives have shown biological activity, we perform a study of antitumor activity in order to
evaluate the cytotoxic activity of these compounds towards the human promyelocytic leukemia cell
line, HLꢀ6018–22. Besides, using compounds with IC50 values lower than 83 ꢁM, we performed
reverse transcription polymerase chain reaction (RTꢀPCR) experiments to determine the effect on the
expression levels of mRNA codifying for antiꢀ and proꢀapoptotic genes, Bclꢀ2 and Bax, and a gene
involved in the inhibition of cell cycle progression, p21WAFꢀ1
.
EXPERIMENTAL
Chemicals
Substituted anilines (RꢀC6H4ꢀNH2 R= 4ꢀOH(1), 4ꢀCH3(2), 4ꢀOCH3(3), 4ꢀH(4), 4ꢀCl(5), 4ꢀBr(6), 4ꢀ
CO2H(7), 4ꢀCOCH3(8), 4ꢀCN(9), 4ꢀNO2(10), 2ꢀOH (11), 2ꢀCl(12), 2ꢀCO2H(13), 2ꢀNO2(14), 3ꢀ
Cl(15)), acetylacetone, sodium nitrite, hydrochloric acid, sodium hydroxide, hydroxilammonium
chloride and acetic acid were procured from SigmaꢀAldrich Corporation and the solvents (methanol,
ethanol, diethylether, acetone CDCl3, and DMSOꢀd6) from commercial sources including Merck,
Fisher and T. J. Baker providers. All βꢀdiketohydrazone precursors, were obtained as previously as
described in literature23 and the structures were checked by IR spectroscopy and, in some cases, by
1H NMR spectroscopy.
Physical measurements

New Journal of Chemistry
Page 4 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
Uncorrected melting points, MP, were determined on a digital STUARD, SMP10 apparatus. The
elemental analysis were obtained in a FISONS, EA 1118 microanalyser using sulfanilamide as
standard. UVꢀvisible spectra were recorded in the 1100ꢀ200 nm range, in quartz cells with 10 mm
length pass, using a Perkin Elmer, Lambda 35 spectrophotometer, diluting the concentrated
solutions, ~1.0×10ꢀ3 mole/L of each compound to around ~1.0×10ꢀ3 mole/L. The infrared spectra,
IR, were obtained in solid state on a Jasco ATR PRO450ꢀS, mounted on a Jasco FT/IRꢀ4200
1
equipment. Depending on the solubility of compounds, the H NMR, 13C NMR and HMBC spectra
were recorded by the standard methods in CDCl3 or DMSOꢀd6 solutions, using 5 mm i.d. glass tubes
and the internal solvent signals as reference in two different equipment i) JOEL, JNMꢀLambda500
1
13
spectrophotometer and ii) a Bruker, Avance AM 400 spectrophotometer. Assignment of H and C
NMR signals was realized according to Figure 1.
Figure 1. Numeration used for 1Hꢀ and 13CꢀNMR assignation.
Crystallography
Highly redundant single crystals Xꢀray diffraction data set of 4, 12 and 15 were collected at room
temperature up on a Bruker AXS SMART APEX CCD diffractometer using monochromatic MoKα
radiation, λ= 0.71069 Ǻ, and a 0.3º separation between frames. Data collection of crystals and
refinement of structures are shown in Table 1, while Table S1 contains the selected bond lengths and
angles and Figures 2 exhibit an ORTEP view of each molecule. Data integration was performed
using SAINT program contained in the diffractometer package. The structures were solved by direct

Page 5 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
methods and Fourier’s difference maps were refined by least squares on F2 with anisotropic
displacement parameters for nonꢀH atoms. All hydrogen atoms were located from difference Fourier
maps and refined isotropically. All calculations to solve the structure and to refine the model
proposed and to obtain results were carried out with the computer programs SHELXSꢀ97 and
SHELXLꢀ97 and SHELXTL/PC24. Crystallographic data (excluding structure factors) for the
structures reported in this paper have been deposited in the Cambridge Crystallographic Data Centre
as supplementary publication, No. 1062571 for compound (12) and 1062570 for compound (15).
Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK. Fax: +44 1223 336 033. Eꢀmail: data_request@ccdc.cam.ac.uk.
Web page: http://www.ccdc.cam.ac.uk. In case of compound 4 a previous report was found.
However the crystallization methodology is different. In this case the ethanol was allowed to slowly
evaporate from the solution. The obtained XꢀRay for 4 is reported in the supplementary information
and for comparison see Bertolasi et. al.25.
Table 1. Crystal data and structure refinement of compounds 4, 12 and 15.
Compound
Empirical formula
Formula weight (g/mol)
Temperature (K)
Wavelength (Å)
4
12
15
C10H9N3O2
203.2
C10H8ClN3O2
237.64
C10H8N3O2Cl
237.64
294(2)
294(2) K
0.71073
294(2)
0.71073
Monoclinic
0.71073
Crystal system
Triclinic
Monoclinic
Space group
C2/1
a= 11.117(4), α= 90
b= 7.955(2), β= 95.76(2)
c= 21.817(4), γ= 90
1919.9(9)
P
a=7.642(2), α=98.77
b=7.933(2), β=104.53
c=9.511(1), γ=98.06
541.9(2)
P21/c
a= 12.577(3), α= 90
b=8.509(8), β= 102.90(2)
c=20.565(6), γ= 90
2145(2)
Unit cell dimension (Å),
(º)
Volume (Å3)
Z
8
2
8
Density (Mg/m3)
1.406
1.456
1.472
Absorption coefficient
(mmꢀ1)
0.102
0.34
0.344

New Journal of Chemistry
Page 6 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
F(000)
848
244
976
Crystal size (mm3)
0.41×0.38×0.32
1.88ꢀ25.97
0.42×0.27×0.09
2.26 to 27.95
0.47×0.45×0.15
1.66ꢀ25.98.
θ range for data
collection(º)
ꢀ13 ≤ h ≤ 13
0 ≤ k ≤ 9
0 ≤ l ≤ 26
1893
ꢀ10≤ h ≤ 9
ꢀ10 ≤ k ≤ 10
0 ≤ l ≤ 12
5299
ꢀ15 ≤ h ≤ 15
0 ≤ k ≤ 10
0 ≤ l ≤ 25
4213
Index ranges
Reflections collected
Independent reflections
Completeness to θ (%)
1893
2605
4213
100
100
100
Max and Min.
Transmission
0.9681 and 0.9594
0.8477 and 0.9699
0.9502 and 0.8551
Fullꢀmatrix leastꢀsquares
Fullꢀmatrix leastꢀsquares on
Fullꢀmatrix leastꢀsquares
Refinement method
on F2
F2
on F2
Data/restrain/parameters
Goodnessꢀofꢀfit on F2
1893/0/122
0.981
2605/0/150
0.913
4213/0/291
1.009
Final R indices
R1 = 0.0459, wR2 = 0.0989
R1 = 0.1056, wR2 = 0.1120
R1 = 0.0497, wR2 = 0.1158
R1 = 0.1192, wR2 = 0.1376
R1 = 0.0615, wR2 = 0.1297
R1 = 0.1267, wR2 = 0.1462
[I>2σ(I)]
R indices (all data)
Largest diff. Peak/hole
(e×Åꢀ3)
0.149/ꢀ0.219
0.210 and ꢀ0.160
0.197/ꢀ0.287
Synthesis. In a 100 mL roundꢀbottomed flask were added 0.01 mmole of any βꢀdiketohydrazone, Rꢀ
C6H4ꢀNHN=C(COCH3)(CO2CH2CH3), Scheme 1, {R= 4ꢀOH(1), 2.62 g; 4ꢀCH3(2), 2.60 g; 4ꢀ
OCH3(3), 2.76 g; 4ꢀH(4), 2.46 g; 4ꢀCl(5), 2.81 g; 4ꢀBr(6), 3.25 g; 4ꢀCO2H(7), 2.90 g; 4ꢀCOCH3(8),
2.88 g; 4ꢀCN(9), 2.71 g; 4ꢀNO2(10), 2.91 g; 2ꢀOH (11), 2.62 g; 2ꢀCl(12), 2.81 g; 2ꢀCO2H(13), 2.90
g; 2ꢀNO2(14), 2.91 g; 3ꢀCl(15), 2.81 g} 25 mL of ethanol, 2 mL of glacial acetic acid and 0.01 mol
(0.69 g) of NH2OH·HCl. The reaction mixture was stirred and heated at reflux during 18 h. Then the
partially precipitated solution was cooled at ꢀ18 ºC by 4 h and the redꢀyellow solid was filtered by
suction, washed with 100ꢀ200 mL of water and dried at 40 ºC in a vacuum oven by 24 h. All
products were recrystallized from ethanol, where compounds 4, 12 and 15 yield single crystals
suitable for diffractometric studies
Analysis

