New 3,4,5-trisubstituted isoxazole derivatives with potential biological properties

Article abstract of DOI:10.1039/c4nj02427c

Synthesis of (E)-3,5-dimethyl-4-(R-phenyldiazenyl)isoxazoles was carried out by reacting β-diketohydrazones (R-C₆H₄-NHNC(COCH₃)₂) with hydroxylammonium chloride, where R = 4-N(CH₃)₂(1), 4-O

Full text of DOI:10.1039/c4nj02427c

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, A. Mesias, X. Zárate and E.
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 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C

New Journal of Chemistry
Page 2 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
New 3,4,5-Trisubstituted Isoxazoles Derivatives with potential
biological properties
a,*
b
c
c
Carlos Bustos , Elies Molins , Juan-Guillermo Cárcamo , Marcelo N. Aguilar , Christian
a
a
d
a
e
Sánchez , Ignacio Moreno-Villoslada , Hiroyuki Nishide , Angela Mesías-Salazar , Ximena Zarate ,
f,*
Eduardo Schott
a
Instituto de Ciencias Químicas, Universidad Austral de Chile, Las Encinas 220, Campus Isla Teja, Valdivia, Chile.
b
Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus de la Universitat Autònoma de Barcelona,
0
8193 Bellaterra, Spain.
c
Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile.
d
Department of Applied Chemistry, School of Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
e
Dirección de Postgrado e Investigacion, Universidad Autónoma de Chile, Av. Pedro de Valdivia 641 , Santiago, Chile.
f
Laboratorio de Bionanotecnologia, Universidad Bernardo O’Higgins, General Gana 1702, Santiago, Chile.
*email: maschotte@gmail.com, Phone/Fax: 56-2-24772243.
Keywords: Isoxazole, cytotoxic effect, synthesis, DFT, X-Ray
Abstract
Synthesis of (E)-3,5-dimethyl-4-(R-phenyldiazenyl)isoxazoles was carried out by reaction of β-
diketohydrazones, of formula R-C
R= 4-N(CH (1), 4-OH(2), 4-CH
CO CH CH (9), 4-COCH (10), 4-CN(11), 4-NO (12), 4-CH CO CH CH (13), 3-Cl(14), 2-OH(15),
6
H
4
-NHN=C(COCH
3 2
) with hydroxilammonium chloride, being
3
)
2
3
(3), 4-OCH (4), 4-H(5), 4-Cl(6) 4-Br(7), 4-CO2H(8), 4-
3
2
2
3
3
2
2
2
2
3

Page 3 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
2
2 2
-Cl(16), 2-NO (17), 2-CO H(18). All compounds were characterized by EA and spectroscopic
methods. The crystalline structure of two compounds was solved by X-rays diffraction method.
DFT and TDDFT computations were performed in order to characterize the molecular geometry
and the molecular orbitals involved in the transitions. Besides, a biological activity study was
performed to evaluate the toxicity of compounds towards human promielocytic leukemia cell line,
HL-60, using MTT reduction method. The IC50 values are in a wide concentrations range, 86 - 755
µM. The isoxazoles (3) and (6) were the most cytotoxic. Expression analysis in HL-60 cells were
carried out with compounds (3) and (6) by RT-PCR, in order to determine the effect on the
WAF-1
expression levels of mRNA codifying for the genes Bcl-2, Bax and p21
. The isoxazole (3)
induced a decrease of the expression of Bcl-2, whereas the isoxazole (6) showed an opposite
behaviour. However, these isoxazoles had no effect on mRNA levels of Bax. On the other hand,
WAF-1
both isoxazoles increased the levels of p21
. These results suggest that the cytotoxic activity of
the isoxazole (3) would be the sum of effects triggered on promotion of apoptosis and cell cycle
arrest, whereas for isoxazole (6) would be mainly through cycle cell arrest.
1
. Introduction
The biological activity of substituted isoxazoles has made them a focus in medicinal chemistry in
1
2
the last years , because they are potent selective agonists of human cloned dopamine D4 receptor ,
3
4
exhibit GABA
A
antagonist activity , as well as analgesic, antiinflamatory , antimicrobial and
5
6,7
8
antifungal activity. Also, they can inhibit COX-2 action and present antinociceptive and
9
anticancer activity.
10
Many synthetic methods have been used to obtain substituted isoxazoles including reactions of
11
12
hydroxylamine with 1,3-dicarbonyl compounds , α,β-unsaturated carbonyl compounds and α,β-
1
3
14
15,16
unsaturated nitriles . The reaction of an oxime-derivative dianion with an ester or amide
also

New Journal of Chemistry
Page 4 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
yields isoxazoles. On the other hand, [3+2] cycloaddition reactions between alkynes and nitrile
1
7,18
oxides have also been developed . However, these methods often require strong bases, strong
mineral acids or high temperatures or provide poor regioselectivity. Functionalizations of isoxazoles
1
9
are also known . Recently, three new methods involving a cascade reaction of nitrocompounds
2
0
21,22
with oxetane-3-one and gold/silver/copper-catalyzed
synthesis of di-substituted and tri-
sustituted isoxazoles by cyclization of alkynyloxime ethers have been reported.
The method reported here allows obtaining isoxazoles under mild conditions by reaction of β-
diketohydrazones with hydroxylammonium chloride. In this work, we present an extensive report
on the synthesis and characterization of a new series of 3,4,5-trisubstituted isoxazoles using (3-(2-
23-27
(4-R-phenyl)hydrazinylidene)pentane-2,4-diones
as precursors, where R= 4-N(CH
3
)
2
(1), 4-
OH(2), 4-CH (3), 4-OCH (4), 4-H(5), 4-Cl(6) 4-Br(7), 4-CO2H(8), 4-CO
3
3
2
CH CH
2
3
(9), 4-
COCH (10), 4-CN(11), 4-NO (12), 4-CH CO CH CH (13), 3-Cl(14), 2-OH(15), 2-Cl(16), 2-
3
2
2
2
2
3
NO
2
2
(17), 2-CO H(18). Crystals suitable for X-Ray diffraction were also obtained for two of the
studied compounds. Furthermore, considering that numerous isoxazole derivatives have shown
2
8-32
antineoplastic activity
, a biological study was performed in order to evaluate the cytotoxic
activity towards the human promielocytic leukemia cell line, HL-60. Moreover, using compounds
with IC values lower than 100 ꢀM, we performed RT-PCR experiments to determine the effect on
50
the expression levels of mRNA that codifies for anti- and pro-apoptotic genes, Bcl-2 and Bax, and a
WAF-1.
gene involved in cell cycle progression, p21
2
2
. Result and discussion
.1. Chemistry

Page 5 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
The chemical route for this reaction is shown in Scheme 1. The presence of the H-bond assisted
by resonance on the carbonyl groups of the β-diketohydrazone, increases its electrophilic character
providing a remarkable difference of the reactivity of both carbonyl groups. Thereby, controlling
the reaction stoichiometry the condensation of one carbonyl group is allowed and the reaction with
hydroxylammonium chloride yields only the mono-oxime as intermediate. Then, in acid media, the
unreacted carbonyl group suffers an addition/displacement reaction to yield the corresponding
isoxazoles. This synthetic approximation was used to improve the regioselectivity of the reaction of
a series of (3-(2-(4-R-phenyl)hydrazinylidene)pentane-2,4-diones with NH
2
OH•HCl, using 1:1
stoichiometric ratio. HOAc was used as catalyst in refluxing EtOH, 18 h. The reaction products,
(
E)-3,5-dimethyl-4-(R-phenyldiazenyl)isoxazoles (1)-(18), Scheme 1, are obtained with high yields
over 65%) and were recrystallized in mixtures EtOH/H O of variable composition. Compounds (7)
(
2
and (17) yielded single crystals, suitable for X-ray diffraction studies. The crystallographic results
23
are in concordance with the previously obtained structures, vide infra .
Scheme 1

New Journal of Chemistry
Page 6 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
2
.2. Description of the structures
Table 1 displays the selected bond distances, angles and torsion angles found for (7a-7d) and
17). Besides, Figures 1 and 2 show an ORTEP views of (7b) and (17), respectively, and the crystal
data and refinement parameters of the structures are shown in Table 2. Both structures were
(
2
3
compared with the structure of compound (18) previously described in the literature , whose
geometrical parameters have been also incorporated in Table 1. Compound (7) crystallizes in the
monoclinic system, space group P2 /n, with cell parameters a= 13.5334(6) Å, b= 12.3004(8) Å, c=
1
2
8.098(2) Å, β= 102.177(6)° and Z= 16. The structure has been refined with final indices, [I
2σ(I)], R1= 0.0789, wR2= 0.1448 and S= 1.056, Table 2. On the other hand, compound (17)
crystallizes in a monoclinic system, space group C2/c, with cells parameters a= 11.765(4) Å, b=
>
1
1.314(6) Å y c= 17.774(12) Å, β= 95.75(4)° and Z= 4 and it has final indices, [I >2σ(I)], R1=
.0597, wR2= 0.1492 and S= 1.026, Table 2. The asymmetric unit of (7) contains four molecules of
0

Page 7 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
isoxazole, (7a)-(7d), where (7a) has a 50 % of disorder on the fragment N(1)-N(2)-C(6) affecting
also the bond distances and angles located in their surroundings, Table 1. In (7a) the N(1)-N(2) and
2
N(2)-C(6) distance values indicate that there is a certain sp distortion in this fragment, which is
corroborated by a significant change in the respective bond angle N(1)-N(2)-C(6), Table 1.
However, the rest of the molecules, (7b)-(7d) and (17), exhibit geometrical parameters similar to
2
3
those found in compound (18) . Both structures contain a C(1)-N(1)-N(2)-C(6) moiety with E
configuration around the -N=N- bridge, with an important degree of π-delocalization, vide infra.
The torsion angles involved in this molecular fragment indicate that the molecules have geometry
basically planar, Table 1. Finally, there are no conventional intermolecular hydrogen bonds and the
entire supramolecular structure is constructed only by weak interactions. The crystallographic
information of the structures described in this article has been included as supplementary material.
Figure 1. ORTEP view of the molecule (7b) of the compound (7) with a probability level of 50%.