Page 7 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
The corresponding spectra for I.R., 1HꢀNMR, 13CꢀNMR and HBMC are included in the
supplementary information for each of the synthetized compounds. A summary of the obtained
results are shown here.
(Z)-4-(2-(4-hydroxyphenyl)hydrazono)-3-methylisoxazol-5(4H)-one (1): Crude yield: 35.6%. Final
Yield: 22.5% MP ºC: 211ꢀ212. Anal. Calc. (%) for C10H9N3O3 (Mι: 219,20 g/mol: C, 54.79; H, 4.14;
N, 19.17; Found (%): C, 54.57; H, 4.25; N, 18.98. UVꢀvisible spectrum in ethanol solution 5 10ꢀ5
mol/L, λmax, nm (logε): 427(4.40); 296(2.72); 252(4.01); 201(4.12). I.R. spectrum in solid state,
(cmꢀ1); ν(OꢀH): 3187s; ν(CꢀH, aliph.): 2822w, 2684w, 2587w; ν(C=O): 1708s; ν(C=N): 1598m;
1
ν(C=C): 1556m; ν(NꢀO): 1000w. H NMR in DMSOꢀd6, δ ppm: 2.23 (s, 3H), 6.81 (d, J = 9.01 Hz,
13
2H), 7.54 (d, J = 8.97 Hz, 2H), 9.76 (s, 1H), 12.80 (s, 1H). C NMR in DMSOꢀd6, δ ppm: 10.01,
116.02, 118.16, 118.68, 133.53, 156.50, 159.63, 163.05.
(Z)-3-methyl-4-(2-p-tolylhydrazono)isoxazol-5(4H)-one (2): Crude yield: 98.9%. Final Yield: 90%
MP: 189ꢀ190 ºC. Anal. Calc. (%) for C11H11N3O2 (Mι: 217,22 g/mol): C, 60.82; H, 5.10; N, 19.34.
Found (%): C, 60.74; H, 5.25; N, 19.72. UVꢀvisible spectrum in ethanol solution 5 10ꢀ5 mol/L, λmax
,
nm (logε): 405 (3.37); 260 (3.94); 252(4.02); 232(4.03); 202(4.40). I.R. spectrum in solid state,
(cmꢀ1); ν(NꢀH): 3204s; ν(CꢀH, arom.): 3023w; ν(CꢀH, aliph.): 2978w, 2963w; 2924w, 2858w;
ν(C=O): 1705s; ν(C=N): 1595w; ν(C=C): 1563s; ν(NꢀO): 993w. 1H NMR in CDCl3, δ ppm: 2.33 (s,
3H), 2.38 (s, 3H), 7.23 (d, J = 8.36 Hz, 2H), 7.32 (d, J = 8.51 Hz, 2H), 12.69 (s, 1H). 13C NMR in
CDCl3, δ ppm: 10.34, 21.20, 116.25, 120.63, 130.52, 137.15, 138.30, 159.49, 165.30.
(Z)-4-(2-(4-methoxyphenyl)hydrazono)-3-methylisoxazol-5(4H)-one (3): Crude yield: 85.2%. Final
Yield: 82.2% MP: 202ꢀ203 ºC. Anal. Calc. (%) for C11H11N3O3 (Mι :231,255 g/mol): C, 56.65; H,

New Journal of Chemistry
Page 8 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
4.75; N, 18.02; Found (%): C, 56.45, H, 5.13, N, 17.97. UVꢀvisible spectrum in ethanol solution 5
10ꢀ5 mol/L, λmax, nm (logε): 420(4.41); 301(3.41); 252(4.02); 201(4.26). I.R. spectrum in solid state,
(cmꢀ1); ν(NꢀH): 3189m; ν(CꢀH, arom.): 3083w, 3040w and 3015w; ν(CꢀH, aliph.): 2961w, 2932w,
1
2896w and 2834m; ν(C=O): 1703s; ν(C=N): 1593m; ν(C=C): 1554s; ν(NꢀO): 990w. H NMR in
CDCl3, δ ppm: 2.32 (s, 3H), 3.85 (s, 3H), 6.97 (d, J = 9.08 Hz, 2H), 7.38 (d, J = 9.12 Hz, 2H), 12.78
(s, 1H). 13C NMR in CDCl3, δ ppm: 10.19, 55.64, 115.10, 117.63, 119.91, 134.01, 158.68, 159.28,
165.39.
(Z)-3-methyl-4-(2-phenylhydrazono)isoxazol-5(4H)-one (4): Crude yield: 87.1% Final Yield:
81.1% MP ºC: 185ꢀ186. Anal. Calc. (%) for C10H9N3O2 (Mι: 203,20 g/mol): C, 59.11; H, 4.46; N,
20.68; Found (%): C, 58.99; H, 4.76; N, 20.67. UVꢀvisible spectrum in ethanol solution 5 10ꢀ5
mol/L, λmax, nm (logε): 396(4.40); 255(4.00); 249(4.05); 200(4.05). I.R. spectrum in solid state,
(cmꢀ1); ν(NꢀH): 3207s, ν(CꢀH, arom.): 3118w, 3056w and 3031w; ν(CꢀH, aliph.): 2978w, ν(C=O):
1709s; ν(C=N): 1594w; ν(C=C): 1572sh and 1561w; ν(NꢀO): 991w. 1H NMR in CDCl3, δ ppm: 2.34
13
(s, 3H), 7.34ꢀ7.18 (m, 1H), 7.46ꢀ7.40 (m, 4H), 12.67 (s, 1H). C NMR in CDCl3, δ ppm: 10.20,
116.12, 121.18, 126.75, 129.82, 140.40, 159.39; 164.95.
(Z)-4-(2-(4-chlorophenyl)hydrazono)-3-methylisoxazol-5(4H)-one (5): Crude yield: 99.2%. Final
Yield: 95.7% MP ºC: 184ꢀ185 ºC. Anal. Calc.(%) for C10H8ClN3O2 (Mι: 237,64 g/mol): C, 50.54; H,
3.39; N, 17.68; Found (%): C, 50.26; H, 3.46; N, 17.55. UVꢀvisible spectrum in ethanol 5 10ꢀ5
mol/L, λmax, nm (logε): 397(4.43); 296(3.35); 250(4.06); 200(4.15). I.R. spectrum in solid state,
(cmꢀ1); ν(NꢀH): 3206s; ν(CꢀH, arom.): 3101w; 3093w, 3077w, 3070w; ν(CꢀH, aliph.): 2927w;
ν(C=O): 1715s; ν(C=N): 1593w; ν(C=C): 1552s; ν(NꢀO): 993m. 1H NMR in CDCl3, δ ppm: 2.33 (s,

Page 9 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
13
3H), 7.36 (d, J = 8.75 Hz), 7.41 (d, J = 8.75 Hz, 2H), 12.64 (s, 1H). C NMR in CDCl3, δ ppm:
10.21; 117.22, 121.81, 129.97, 132.04, 139.03, 159.28, 164.84.
(Z)-4-(2-(4-bromophenyl)hydrazono)-3-methylisoxazol-5(4H)-one (6): Crude yield: 69.2%. Final
Yield: 62.4% MP 194ꢀ195 ºC. Anal. Calc. (%) for C10H8BrN3O2 (Mι: 282,09g/mol): C, 42.58; H,
2.86; N, 14.90; Found (%): C, 42.47; H, 3.02; N, 14.70. UVꢀvisible spectrum in ethanol solution 5
10ꢀ5 mol/L, λmax, nm (logε): 399(4.45); 296(2.28); 251(4.05); 201(4.19). I.R. spectrum in solid state,
(cmꢀ1); ν(NꢀH): 3206s; ν(CꢀH, arom.): 3097w, 3069w and 3011w; ν(CꢀH, aliph.): 2926w; ν(C=O):
1
1715s; ν(C=N): 1591w; ν(C=C): 1559s; ν(NꢀO): 992w. H NMR in CDCl3, δ ppm: 2.33 (s, 3H),
13
7.30 (d, J = 2.13 Hz, 2H), 7.56 (d, J = 2.13 Hz, 2H), 12.62 (s, 1H). C NMR in CDCl3, δ ppm:
10.20, 117.51, 119.72, 121.92, 132.89, 139.50, 159.29, 164.82.
(Z)-4-(2-(3-methyl-5-oxoisoxazol-4(5H)-ylidene)hydrazinyl)benzoic acid (7): Crude yield: 63.1%.
Final Yield: 58.5% MP dec. at 226 ºC. Anal. Calc. (%) for C11H9N3O4 (Mι : 247,21 g/mol): C,
53.44; H, 3.67; N, 17.00; Found (%): C, 53.57; H, 4.86; N, 16.88. UVꢀvisible spectrum in ethanol
solution 5 10ꢀ5 mol/L, λmax, nm (logε): 396(4.51); 256(4.08); 211(3.97); 201(4.14). I.R. spectrum in
solid state, (cmꢀ1); ν(NꢀH): 3200w, ν(OꢀH): 3400ꢀ2200 range; ν(C=O): 1714s and 1673s; ν(C=N):
1
1606m; ν(C=C): 1556s; ν(NꢀO): 997w. H NMR in DMSOꢀd6, δ ppm: 2.26 (s, 3H), 7.75 (dd, J =
4.69, 2.13 Hz, 2H), 7.99 (dd, J = 4.69, 2.13 Hz, 2H), 12.78 (s, 1H), 12.92 (s, 1H). 13C NMR in
DMSOꢀd6, δ ppm: 9.99, 116.37, 122.05, 127.55, 130.73, 144.86, 159.97, 161.88, 166.58.
(Z)-4-(2-(4-acetylphenyl)hydrazono)-3-methylisoxazol-5(4H)-one (8): Crude yield: 93.4%. Final
Yield: 87.5% MP 198ꢀ199 ºC. Anal. Calc. (%) for C12H11N3O3 (Mι: 245,23 g/mol): C, 58.77; H,

New Journal of Chemistry
Page 10 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
4.52; N, 17.13; Found (%) C, 58.57; H, 4.35; N, 16.99. UVꢀvisible spectrum in ethanol solution 5
10ꢀ5 mol/L, λmax, nm (logε): 399(4.41); 276(3.89); 254(3.77); 246(3.79); 201(4.31). I.R. spectrum in
solid state, (cmꢀ1); ν(NꢀH): 3207w; ν(CꢀH, arom.): 3097w, 3060w, and 3002w; ν(CꢀH, aliph.):
1
2925w; ν(C=O): 1712s and 1678s; ν(C=N): 1601w and 1591w; ν(C=C): 1564w; ν(NꢀO): 996w. H
NMR in CDCl3, δ ppm: 2.36 (s, 3H), 2.62 (s, 3H), 7.48 (d, J = 8.76 Hz, 2H), 8.05 (d, J = 8.76 Hz,
2H), 12.65 (s, 1H). 13C NMR in CDCl3, δ ppm: 10.23, 26.51, 115.72, 123.22, 130.35, 134.82,
143.87, 159.35, 164.41, 196.39.
(Z)-4-(2-(3-methyl-5-oxoisoxazol-4(5H)-ylidene)hydrazinyl)benzonitrile (9): Crude yield: 96.4%.
Final Yield: 91.3% MP ºC: 185ꢀ186. Anal. Calc. (%) for C11H8N4O2 (Mι: 228,21g/mol) C, 57.89; H,
3.53; N, 24.55; Found (%): C, 57.70; H, 3.79; N, 24.12. UVꢀvisible spectrum in ethanol solution 2.5
10ꢀ5 mol/L, λmax, nm (logε): 391(4.50); 254(4.09); 201(4.15). I.R. spectrum in solid state, (cmꢀ1);
ν(NꢀH): 3210s; ν(CꢀH, arom.): 3091w 3064w; ν(CꢀH, aliph.): 2970w; ν(C≡N): 2223s; ν(C=O):
1720s; ν(C=N): 1589s; ν(C=C): 1560s; ν(NꢀO): 993m. 1H NMR in CDCl3, δ ppm: 2.36 (s, 3H), 7.52
13
(d, J = 8.78 Hz, 2H), 7.74 (d, J = 8.75 Hz, 2H), 12.59 (s, 1H). C NMR in CDCl3, δ ppm: 10.22,
109.34, 116.23, 118.20, 124.01, 133.94, 143.66, 159.26, 164.13.
(Z)-3-methyl-4-(2-(4-nitrophenyl)hydrazono)isoxazol-5(4H)-one (10): Crude yield: 97.9%. Final
Yield: 91.2% MP ºC: 177ꢀ178. Anal. Calc. (%) for C10H8N4O4 (Mι: 248,19 g/mol): C, 48.39; H,
3.25; N, 22.57; Found (%): C, 42.81; H, 3.51; N, 22.58. UVꢀvisible spectrum in ethanol solution 2.5
10ꢀ5 mol/L, λmax, nm (logε): 400(4.59); 297(3.74); 222(3.99); 202(4.20). I.R. spectrum in solid
state, (cmꢀ1); ν(NꢀH): 3205s; ν(CꢀH, arom.): 3108w, 3092 and 3009w; ν(CꢀH, aliph.): 2933w;
ν(C=O): 1720s ν(C=N): 1611w; ν(C=C): 1557s; ν(NꢀO): 994m. 1H NMR in DMSOꢀd6, δ ppm: 2.27