New Journal of Chemistry
Page 8 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
Figure 2. ORTEP view of compound (17) with a probability level of 50%
Table 1. Selected bond lengths (Å), bond angles (º) and torsion angles (º) in the asymmetric unit of
23
the four molecules of (7) and (17) compared with the structural data of (18) obtained from
literature, where (*) shows the disorder found in (7a).
Bond/Angle
C(2)-C(1)
7a
7b
7c
7d
17
18
1.372(8)
1.399(7) 1.424(6) 1.385(7) 1.351(4) 1.347(3)
1.413(6) 1.424(6) 1.414(6) 1.385(4) 1.403(3)
1
.411(10)
1.500(14)
1.413(8)
1.330(7)
1.321(7)
C(1)-N(1)
*
C(1)-C(5)
C(2)-O(3)
C(5)-N(4)
1.368(7) 1.370(7) 1.399(7) 1.429(4) 1.418(4)
1.320(6) 1.326(6) 1.338(6) 1.339(4) 1.352(3)
1.330(6) 1.317(6) 1.314(6) 1.300(4) 1.301(3)

Page 9 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
1
.437(14)
N(2) -C(6)
N(1)-N(2)
1.432(6) 1.427(6) 1.430(7) 1.423(4) 1.431(3)
1.258(6) 1.259(6) 1.264(6) 1.257(3) 1.254(3)
*
1.63(3)
1
.249(13)
*
1.253(16)
1.450(7)
114.2(7)
105.7(5)
O(3)-N(4)
1.446(6) 1.423(6) 1.435(7) 1.426(4) 1.424(3)
130.2(5) 131.9(5) 120.6(5) 122.5(3) 121.1(2)
106.0(5) 106.2(4) 105.9(4) 104.8(3) 105.8(2)
C(2)-C(1)-N(1)
C(2)-C(1)-C(5)
1
40.0(7)
N(1)-C(1)-C(5)
123.6(5) 121.8(5) 133.4(5) 132.7(3) 133.1(2)
*
105.4(7)
O(3)-C(2)-C(1)
N(4)-C(5)-C(1)
110.3(6)
110.1(6)
109.3(5) 109.4(5) 109.3(5) 109.9(3) 109.4(2)
111.1(5) 109.3(5) 110.3(5) 111.2(3) 110.6(2)
1
12.3(10)
N(2)-N(1)-C(1)
N(1)-N(2)-C(6)
114.9(5) 114.2(5) 113.0(5) 114.0(3) 113.2(2)
114.2(5) 115.0(4) 112.9(5) 113.7(3) 113.0(2)
124.9(5) 124.2(5) 114.6(5) 117.3(3) 119.2(2)
9
9.4(11)
09.4(12)
92.9(15)
03.0(13)
1
*
1
C(7)-C(6)-N(2)
*
147(2)
C(7)-C(6)-N(2)-N(1)
173(3)
4.9(8)
3.4(8) 171.1(5) 176.5(3) 153.8(2)

New Journal of Chemistry
Page 10 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
-
-
C(6)-N(2)-N(1)-C(1)
N(2)-N(1)-C(1)-C(5)
N(2)-N(1)-C(1)-C(2)
178(3)
179.5(5)
179.0(5)
-6.7(9)
178.7(4)
-7.6(9)
176.6(2)
1
1
79.1(5)
178.2(3)
-
-10(2)
5.7(5)
7.3(4)
-
79.8(5)
-
174.6(9)
4.4(9) 174.5(5)
176.7(3) 173.1(2)
2
.3. Pauling’s bond orders studies
In order to know the π-delocalization degree of the fragment C(2)-C(1)-N(1)-N(2)-C(6) in
compounds (7b)-(7d), (17) and (18), the Pauling's bond orders, n, and the conjugation parameters,
, were calculated using the structural data shown in the Table 1. The bond orders found for n
C(2)-C(1)), n (C(1)-N(1)), n (N(1)-N(2)), and n (N(2)-C(6)) have intermediates values between
η
1
(
2
3
4
ideal orders 1 and 2 for single and double bonds, respectively; which is in concordance with the
presence of a delocalized system, Table 2.
Table 2. Pauling’s bond orders, n, and π-delocalization parameters, η, of compounds (7b)-(7d), (17)
and (18) calculated for the fragments C(2)-C(1)-N(1)-N(2)-C(6). (7a) was not included due to
disorder.
Compound
n1
n2
n3
n4
%η
1,2
%η
2,3
%η
3,4
(7b)
1.4504
1.2000
1.7240
1.1283
38
76
20

Page 11 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
(
7c)
1.3368
1.5181
1.6961
1.7184
1.1581
1.1964
1.3152
1.2418
1.7184
1.6906
1.7297
1.7467
1.1468
1.1356
1.1619
1.1319
41
34
31
26
78
75
71
75
21
22
22
19
(7d)
(
17)
18)
(
The n
N(2). However n
character respect to the bond N(1)-N(2). On the contrary, an increase of both n
1
and n
3
values show a decrease of the double bond character of the C(2)-C(1) and N(1)-
value shows that the C(2)-C(1) bond has a higher decrease of double bond
and n values
1
2
4
respect to the formal single bonds C(1)-N(1) and N(2)-C(6) were found. These observations
indicate an intense π-delocalization on the complete fragment C(2)-C(1)-N(1)-N(2)-C(6). The π-
delocalization is corroborated by the
N(2), and N(1)-N(2)-C(6), whose π-delocalization values %
ranges of 26-41 %, 71-78 %, and 19-22 %, respectively, Table 2. The increase of %
η
values found for sub fragments C(2)-C(1)-N(1), C(1)-N(1)-
3,4 takes values in the
1,2 values
η
1,2, %η1,2, and %η
η
respect to %η3,4 indicate that the π-delocalization is more intense in the sub fragment C(1)-N(1)-
N(2) which is part of the isoxazole ring. These observed features are in concordance with the
structural data discussed above and with the basically planar geometry for the fragment C(2)-C(1)-
N(1)-N(2)-C(6) deduced from the torsion angles shown in Table 1.
2
.4. Spectroscopic studies
The UV-visible absorption spectra of compounds (1)-(18) were registered in ethanol solutions
and exhibit one main π*←
π absorption band placed in the ranges of 392-315 nm. This transition is
very intense, log :4.36-4.18, probably due to a π*←π absorption that emerge from the π-delocalized
ε