Page 11 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
(s, 3H), 7.86 (d, J = 9.27 Hz, 2H), 8.29 (d, J = 9.26 Hz, 1H), 12.85 (s, 1H). 13C NMR in DMSOꢀd6,
δ ppm: 10.02, 116.86, 123.83, 125.28, 143.96, 146.87, 160.07, 161.30.
(Z)-4-(2-(2-hydroxyphenyl)hydrazono)-3-methylisoxazol-5(4H)-one (11): Crude yield: 80.0%.
Final Yield: 71.4% MP: 209ꢀ210 ºC. Anal. Calc. (%) for C10H9N3O3 (Mι: 219,20 g/mol): C, 54.79;
H, 4.14; N, 19.17; Found (%): C, 52.89; H, 4.68; N, 18.08. UVꢀvisible spectrum in ethanol solution
2.5 10ꢀ5 mol/L, λmax, nm (logε): 425(4.36); 245(3.91); 202(4.13). I.R. spectrum in solid state,
(cmꢀ1); ν(NꢀH): 3194w; ν(OꢀH): 3400ꢀ2400 range; ν(C=O): 1717s; ν(C=N): 1599w; ν(C=C): 1536s;
1
ν(NꢀO): 1001m. H NMR in DMSOꢀd6, δ ppm: 2.26 (s, 3H), 6.94 (dd, J = 7.48, 1.26 Hz, 1H), 6.98
(d, J = 8.12,1H), 7.11 (dd, J = 7.43, 1.58 Hz, 1H), 7.60 (d, J = 8.07, 1H), 10.79 (s, 1H), 12.75 (s,
1H). 13C NMR DMSOꢀd6, δ ppm: 9.76, 114.62, 115.84, 120.22, 120.60, 127.04, 128.26, 146.30,
159.24, 164.19.
(Z)-4-(2-(2-chlorophenyl)hydrazono)-3-methylisoxazol-5(4H)-one (12): Crude yield: 96.3%. Final
Yield: 85.6% MP: 165ꢀ166 ºC. Anal. Calc. (%) for C10H8ClN3O2 (Mι: 237,64 g/mol): C, 50.54; H,
3.39; N, 17.68; Found (%): C, 50.24; H, 3.54; N, 17.55. UVꢀvisible spectrum in ethanol solution 2.5
10ꢀ5 mol/L, λmax, nm (logε): 395(4.20); 260(3.71); 252(3.80); 202(4.04). I.R. spectrum in solid
state, (cmꢀ1); ν(NꢀH): 3204m; ν(CꢀH, arom.): 3090w, 3067w and 3025w; ν(CꢀH, aliph.): 2928w;
1
ν(C=O): 1719s; ν(C=N): 1595w and 1586w; ν(C=C): 1556s; ν(NꢀO): 991w. H NMR in CDCl3, δ
ppm: 2.35 (s, 3H), 7.18 (dd, J = 7.99, 1.46 Hz, 1H), 7.37 (t, J = 7.82, 1H), 7.44 (d, J = 8.05, 1H),
7.79 (d, J = 8.25, 1H), 12.95 (s, 1H). 13C NMR CDCl3, δ ppm: 10.27, 116.13, 122.090, 123.33,
126.74, 128.26, 130.00, 137.07, 159.23,164.37. 4

New Journal of Chemistry
Page 12 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
(Z)-2-(2-(3-methyl-5-oxoisoxazol-4(5H)-ylidene)hydrazinyl)benzoic acid (13): Crude yield: 99.2%.
Final Yield: 95.7% MP: 220ꢀ221 ºC. Anal. Calc. (%) for C11H9N3O4 (Mι: 247,21 g/mol): C, 53.44;
H, 3.67; N, 17.00; Found (%): C, 53.49; H, 3.82; N, 16.87. UVꢀvisible spectrum in ethanol solution
2.5 10ꢀ5 mol/L, λmax, nm (logε): 399(4.42); 264(3.98); 226(4.08); 206(4.30). I.R. spectrum in solid
state, (cmꢀ1); ν(OꢀH): 3300ꢀ2300 range; ν(C=O): 1733s and 1675s; ν(C=N): 1603m and 1586w;
1
ν(C=C) 1551s; ν(NꢀO): 994s. H NMR in CDCl3, δ ppm: 2.26 (s, 3H), 7.30 (dd, J = 7.46, 1.03 Hz,
1H), 7.70 (dd, J = 7.39, 1.19 Hz, 1H), 7.92 (d, J = 8.23 Hz, 1H), 7.99 (d, J = 7.91, 1H). 13C NMR in
CDCl3, δ ppm: 9.91, 115.02, 116.02, 122.99, 124.90 , 131.26, 134.53, 142.55, 159.659, 162.13,
168.23.
(Z)-3-methyl-4-(2-(2-nitrophenyl)hydrazono)isoxazol-5(4H)-one (14): Crude yield: 97.1%. Final
Yield: 90.2% MP: 180ꢀ181 ºC. Anal. Calc. (%) for C10H8N4O4 (Mι: 248,19 g/mol): C, 48.39; H,
3.25; N, 22.57; Found (%): C, 48.45; H, 3.36; N, 22.45. UVꢀvisible spectrum in ethanol solution 2.5
10ꢀ5 mol/L, λmax, nm (logε): 410(4.32); 344(4.09); 281(3.99); 201(4.38). I.R. spectrum in solid
state, (cmꢀ1); ν(NꢀH): 3191m; ν(CꢀH, arom.): 3098w, 3077w; ν(C=O): 1728s; ν(C=N): 1610s;
1
ν(C=C): 1568s and 1556s; ν(NꢀO): 991w. H NMR in DMSOꢀd6, δ ppm: 2.32 (s, 3H), 7.43 (dd, J =
7.32, 1.22 Hz, 1H), 7.90 (dd, J = 7.32, 1.40 Hz, 1H), 8.13 (d, J = 8.49, 1H), 8.30 (d, J = 8.42, 1H),
13.86 (s, 1H). 13C NMR in DMSOꢀd6, δ ppm: 9.94, 116.88, 125.20, 126.00, 126.03, 135.35, 136.81,
136.59, 159.73, 162.35.
(Z)-4-(2-(3-chlorophenyl)hydrazono)-3-methylisoxazol-5(4H)-one (15): Crude yield: 95.5%. Final
Yield: 89.7% MP: 161ꢀ162 ºC. Anal. Calc. (%) for C10H8ClN3O2 (Mι: 237,64 g/mol): C, 50.54; H,
3.39; N, 17.68; Found (%): C, 50.27; H, 4.46; N, 17.26. UVꢀvisible spectrum in ethanol solution 2.5

Page 13 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
10ꢀ5 mol/L, λmax, nm (logε): 390(4.22); 260sh(3.91); 252(4.00); 202(4.28). I.R. spectrum in solid
state,
(cmꢀ1); ν(NꢀH): 3207s; ν(CꢀH, arom.): 3132w, 3088w, 3068w, 3031w; ν(CꢀH, aliph.):
2928w; ν(C=O): 1716s; ν(C=N): 1586sh; ν(C=C): 1564s; ν(NꢀO): 991w. 1H NMR in CDCl3, δ ppm:
2.33 (s, 3H), 7.24ꢀ7.19 (m, 2H), 7.35 (t, J = 8.06 Hz, 1H), 7.48 (t, J = 2.00 Hz, 1H), 12.55 (s, 1H).
13C NMR in CDCl3, δ ppm: 10.22, 114.41, 115.95, 122.31, 126.51, 130.82, 135.94, 141.54, 159.32,
164.64.
Biological studies
Tissue Culture: The HLꢀ60, promyelocytic leukemia cell line, was purchased from the American
Type Culture CollectionꢀATCC (Manassas, VA). This cell line was collected from peripheral blood
cells of a 36ꢀyear old Caucasian female with acute promyelocytic leukemia (APL) and they grow as
a suspension culture. The predominant cell population consists of neutrophilic promyelocytes26,27. In
the laboratory, these cells were stored under liquid nitrogen until use. Then, they were thawed by
gentle stir of their containers (vials) during 2 minutes in a water bath at 37 ºC. After thawing, the
content of each vial of cells was transferred into a 25 cm2 tissue culture flask, Tꢀ25, diluting with 10
mL of RPMI 1640 containing 1 mmol/L Lꢀglutamine (GIBCO/BRL, Gaithersburg, MD),
supplemented with 10% (v/v) of fetal bovine serum (FBS) and 1% (w/v) penicillin/streptomycin.
The Tꢀ25 flask (2 x 106 viable cells) was observed under the microscope, followed by incubation in
a humidified 5% CO2 at 37 °C. Three times a week, the culture cells was diluted under same
conditions in order to maintain a density of 5 x 105 cells/mL and harvested in the exponential phase
of growth. The cell viability was assessed by the Trypan Blue exclusion test (Life Technologies) and
manually counted using a hemocytometer.