New Journal of Chemistry
Page 12 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
-C=C-N=N-C- chromophore present in each isoxazole molecule, see Scheme 1 and TDDFT
calculations. This transition does not show correlation with the Hammett equation, log vs σp (data
λ
not shown). Other intense π*←π absorption bands located at higher energies have been attributed to
the internal absorption of the phenyl rings.
The IR spectra of compounds (1)-(18), recorded in solid state, exhibit three typical stretching
-1
absorption between 1595-1619, 1600-1490 and 1416-1403 cm , attributed to the ν(C=N) and/or
ν(N=N), ν(C=C) and ν(N-O) chromophores, respectively. Moreover, the spectra also show the weak
-
absorptions corresponding to the aromatic and aliphatic stretching modes, ν(C-H), around 3000 cm
1
,
respectively and the characteristic stretching absorptions attributed to the substituent groups.
1
13
Figure 3. Labelling of atoms for the assignment of H and C NMR signals in the spectra.
1
The outstanding features of the H NMR spectra of (1)-(18) are two singlets placed at 2.42-2.57
1 5
and 2.70-2.80 ppm ranges attributed to the protons linked to the carbon atoms C and C ,
respectively, Figure 3. Besides, these compounds exhibit the resonances attributed to the aromatic
protons in the 6.67-8.14 ppm range. The corresponding signals due to the substituent groups are
also observed.

Page 13 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
1
3
On the other hand, the C NMR spectra show all resonances corresponding to the isoxazole
carbon atoms, the phenyl rings and the resonances that emerge from each substituent group. An
important aspect of these spectra is the almost invariable chemical shifts of the carbon atoms C , C ,
1
2
C , C and C , whose positions are in the narrow ranges of 12.29-11.46, 154.75-152.84, 133.50-
3
4
5
1
31.45, 171.90-166.49 and 11.98-11.98 ppm, respectively.
Additionally, the HMBC spectra were recorded in order to assign the carbon and hydrogen
resonances of the isoxazole ring. As an example, Figure 4 shows the HMBC spectrum of compound
2) that show the common interaction pattern present in the isoxazole rings. In fact, the protons of
(
1
5
the CH - group interact with C and C , while protons of the CH - group interact with carbon C
3
2
3
3
4
3
and also C . In this figure it is possible to observe also the H-C interactions attributed to the phenyl
ring.
Figure 4. HMBC spectrum of compound (2) in DMSO-d showing the H-C interactions.
6

New Journal of Chemistry
Page 14 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
2
.5. Computational study
To get further in the characterization of compounds (1)-(18), we carried out theoretical
calculations in order to give a better explanation to some previously mentioned facts. It is possible
to observe that in the optimized molecular structures, see Table S1, the bond lengths and angles are
unaffected by the change of the -R substituent. The substituents were changed from electron-
withdrawing to electron-donor groups in the different positions of the phenyl rings. The observed
changes are about 0.03 Å and 10°. The bond lengths that show more variation correspond to those
bonds involved in the substituted phenyl rings.
In order to assure that each optimized structure corresponds to energy minimum, vibrational
frequencies were calculated. Only positive frequencies were observed with a very good
concordance between the experimental and the theoretical IR spectra, see Figure 5 for compound
(2). In the characterization of these vibrational frequencies we found that the signals located in the
-1
range 1595-1619 cm of the experimental IR spectra, correspond to the chromophores -C=N- and -
N=N-, see Table S2.

Page 15 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
1
00
80
60
40
20
0
3550
3050
2550
2050
1550
1050
550
-1
Wavenumber (cm )
Theoretical
Experimental
Figure 5. Experimental and theoretical plot of the obtained IR spectra for (2).
To perform a characterization of the UV-vis spectra of (1)-(18) TD-DFT computations were
performed. All the calculated transitions are depicted in the supplementary information, Table S3,
and a superimposition of the UV-VIS spectra of (2) and the calculated transition is shown in Figure
6
. These computations indicate that the transition, located in the range 313-393 nm, correspond to
the transition between HOMO-LUMO. These two involved orbitals are delocalized over the whole
chain of each compound (see Supplementary Information). The absence of Hammett correlation
could be explained considering the level of localization and the composition of these molecular
orbitals, which does not depend on the nature of the substituent, placed on the azo phenyl group of
compounds (1)-(18).