New Journal of Chemistry
Page 14 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
Cytotoxicity/MTT Assay: This colorimetric assay measures the reduction of 3ꢀ(4,5ꢀ
dimethylthiasolꢀ2ꢀyl)ꢀ2,4,ꢀdiphenyltetrazolium bromide (MTT) by mitochondrial succinate
dehydrogenase. The MTT enters the cells and passes to the mitochondria where is reduced to
formazan, an insoluble colored product. In order to dissolve the formazan, the cells are then treated
with an organic solvent (DMSO or isopropanol) and the obtained solution is measured
spectrophotometrically between 530ꢀ650 nm range28,29
.
Aliquots of 200 ꢁL of HLꢀ60 cell suspension (5x 105 cells/mL) were seeded into 96 well
polystyrene tissue culture plates, an aliquot of 1.0 ꢁL of isoxazolone (2.0, 5.0, 8.0, 12.0, 20.0, 30.0,
40.0, 60.0, 80.0 and 100.0 ꢁM) and, carefully, were also added to each well using DMSO:EtOH
(1:4) as solvent and vehicle control. Cells incubated in culture medium alone served as a control for
cell viability (untreated wells). Cells were placed in the humidified 5% CO2 incubator at 37°C for
24 hours. After incubation, 10 ꢁL aliquots of MTT solution (5 mg/mL in PBS) were added to each
well and reꢀincubated for 4 hours at 37° C, followed by low centrifugation at 800 rpm for 5 minutes.
Then, the 200 ꢁL of supernatant culture medium were carefully aspirated and 200 ꢁL aliquots of
dimethylsulfoxide (DMSO) were added to each well in order to dissolve the formazan crystals,
followed by incubation during 10 minutes. The culture plate was placed on an Emax model microꢀ
plate reader (Molecular Devices) and the absorbance was measured using a 650 nm filter. The
amount of color produced is directly proportional to the number of viable cells. All assays were
performed four times with three replicates each one processed independently and means ± SD
values were used to estimate the cell viability. Cell viability rate was calculated as the percentage of
MTT absorption as follows:
% survival = (mean experimental absorbance/mean control absorbance) × 100.

Page 15 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
The isoxazolone concentration was plotted against the corresponding percentage (%) of cell viability
obtained with MTT assays, and the 50% inhibitory concentration (IC50) was calculated by nonꢀlinear
regression using fitting with Sigmaplot©11 program from Systat Software, Inc. As positive control
Paclitaxel was used, see Figure S1.
Semiquantitive duplex RTꢀPCR: Total RNA was isolated from cells using Chomczynski’s
method30 and quantified by measuring of its optical density at 260 nm. First strand cDNA was
synthesized with 2 µg of total RNA in a buffer containing 100 pmol of oligo dT, dNTPs 0.25 mM,
DTT 10 mM, 20U of RNase Inhibitor and 50U of MꢀMLV Reverse Transcriptase (Invitrogen). The
mixture was incubated for 1 h at 42◦ C. The PCR amplifications were carried out using GoTaq
Green Master Mix (Promega). For quantitative analysis of Bclꢀ2, p21WAFꢀ1, and Bax mRNAs,
human glyceraldehydeꢀ3ꢀphosphate dehydrogenase (G3PDH) gene served as the internal control for
calculation of the densitometry results. The primers were designed based on the sequences of the
gene bank:
Bclꢀ2
(F), 5`ꢀTGCACCTGACGCCCTTCACꢀ3`
(R), 5`ꢀAGACAGCCAGGAGAAATCAAACAGꢀ3`
(F), 5`ꢀACCAAGAAGCTGAGCGAGTGTCꢀ3`
(R), 5`ꢀACAAAGATGGTCACGGTCTGCCꢀ3`
(F), 5`ꢀGGꢀGGACAGCAGAGGAAGACꢀ3`
(R), 5`ꢀCGGCGTTTGGAGTGGTAGAꢀ3`
(F), 5`ꢀACCCAGAAGACTGTGGATGGꢀ3`
(R), 5`ꢀCCCCTCTTCAꢀAGGGGTCTACꢀ3`
Bax
p21WAFꢀ1
GAPDH

New Journal of Chemistry
Page 16 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
The PCR products were separated using 1.5% agarose gels. After the gels were stained with
ethidium bromide, gel images were obtained and the densities of the products were quantified using
GelꢀPro Analyzer 4.0 Software (Media Cybernetics, USA). All experiments were repeated at least
four times. Figures showing the images of the electrophoresis of PCR products correspond to one of
several repetitions of the three independent experiments performed, and were chosen because it
clearly shows the bands under study; the images used for quantification do not necessarily
correspond to those shown, because unsaturated images were chosen for proper quantification.
Statistical Analysis: Data were compared by oneꢀway analysis of variance Student’s tꢀtest to
determine statistical significance (Sigmaplot©11). Each experiment is performed in triplicate on 4
occasions. Results are expressed as mean ± SD. Differences with P values of < 0.05 were considered
significant.
Computational Details. The Gaussian 09 computational package31 was used to perform groundꢀ
state and transitionꢀstate geometry optimization calculations employing Becke’s threeꢀparameter
hybrid exchange functional and the Lee−Yang−Parr nonlocal correlation functional B3LYP32–34 and
the 6ꢀ31G* basis set was used for C, N, O, Cl, Br, and H atoms35. The Hessian was computed of all
investigated compounds in order to characterize them as minima and no imaginary values were
found. Timeꢀdependent DFT (TDDFT) calculations were also performed using this methodology,
and the first 60 singlet excited states were calculated. Calculations by the firstꢀprinciples method
were used to obtain accurate excitation energies and oscillator strengths. We modelled the effect of
the solvent with the polarizable continuum model (PCM) using ethanol as solvent36 for the
optimization and TDDFT calculations, see Figure S2 for all the FMO involved in the transitions.