New Journal of Chemistry
Page 16 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
Figure 6. Experimental and theoretical plot of the obtained UV-vis spectra for compound (2).
2
.6. Biological activity studies
2
.6.1. Cytotoxic effect of isoxazoles: Compounds (1)-(18) were subjected of in vitro biological
studies by incorporating them to cultures of leukemia HL-60 cells. The cytotoxic effect was
measured after exposure of the cells to the isoxazoles for 24 h, and was expressed as the relative
change on the cell viability compared to the control sample (untreated cells). The IC values were
5
0
obtained from dose-response curves and they fall in a wide range of concentrations, see Table 3.
Isoxazoles (3) and (6) were the most active, with IC50 values of 95.4 and 85.6 µM, respectively.
Interestingly both compounds are functionalized in the same position with substituents of similar
33
size . Both mentioned compounds which showed the highest cytotoxic activity toward HL-60 cells
were subject of additional biological studies.

Page 17 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
Table 3. In vitro activity, IC50 values, of the isoxazoles toward HL-60 cells
Isoxazole
Substituent (R)
p-N(CH
p-OH
IC50 (µM)
285.0
146.1
95.4
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
3 2
)
p-CH
3
3
p-OCH
153.6
151.4
85.6
p-H
p-Cl
p-Br
340.7
216.8
452.9
109.9
165.5
223.8
192.1
754.9
516.3
129.5
103.6
102.9
p-CO H
2
p-CH CO H
2
2
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(18)
(19)
p-COCH3
p-CN
p-NO
CH CH
m-Cl
2
p-CO
2
2
3
o-OH
o-Cl
o-CO
2
H
o-NO
2
WAF-1
2
.6.2. Effect of the isoxazoles on expression of Bcl-2, Bax and p21
: Due to the interesting
cytotoxic activity of (3) and (6) on HL-60 cells, we also explored their effect on the expression
genes involved in apoptosis and cell cycle progression. Apoptosis is an active process of cell death
that occurs in response to a variety of agents including ionizing radiation or anticancer
34
chemotherapeutic drugs . This implicates the balanced transcriptional control of anti-apoptotic and
pro-apoptotic genes, such as Bax and Bcl-2. We have also included in this study a gene associated

New Journal of Chemistry
Page 18 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
WAF-1
with the inhibition of the proliferation, p21
. It is now well established that cyclins play a
positive role in promoting cell cycle transitions via their ability to associate and activate their
3
5-37
cognate cyclin dependent kinases (Cdks)
implicated in the control of the G1 to S phase transition in mammals
potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclins
. Cdk2 associates with cyclins A, D and E has been
35-37
WAF1
. The protein p21
is a
3
8,39
complexes A-Cdk2, E-Cdk2, D1-Cdk4 and D2-Cdk4
. Thus, an increase of expression of
WAF-1
p21
plays an important role in stopping of the cell cycle.
In order to determine the expression of Bax and Bcl-2 on the mRNA level, we performed a semi-
quantitative duplex RT-PCR. Treatment of HL-60 cells with (3) at concentrations of IC50, 95.4 µM,
during 24 h, induces an important decrease on the expression of Bcl-2 to around 12 % relative to the
control (P< 0.05), Figures 7A (lane 4) and 7D. Meanwhile, treatment with (6) at concentrations of
IC , 85.6 µM during 24 h, increased the expression of Bcl-2 to 203% relative to the control,
5
0
Figures 7A (lane 3) and 7D. Besides, under these experimental conditions, both (3) and (6) have no
effect on mRNA levels of Bax, Figures 7B (lanes 4 and 3) and 7E, respectively. On the other hand,
WAF-1
we found that (3) and (6) increased the levels of p21
mRNA expression to 340% and 365%
WAF-1
relative to the control, respectively, Figures 7C (lanes 4 and 3) and 7F. The increase on the p21
mRNA expression indicates that the isoxazoles (3) and (6) would induce the arrest of cell cycle on
the HL-60 cells. Thus, taken together the results suggest that the cytotoxic effect of (3) would be the
sum of a decrease in Bcl-2, an anti-apoptotic protein, hence promoting the apoptosis, and by arrest
WAF-1
of cell cycle through the increase of p21
. Meanwhile the cytotoxic ability of (6) would be
WAF-1
directed mainly by inhibition of the cell cycle through the increase of p21
.

Page 19 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
Figure 7. Effect of the (3) and (6) on mRNA expression levels of Bcl-2 (A and D), Bax (B and E)
WAF-1
and p21
(C and F) in HL-60 cells examined by semiquantitive duplex RT-PCR. PCR products
were separated using 1.5% agarose gel electrophoresis, the arrows indicate the PCR products for
WAF-1
GAPDH (600 bp), Bcl-2 (293 bp), Bax (332 bp) and p21
(156 bp). Lane 1, untreated control;
line 2, vehicle control; lane 3, treatment with 86 µM isoxazole (6); lane 4, treatment with 95 µM
isoxazole (3). The PCR products were quantified relative to the internal standard GAPDH (D, E and
F). The results are shown as mean ± standard deviations (SD) and correspond to the percentage
change compared to the control sample assigned as 100%. Differences with P values of < 0.05 (*)
or P values of < 0.01 (***) were considered significant.
3
. Conclusions
A one-pot synthesis of a family of new 3,4,5-trisubstituted isoxazoles has been done with good
yields, by treatment of β-diketohydrazones of the type (3-(2-(4-R-phenyl)hydrazinylidene)pentane-
,4-dione, with hydroxylammonium chloride in ethanol and acetic acid as catalyst. All compounds
2
1
13
were characterized using elemental analysis and spectroscopic methods, (UV-vis, IR, H NMR, C
NMR, and HMBC). The crystalline and molecular structure of (E)-3,5-dimethyl-4-((4-
bromophenyl)diazenyl)isoxazole (7) and (E)-3,5-dimethyl-4-((2-nitrophenyl)diazenyl)isoxazole

New Journal of Chemistry
Page 20 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
(17) were determined by X-ray diffraction methods. From the Pauling bond order, which were
calculated from the crystallographic structure, it was possible to corroborate the delocalization of
the system. The analytical, spectroscopic and structural data are in concordance with the proposed
structures.
On the other hand, the cytotoxic activity of the most biologically active compounds was
evaluated on human promyelocytic leukemia cells, HL-60. Compounds (3), (E)-3,5-dimethyl-4-(p-
tolyldiazenyl)isoxazole, and (6), (E)-4-((4-chlorophenyl)diazenyl)-3,5-dimethylisoxazole, are the
compounds that exhibit significant cytotoxic activity, with IC50 values below 100 ꢀM and thus they
would emerge as new potential molecules for further antineoplasic studies. In this context, the effect
of both compounds on the expression of genes involved in apoptosis and in the cell cycle control
has been evaluated. Compound (3) has an apoptotic effect, since the level of Bcl-2 mRNA decreases
WAF-1
to approximately 12%. Importantly, both isoxazoles have an effect on the expression of p21
,
with an increase over 340% of the mRNA levels, suggesting a substantial anti-proliferative activity.
4
. Experimental
4
.1 Chemicals
Substitutes anilines, R-C
6
H
4
-NH
H (8), 4-CO
(13), 3-Cl (14), 2-OH (15), 2-Cl (16), 2-NO
2
(R= 4-N(CH
3
)
2
(1), 4-OH (2), 4-CH
3
(3), 4-OCH
3
(4), 4-H(5),
4
-Cl (6) 4-Br (7), 4-CO
CO CH CH
2
2
CH CH
2
3
(9), 4-COCH
3
(10), 4-CN (11), 4-NO
2
(12), 4-
CH
2
2
2
3
2
(17), 2-CO H (18)), acetylacetone,
2
sodium nitrite, acetic acid, hydrochloric acid, sodium hydroxide and hydroxylammonium chloride
were procured from Sigma-Aldrich Corporation and solvents, methanol, ethanol, diethylether,
acetone CDCl
3
, and DMSO-d , from the usual commercial sources, Merck, Fisher and T. J. Baker
6

Page 21 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
6 4 3 2 3 2
providers. All β-diketohydrazone precursors, R-C H -NHN=C(COCH ) (R= 4-N(CH ) (1), 4-OH
(
2), 4-CH (3), 4-OCH (4), 4-H (5), 4-Cl (6) 4-Br (7), 4-CO H (8), 4-CO CH CH , (9), 4-COCH
3
3
2
2
2
3
3
(10), 4-CN (11), 4-NO
2
(12), 4-CH
2
2
CO CH
2
CH
3
(13), 3-Cl (14), 2-OH (15), 2-Cl (16), 2-NO
2
(17),
24-27
2
-CO
2
H (18)} were obtained using the method recommended in literature
and the structures
were checked by IR spectroscopy and, in some cases, by NMR spectroscopy.
4
.2 Physical measurements
Uncorrected melting points, MP, were determined on digital STUARD, SMP10 apparatus.
Elemental analysis, EA; were obtained from a FISONS, EA 1118 microanalyses using
sulfanilamide as standard. UV-Vis spectra were recorded in the 1100-200 nm range, in quartz cells
with 10 mm length pass, using a Perkin Elmer, Lambda 35 spectrophotometer, and concentrated
-3
-5
solutions, ~1.0×10 mole/L, of each compound, diluting around ~1.0×10 mole/L. The infrared
spectra, IR, were obtained in solid state on an ATR Jasco, PRO450-S, mounted on a Jasco, FT/IR-
1
13
4
200 equipment. Depending of the solubility of compounds, H NMR, C NMR and HMBC
spectra were recorded by the standard methods in CDCl or DMSO-d solutions, using 5 mm i.d.
3
6
glass tubes and the internal solvent signals as reference, in two different equipments i) JOEL, JNM-
Lambda500 spectrophotometer and ii) a Bruker, Avance AM 400 spectrophotometer. Assignment
1
13
of H and C NMR signals was realized in accord with Figure 3.
4
.3 Data collection
Single crystal X-ray diffraction data set for (17) was collected at room temperature up to a max
2
θ
of ca. 52º on an Enraf-Nonius CAD4 diffractometer using monochromatic MoKα radiation, λ=
0
.71069 Ǻ and ω-2ϑ scan mode. For (7), the experiment was done on a Bruker-CCD Smart 1 K

New Journal of Chemistry
Page 22 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
diffractometer using also MoKα radiation. Crystal data and structure refinement are shown in Table
, while Table 1 contains the selected bond lengths and angles and Figures 1 and 2 exhibit ORTEP
4
40
views of the molecules. Data processing was performed using the WinGX package . The structures
4
1
2
were solved by direct methods using SIR-2004 and refined by least squares on F with anisotropic
4
2
displacement parameters for non-H atoms using SHELXL97 . All hydrogen atoms were located
from difference Fourier maps and refined as riding on their parent atoms. Crystallographic data for
the structures reported in this paper have been deposited in the Cambridge Crystallographic Data
Centre as supplementary publication, CCDC No. 882.688 and Nº 882.689 for compounds (7) and
(17), respectively. 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. E-mail:
data_request@ccdc.cam.ac.uk. Web page: http://www.ccdc.cam.ac.uk.
Table 4. Crystal data and structure refinement parameters of the compounds (7) and (17).
7
17
Empirical formula
Formula weight
Temperature
C H Br N O
C H N O
3
11
10
1
12
1
11 10
4
280.12
246.23 g/mol
294(2) K
0.71073 Å
Monoclinic
C2/c
294(2) K
0.71073 Å
Wavelength
Crystal system
Space group
Monoclinic
P2 /n
1
a = 13.5334(6) Å, α=
0°
a = 11.765(4) Å , α=
Unit cell dimensions
9
90°