Page 17 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
RESULTS AND DISCUSSION
Synthesis
Under inert atmosphere and prolonged reflux in ethanol the precursor compounds βꢀ
hydrazodiketone, RꢀC6H4NHN=C(COCH3)(COOCH2CH3) (R= 4ꢀOH (1), 4ꢀOCH3 (2), 4ꢀCH3 (3),
4ꢀH (4), 4ꢀCl (5), 4ꢀBr (6) 4ꢀCO2H (7), 4ꢀCOCH3 (8), 4ꢀCN (9), 4ꢀNO2 (10), 2ꢀOH (11), 2ꢀCl (12),
2ꢀCO2H (13), 2ꢀNO2 (14), 3ꢀCl (15)) reacts with hydroxylamine hydrochloride affording in good
yield a series of yellowꢀorange oxazolones, 1ꢀ15, see Scheme 1. The low yield observed for 1 might
be explained due to the presence of the hydroxyl substituent group in para position which enhances
the solubility of this compound. In concordance with the scheme, we propose that these reactions
form an intermediate oxime that after a long time refluxed suffers a subsequent cyclization reaction
yielding the corresponding product. The information contrasts with the literature, where it becomes
established that the hefts of reactions require strong bases, strong mineral acids, high temperatures14–
16. Contrarily, the reactions presented here occur in one step, under mild conditions of reflux in
ethanol during 18 H, catalyzed by a weak organic acid, HOAc, and with high regioselectivity. The
purification was performed in every case via a recrystallization from hot ethanol. All the synthetized
compounds were characterized using elemental analysis and spectroscopic techniques vide infra.
Additionally, suitable crystals for XꢀRay diffraction were obtained and it was possible to resolve the
structure of 4, 12, 15. Compound 4 was previously reported25 and our structure is in concordance
with this previous report, whereas the crystallization in this case was performed allowing the
solution to slowly evaporate the ethanol.

New Journal of Chemistry
Page 18 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
Scheme 1. Proposed reaction mechanism.
Structure description
For compounds 4, 12 and 15 suitable crystals were obtained for XꢀRay diffraction (as shown in
ꢀ
Figure 2). Those compounds showed C2/c, P1, P2(1)/c spatial groups, respectively. The unitary cell
of the compounds showed Z=8, Z=2 and Z=8, respectively. An ORTEP diagram for each compound
can be found in Figure 2. Surprisingly, compound 15 showed 4 pairs of isoxazolone molecules
slightly different, which are labeled as 15a and 15b. Table S1 showed the most important distances
and angles for these molecules. In the theoretical section a comparison between these experimental
distances and the calculated distances will be made. The observed distances for the studied
compounds are in good agreement with similar reported molecules37–39. For further details please
see the SI. All the molecules showed almost a planar structure, with a dihedral angle smaller than 3°.

Page 19 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
Figure 2. ORTEP diagram with a 50% of probability for compounds 4, 12 and 15.
Spectroscopic studies
The UVꢀvisible spectra of compounds 1ꢀ15, see Table 2, registered in ethanol solutions exhibit two
principal absorption bands in the range 427ꢀ222 nm. We believe that the first observed band, λ1,
located in 427ꢀ390 nm range, is associated with a π*←π transition due to the conjugated aromatic
heterocyclic system. The second observed band, λ2, placed in 343ꢀ254 nm range, probably, is due to
π*←π transition centered in the chromophore ꢀNꢀNꢀ. Other high energy absorption bands, located
around 250 nm and lower, may be due to internal transition in the aromatic rings. The expected
behaviour for the first three calculated transitions was corroborated via theoretical calculations, vide
infra.
Table 2. UVꢀvisible absorption bands (in nm) of 1ꢀ15 in ethanol solution, calculated extinction
coefficient (logε) in parentheses and their corresponding Hammett parameters (σ).

New Journal of Chemistry
Page 20 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
Comp.
1
σ
λ1
λ2
λ3
λ4
λ5
ꢀ0.37 427(4.40) 300(3.43) 252(4.00)
ꢀꢀꢀ
ꢀꢀꢀ
2
ꢀ0.27 421(4.42) 303(3.42) 252(4.02) 237h(4.02)
ꢀꢀꢀ
3
4
5
ꢀ0.17 405(4.37) 260h(3.94) 253(4.02)
396(4.40) 255(3.99) 249(4.05)
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
0
0.23 397(4.42) 258h(3.95) 250(4.05) 238h(3.96)
0.23 399(4.45) 260h(3.97) 251(4.06) 237h(3.97)
6
7
ꢀꢀꢀ
ꢀꢀꢀ
0.45 396(4.50) 265h(4.04) 256(4.07)
ꢀꢀꢀ
8
9
10
0.5
399(4.36) 276(3.89) 254(3.89) 246(3.79) 222(3.94)
0.66 391(4.50) 254(4.09)
0.78 400(4.59) 297(3.73)
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
11
12
13
14
15
425(4.37) 259h(3.78) 245(3.91)
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
395(4.17) 260(3.70) 252(3.80) 246h(3.79)
399(4.41) 273h(3.86) 264(3.97) 227(4.07)
410(4.32) 343(4.09) 281(3.99) 248h(4.10)
390(4.32) 259h(3.91) 252(3.99)
ꢀꢀꢀ
A Hammett correlation plot was performed for all the observed UVꢀVis transitions, but no
correlation was observed (data not shown). Nevertheless, a trend is observed for the transitions,
where λ1 and λ2 show a bathochromic shift which might be due to the effect that has the R group
over the azo ring.
The infrared spectra of 1ꢀ15 could be found in the SI. These spectra on KBr disc exhibit the typical
aromatic and aliphatic ν(CꢀH) absorptions bands, around 3000 cmꢀ1, respectively. On the other hand,
at least three intense absorption bands emerge in the range 1611ꢀ1495 cmꢀ1. Those signals are typical

Page 21 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
for the ν(C=N) and ν(C=C) chromophores presents on the oxazolone and aromatic rings. The
substituent groups, R, of compound 7, 8 and 13 show the characteristic carbonyl stretching mode,
ν(C=O), at 1673, 1678 and 1676 cmꢀ1, respectively. The ν(NꢀO) stretching mode of the oxazolone
ring may be found closely around 1250 cmꢀ1. The most remarkable signal correspond to the ν(NꢀH)
signal that can be observed in almost all the compound located between 3188ꢀ3212 cmꢀ1, however in
case of compounds 1, 11 and 13 this band is not observed due to the presence of the intense ν(OꢀH)
band at the same frequency. In case of compound 4 despite the presence of the intense ν(OꢀH)
signal, the ν(NꢀH) was observed at higher frequency. This spectral information is in concordance
with those given previously in literature40.
1
Tables 3 and S3 shows the chemical shift and the assignment of each signals observed in the Hꢀ
13
NMR and CꢀNMR spectra of compounds 1ꢀ15, respectively. Both series of spectra were recorded
in CDCl3 or DMSOꢀd6 solutions and all the signals were successfully assigned with help of the
HMBC spectra. Figure 3 exhibits a typical HMBC spectrum obtained for 3. For compound 13 the
signals for NꢀH and 2ꢀCOOH were not clearly observed, however they are attributed as a broad
signal between 2.50ꢀ7.00 ppm. For the rest of the compounds all the expected signals were
observed. The first signal attributed to the ꢀCH3 group, located between 2.35ꢀ2.23 ppm. The
aromatic signals were observed between 6.66ꢀ8.07 ppm. In case of the NꢀH signal it was observed
for all compounds over 11.16 ppm. Furthermore, the signal expected for the substituent over the azo
benzene ring was also observed at different locations, depending on the character of the substituent,
see Table 3. The integrals for all the mentioned signals are in concordance with the number of
13
expected protons. In case of the CꢀNMR signals, it was possible to do the assignation of all the
expected signals for all the synthetized compounds. The most relevant finding in this sense is the
location of the carbons located in the oxazolone ring, which are common to all the studied
compounds for C1, C2, C3 and C4, see Table S2. The conjugation of the fragment ꢀN=CꢀC=NꢀNHꢀ

New Journal of Chemistry
Page 22 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
that binds the azophenyl substituted ring is interrupted in the ꢀNHꢀAr. This situation prevents any
important influence in any measured spectroscopical property.
Figure 3. HMBC spectrum of compound 3 in CDCl3 showing the HꢀC interactions.
Table 3. Chemical shift signals (δ: ppm) in the 1HꢀNMR spectra of 1ꢀ15 in CDCl3.