Page 23 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
b = 12.3004(8) Å, β=
b = 11.314(6) Å, β=
1
02.177(6)°
c = 28.098(2) Å γ =
0°
95.75(4)°
c = 17.774(12) Å, γ=
9
90°
Volume
4572.1(5) Å3
16
2354(2) Å3
8
Z
Density (calculated)
Absorption coefficient
F(000)
1.628 Mg/m3
3.578 mm-1
2240
1.390 Mg/m3
0.105 mm-1
1024
0.38 × 0.34 × 0.23
Crystal size
0.42 x 0.36 x 0.12 mm3
2.88 to 29.36°
mm3
θ
range for data collection
2.30 to 25.98°
-14 ≤ h ≤ 14,
0 ≤ k ≤ 13,
0 ≤ l ≤ 21
2703
-
17 ≤ h≤ 18,
Index ranges
-16 ≤ k ≤ 15,
-
38 ≤ l ≤ 38
Reflections collected
Independent reflections
Completeness to
50022
11446 [R(int)= 0.0931] 2303 [R(int) = 0.0553]
θ
= 29.36°, 91.0 %
θ = 25.98°, 100.0%
Refinement method
Full-matrix least-
Full-matrix least-

New Journal of Chemistry
Page 24 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
squares on F2
11446/246/594
1.056
squares on F2
2303/0/165
1.017
Data/restraints/parameters
Goodness-of-fit on F2
R1= 0.0789, wR2=
R1= 0.0576, wR2=
Final R indices [I >2σ(I)]
0
.1448
R1= 0.1773, wR2=
.1790
0.1374
R1= 0.1436, wR2=
R indices (all data)
0
0.1615
Largest diff. peak and
hole
0.574 and -0.555 e·Å-3 0.216 and -0.150 e·Å-3
4
.4. Pauling’s bond orders, n, and conjugation parameters, η.
The π-electronic delocalization on the fragment C(2)-C(1)-N(1)-N(2)-C(6) of compounds (7b)-
7d) and (17) was evaluated in terms of Pauling’s bond order, n, taking three separated sub
fragments, C(2)-C(1)-N(1), C(1)-N(1)-N(2) and N(1)-N(2)-C(6), for obtaining the paremeters.
The standard single bond length in crystalline state found in C , dC-C: 1,513 Ǻ, CH NH
.469 Ǻ and NH NH , dN-N: 1.425 Ǻ; were obtained from literature and the corresponding
crystallographic data from the Table 1. These data were used for obtaining n in C(2)-C(1); n in
C(1)-N(1); n in N(1)-N(2) and n in N(2)-C(6) using the Pauling’s equation, [43] - R(n)/2=
(
η
2
H
6
3
2
, dC-N:
26,27
1
2
2
1
2
ꢀ
3
4
0
n
η
.353logn. Conjugation parameters,
)+(n -1)]×100, % )+(n
η, were evaluated in terms of percentage, as %η1,2= ½[(2-
1
2
η2,3= ½[(2-n
2
3
-1)]×100 and %
η
3,4= ½[(2-n
3
)+(n
4
-1)]×100, were
η
1,2
,
η2,3 and
+
-
3,4 are by definition equal to 0, 1 and 0.5 for non-delocalized C=C-N=N-C, ionic C-C=N-N=C

Page 25 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
and fully delocalized fragment
, respectively. The results are shown in Table 3 and
23
compared with the analogous fragment of the compound (18) available in literature . Because of
the disorder found in the crystallographic data of the molecule (7a) the corresponding data were not
considered in these calculations.
4
.5. Synthesis
6 4
In a 100 mL round-bottomed flask were added: 0.01 mole of any β-diketohydrazone, R-C H -
3 2 3 2 3
NHN=C(COCH ) {m, R= 2,47 g, 4-N(CH ) (1); 2.20 g, 4-OH(2); 2.18 g, 4-CH (3); 2.34 g, 4-
OCH (4); 2.04 g, 4-H(5); 2.39 g, 4-Cl(6); 2.83 g, 4-Br(7); 2.48 g, 4-CO H(8); 2.76 g, 4-
3
2
CO
CH
2
CH
2
CH
CH
3
(9); 2.46 g, 4-COCH
3
(10); 2.29 g, 4-CN(11); 2.49 g, 4-NO
2
(12); 2.90 g, 4-
(17)};
2
CO
2
2
CH (13); 2.39 g, 3-Cl(14); 2.20 g, 2-OH(15); 2.39 g, 2-Cl(16); 2.49 g, 2-NO
3
2
2
2
.48, 2-CO H(18)}, 30 mL de ethanol, 2 mL of glacial acetic acid and 0.01 mole (0.69 g) of
NH OH·HCl. The reaction mixture was heated at reflux temperature stirring magnetically during 18
2
h. For precipitation of each compound the mixture was cooled to room temperature and then cooled
at -18 ºC during 2 h or by addition of around 25 mL of water. The yellow-orange solids were
filtered by suction, washed with abundant quantity of water and dried under vacuum at 40 ºC by 24
h. All compounds were recrystallized from ethanol/H
Compound (13) may be also recrystallized from a 5:1 acetone/H
17) yield single crystals suitable for X-ray studies by recrystallization from 3:1 and 2:1 EtOH/H
mixtures, respectively.
2
O mixtures of variable composition.
2
O. Finally, compounds (7) and
(
2
O