Page 23 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
CH3
H(Ar)
7.69 – 7.38 (m, 2H)
NꢀH
H(R)
2.23 (s, 3H)
12.80 (s, 1H) 9.76 (s, 1H)
12.78 (s, 1H) 3.84 (s, 3H)
12.69 (s, 1H) 2.38 (s, 3H)
1
2
6.95 – 6.66 (m, 2H)
7.40 – 7.35 (m, 2H)
2.32 (s, 3H)
2.32 (s, 3H)
2.34 (s, 3H)
2.33 (s, 3H)
2.33 (s, 3H)
2.26 (s, 3H)
2.35 (s, 3H)
2.35 (s, 3H)
2.27 (s, 3H)
6.99 – 6.94 (m, 2H)
7.32 (d, J = 8.5 Hz, 2H)
7.23 (d, J = 8.4 Hz, 2H)
7.47 – 7.40 (m, 4H) )
7.28 (m, 1H)
3
12.67 (s, 1H)
12.64 (s, 1H)
12.65 (s,1H)
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
4
7.43 – 7.39 (m, 2H)
5
7.38 – 7.34 (m, 2H)
7.64 – 7.47 (m, 2H)
6
7.34 – 7.28 (m, 2H)
8.02 – 7.96 (m, 2H)
12.78 (s, 1H) 12.93 (s, 1H),
12.64 (s, 1H) 2.61 (s, 3H)
7
7.78 – 7.72 (m, 2H)
8.07 – 8.02 (m, 2H)
8
7.50 – 7.46 (m, 2H)
7.73 (d, J = 8.8 Hz, 2H)
7.51 (d, J = 8.8 Hz, 2H)
8.34 – 8.26 (m, 1H)
12.58 (s, 1H),
11.16 (s, 1H),
ꢀꢀꢀ
ꢀꢀꢀ
9
10
7.91 – 7.83 (m, 1H)
7.60 (dd, J = 8.1, 1.5 Hz 1H)
7.15 – 7.08 (m, 1H)
2.26 (s, 3H)
2.34 (s, 3H)
12.75 (s, 1H) 10.79 (s, 1H),
11
12
7.01 – 6.91 (m, 2H)
7.78 (dd, J = 8.3, 1.2 Hz, 1H)
7.43 (dd, J = 8.0, 1.2 Hz, 1H)
7.40 – 7.33 (m, 1H)
12.94 (s, 1H)
No Obs.
ꢀꢀꢀ
7.18 (td, J = 8.0, 1.5 Hz, 1H)
7.99 (dd, J = 7.9, 1.3 Hz, 1H)
7.92 (d, J = 8.2 Hz, 1H)
7.74 – 7.66 (m, 1H)
2.26 (s, 3H)
No Obs.
13
7.38 – 7.22 (m, 1H)
8.29 (d, J = 8.4 Hz, 1H)
8.12 (d, J = 8.4 Hz, 1H)
7.90 (t, J = 7.9 Hz, 1H)
7.43 (t, J = 7.8 Hz, 1H)
7.49 (t, J = 2.0 Hz, 1H)
7.36 (td J = 8.0, 5.2 Hz, 1H)
7.23 (dddd, J = 10.6, 8.0, 2.0, 0.8 Hz, 2H)
2.32 (s, 3H)
2.34 (s, 1H)
13.85 (s, 1H)
12.56 (s, 1H),
ꢀꢀꢀ
ꢀꢀꢀ
14
15

New Journal of Chemistry
Page 24 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
Theoretical calculations
In order to perform a better description of the studied compounds DFT and TDDFT calculations
were performed for all the synthetized compounds. All the calculated geometrical parameters show a
good agreement with the obtained XꢀRay diffraction distances, see Table 4. When all the studied
compounds are compared, it can be observed that the calculated bond distances remain almost
unaltered as the substituent is changed over the azophenyl ring. Furthermore all the molecules show
a planar structure.
Table 4. Table 4. Selected bond distances for compounds 1ꢀ15 in Angstroms, compared with the
corresponding distances of the XꢀRay diffraction in brackets and the XRD values of each
measurement in parentheses.
Comp.
CꢀR
1.368
C6ꢀN2
1.407
1.406
1.407
N2ꢀN1
1.308
1.309
1.307
N1ꢀC1
1.317
1.315
1.317
<N1N2HO2
O3ꢀN4
1.449
0
1
2
1.51
0
1.449
1.364
0
1.449
3
1.085 [0.930]
1.755
1.406 [1.420(2)] 1.310 [1.313(2)] 1.314 [1.315(2)]
0 [4.12]
1.449 [1.475(3)]
1.45
4
1.404
1.403
1.401
1.401
1.4
1.311
1.311
1.314
1.314
1.316
1.317
1.311
1.313
1.313
1.31
0
5
1.901
0
1.45
6
1.484
0
1.45
7
1.498
1.311
1.309
1.308
1.313
0
1.45
8
1.433
0
1.45
9
1.467
1.398
1.403
0
0
1.451
10
11
12
13
14
15
1.37
1.448
1.751 [1.732(3)] 1.404 [1.395(3)] 1.312 [1.307(2)] 1.313 [1.296(3)]
0 [4.22]
0
1.452 [1.470(3)]
1.449
1.54
1.406
1.399
1.31
1.314
1.306
1.474
1.317
-3.6
1.454
1.756 [1.738(4)*] 1.404 [1.414(4)*] 1.312 [1.307(3)*] 1.312 [1.305(5)*]
0 [3.65*]
1.45 [1.537(4)*]
*Mean measured distance.
The isosurface of the frontier molecular orbitals were plotted for all the studied compounds, see
Figure S1 in the SI. As can be observed the localization of those orbitals remains unaltered as the

Page 25 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
substituent is changed in the azophenyl ring from electronꢀdonating to electronꢀwithdrawing
substituent. The HOMO is in all cases mainly localized over the two rings (oxazolone and azophenyl
ring). On the other hand the LUMO is mainly localized over the oxazolone ring and over the =Nꢀ
NHꢀ bridge. The HOMOꢀLUMO gap (HLG) does not vary dramatically between the studied
compounds, as might be expected due to the similarity observed in the UVꢀVis spectra, vide supra
.
In this sense the HLG has a value of around 3.3eV for all the studied compounds.
In order to perform a better characterization of the UVꢀVis spectra, TDDFT calculations were
performed, see Table S3 in the SI. As can be observed, in all the studied compounds three main
absorption bands are observed. In the TDDFT calculations the same number of transitions was
observed. The first calculated transition corresponds in every case to a HOMOꢀLUMO transition
(π*←π). Almost every second calculated transition goes from an occupied orbital to the LUMO+1
(π*←π). The third calculated transition does not show any trend. As observed in the first and second
transition a small dependency on the change of the substituent over the azophenyl ring is observed,
which explains the small variation between the observed UVꢀVis absorption profiles.
To make the first approach of the reactivity that this compounds might show, the estimated reactivity
indexes (electronic chemical potential (
reported in Table 5. In all cases no clear tendency on the variation of the global index of reactivity
was observed. is a measure of the resistance of a chemical species to change its electronic
configuration. It is thought as an indicator, together with electronegativity, of the chemical reactivity
and stability of systems. The highest value for is shown by 14, which has the ꢀnitro substituent,
which is the most electronꢀwithdrawing substituent. On the other hand, the smallest value is shown
by 1 which has the most electronꢀdonating substituent. characterizes the tendency of escaping of
electrons from the equilibrium system; it is then related to the electronic charge rearrangement
ꢀ), chemical hardness (η) and electrophilicity (ω
))41–43 are
η
η
o
ꢀ

New Journal of Chemistry
Page 26 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
associated to any chemical process. The lowest
substituent, and the highest is shown by 3.
ꢀ is shown by 14, which has the most electron donor
ꢀ
Table 5. HOMO and LUMO energies, HLG and reactivity indexes, electronic chemical potenꢀ
tial (
ꢀ
), chemical hardness (
η
) and electrophilicity (
ω), all in eV.
Comp. HOMO LUMO HLG
ꢁ
η
ω
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
-6.18 -2.89 3.29 -4.54 1.65
-6.33 -2.82 3.51 -4.58 1.76
-6.07 -2.86 3.21 -4.47 1.61
6.25
5.96
6.21
6.48
6.94
6.99
7.43
7.43
7.88
8.55
6.17
6.49
6.59
7.42
6.93
-6.5
-3.01 3.49 -4.76 1.75
-6.57 -3.16 3.41 -4.87 1.71
-6.54 -3.17 3.37 -4.86 1.69
-6.8
-3.34 3.46 -5.07 1.73
-6.75 -3.33 3.42 -5.04 1.71
-6.95 -3.49 3.46 -5.22 1.73
-7.13 -3.7
-6.27 -2.88 3.39 -4.58
-6.6 -3.03 3.57 -4.82 1.79
-6.54 -3.05 3.49 -4.8 1.75
-6.98 -3.37 3.61 -5.18 1.81
-6.7 -3.18 3.52 -4.94 1.76
3.43 -5.42 1.72
1.7
Electrophilicity index,
ω
, could give information comparing two molecules in which one is an
. In this case the highest
was found for compound 10, which has in position para a nitro group which is the most
electrophile (or nucleophile) and this is indicated by a higher (or lower)
value of
ω
ω
electronꢀwithdrawing substituent. On the other hand the smallest value of
ω was found for
compound 2.
Biological essays
Compounds 1 to 15 were subjected to in vitro biological studies by incorporating them to cultures of
leukemia HLꢀ60 cells. The cytotoxic effect was measured after exposure of the cells to the

Page 27 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
isoxazolones for 24 h, and this effect was expressed as the relative change on the cell viability
compared to the control samples (untreated and vehicle treated cells). The IC50 values were obtained
from doseꢀresponse curves and they fall in a wide range of concentrations, see Table 6. The
isoxazolones 5, 10, 11 and 15 were the most active, with IC50 values between 73 and 83 µM. Each
of four compounds mentioned, which showed the highest cytotoxic activity toward HLꢀ60 cells,
were subject of additional biological studies.
Table 6. Cytotoxic activity of the isoxazolones (IC50 values) on HLꢀ60 cells and Standard Deviation
in parenthesis.
IC50 (µM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
111 ( 14 )
606 ( 88 )
283 ( 76 )
160 ( 32 )
81 ( 10 )
167 ( 42 )
142 ( 21 )
ND ( ND )
124 ( 42 )
73 ( 7 )
83 ( 24 )
168 ( 37 )
414 ( 41 )
ND ( ND )
77 ( 6 )
*ND: Not Determined.
Effect of the isoxazolones on the expression of Bclꢀ2, Bax and p21WAFꢀ1. We also explored the effect
of isoxazolones 5, 10, 11 and 15 on the expression genes involved in apoptosis and cell cycle
progression. Apoptosis is an active process, named also programmed cell death, which occur in cells

New Journal of Chemistry
Page 28 of 35
View Article Online
DOI: 10.1039/C5NJ02604K
of multicellular organism in response to a variety of agents including heat, radiation, nutrient
deprivation, viral infection, hypoxia, increased intracellular calcium concentration, or chemical
exposition (including anticancer drugs)44. This implicates, inter alia, the balanced transcriptional
control of antiꢀapoptotic and proꢀapoptotic genes, such as Bax and Bclꢀ2. Within the
antiproliferative mechanisms is also important to check the expression levels of p21WAFꢀ1, a genic
product associated with the inhibition of the proliferation by cell cycle arrest; which we have also
included in this study45–47. The protein p21WAFꢀ1 is a potent inhibitor of cyclinꢀdependent kinases
(CDKs), in general also named cyclinꢀdependent kinase inhibitor (CKI) and can inhibit the
phosphorylation of tumor suppressor proteins such as retinoblastoma protein (Rb); allowing these
last exercise its oversight role on cell proliferation48,49. Thus, a stimulus that increase the expression
of p21WAFꢀ1 signalize toward the cell cycle arrest and is therefore antiproliferative.
In order to determine the effect of isoxazolones 5, 10, 11 and 15 on Bax, Bclꢀ2 and p21WAFꢀ1 mRNA
expression level, we performed a semiꢀquantitative duplex RTꢀPCR. Treatment of HLꢀ60 cells
during 24 h with compounds 5 and 10 at a concentration similar to its IC50 values respective, 80 and
70 µM, induced an important decrease on the expression of Bclꢀ2 to around 47% (Figures 4ꢀA, lane
3; and Figures 4 D, 5) and 43% (Figures 4 A, lane 4; and Figures 4 D, 10) relative to the control (P<
0.05), respectively. Meanwhile, treatment with compounds 11 and 15 at concentrations of 80 and 75
µM during 24 h, increased the expression of Bclꢀ2 to 309% (Figures 4 A, lane 5; and Figures 4 D,
10) and 207% (Figures 4 A, lane 6; and Figures 4 D, 15) relative to the control (P< 0.05),
respectively. Meanwhile, compounds 5, 10 and 15 elicited no statistically significant increases to
164, 124 and 119% on Bax mRNA levels, respectively (Figures 4 B, lanes 3, 4, and 6; and Figures 4
E, 5, 10 and 15). Conversely, isoxazolone 11 induced a decrease to 54% of Bax mRNA levels
(Figures 4 B, lane 5; and Figures 4 E, 11) relative to the control (P< 0.05). On the other hand, we

Page 29 of 35
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ02604K
found that isoxazolones 5, 10, 11 and 15 elicited no statistically significant decrease of the levels of
p21WAFꢀ1 mRNA expression to 58, 81, 79 and 74% relative to the control, respectively (Figures 4 C,
lanes 3ꢀ6; and Figures 4 F, 5, 10, 11 and 15).
These results suggest that the cytotoxic activity of the isoxazolones 5 and 10 would be the sum of
positive modulation on signals to promotion of apoptosis and negative modulation on antiꢀapoptotic
mechanisms, without a clear consequence on cell cycle. While, the cytotoxic effect observed for
isoxazolones 11 and 15 seems to be through mechanisms other than those analyzed in this work.
Furthermore, for this family of compounds, it is observed that the electrophilicity index values
calculated by means of DFT get smaller while the biological activity gets bigger, which might
correlate the observed activity against cancer cell lines and the calculated reactivity indexes.
Figure 4. Effect of the isoxazolones 5, 10, 11 and 15 on mRNA expression levels of Bclꢀ2 (A and
D), Bax (B and E) and p21WAFꢀ1 (C and F) in HLꢀ60 cells examined by semiquantitive duplex RTꢀ
PCR. PCR products were separated using 1.5% agarose gel electrophoresis, the arrows indicate the
PCR products for GAPDH (600 bp, upper arrow in all cases), Bclꢀ2 (293 bp), Bax (332 bp) and
p21WAFꢀ1 (156 bp). Lane 1, untreated control (C); line 2, vehicle control (V); lane 3, treatment with