New Journal of Chemistry
Page 26 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
4
.6. Analysis
4
.6.1. Compound (1) (E)-4-((3,5-dimethylisoxazol-4-yl)diazenyl)-N,N-dimethylaniline: Yield: 39
. Recryst. from EtOH/H O 2:1. MP: 129-130 ºC. EA: for C13 O (Mι: 244.29 g/mole):
Calc(%): C, 63.91; H, 6.60; N, 22.93; Found(%): C, 63.83; H, 6.69; N, 22.78. UV-vis in EtOH
%
2
16 4
H N
-5
-1
4
.09×10 mole/L, λmax, nm(logε): 423sh; 392(4.18); 328(3.47); 315(3.45); 262(3.93). IR, ν(cm ):
ν(R-H): 2986w, 2908m, 2818w; ν(N=N) or ν(C=N): 1600s; ν(C=C): 1561m, 1517m; ν(N-O):
1
1
409m. H NMR (495 MHz in CDCl
3
) δ ppm: 2.52 (s, 3H, H
1
), 2.70 (s, 3H, H
5
), 3.07 (s, 6H,
) δ
, C
1
3
N(CH
3
)
2
), 6.77-6.69 (m, 2H, Ar-H) 7.80 – 7.69 (m, 2H, Ar-H). C NMR (495 MHz in CDCl
: 12.10; C : 154.36; C : 132.34; C :166.49; C : 11.72; C , C : 144.05, 152.28; C
: 40.40 N(CH
3
ppm: C
1
2
3
4
5
6
9
7
8
:
1
24.12. 111.57; C
R
3 2
) .
4
.6.2. Compound (2) (E)-4-((3,5-dimethylisoxazol-4-yl)diazenyl)phenol: Yield: 92 %. Recryst.
O 2:1. MP: 170-171 ºC. EA: for C11 (Mι: 217.22 g/mole): Calc(%): C,
0.82; H, 5.10; N, 19.34; Found(%): C, 60.81; H, 5.23; N, 19.26. UV-vis in EtOH 4.72×10
from EtOH/H
2
11 3 2
H N O
-5
6
-1
mole/L, λmax, nm(logε): 420sh; 346(4.35); 246(4.09). IR, ν(cm ): ν(O-H): 3142s; ν(Ar-H): 3087w,
3
1
2
030w; ν(R-H): 2942w, 2841w; ν(N=N) or ν(C=N): 1617s; ν(C=C): 1586s y 1508m; ν(N-O):
1
411s. H NMR (495 MHz in DMSO-d
6
) δ ppm: 2.43 (s, 3H, H
1
), 2.70 (s, 3H, H
5
), 6.94-6.87 (m,
(-OH)). C NMR (495 MHz in DMSO-d ) δ
: 168.03, C : 11.25; C , C : 145.54, 160.56; C , C
13
H, Ar-H), 7.74-7.66 (m, 2H, Ar-H), 10.22 (s, 1H, H
: 11.64; C : 153.14; C : 131.45; C
24.09, 115.84.
R
6
ppm: C
1
2
3
4
5
6
9
7
8
:
1

Page 27 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
4
.6.3. Compound (3) (E)-3,5-dimethyl-4-(p-tolyldiazenyl)isoxazole: Yield: 93 %. Recryst. from
EtOH/H O 2:1. MP: 78-79 ºC. EA: for C H N O (Mι: 215.25 g/mole) Calc(%): C, 66.96; H, 6.09;
2
12 13
3
-5
N, 19.52. Found(%): C, 67.04; H, 6.18; N, 19.58. UV-vis in EtOH 6.37×10 mole/L, λmax, nm
-1
(logε
): 423(2.93); 323(4.30); 312sh; 240(4.03); 234(4.06); 228sh. IR, ν(cm ): ν(Ar-H): 3087w,
3
1
026w; ν(R-H): 2992w, 2923w; ν(N=N) or ν(C=N): 1610s; ν(C=C): 1589m y 1497m; ν(N-O):
1
407s. H NMR (495 MHz in CDCl
3
) δ ppm: 2.42 (s, 3H, H
R
3
1
(CH -)), 2.53 (s, 3H, H ), 2.74 (s, 3H,
13
H ), 7.30-7.27 (m, 2H, Ar-H), 7.71 (d, J = 8.30 Hz, 2H, Ar-H). C NMR (495 MHz in CDCl ) δ
5
3
ppm: C
1
: 12.19; C
2
: 153.99; C
3
: 132.50; C
4
: 168.92; C
5
: 11.79; C , C
6 9
: 141.36, 151.17; C
7 8
, C :
1
22.28, 129.83; C : 21.58.
R
4
.6.4. Compound (4) (E)-4-((4-methoxyphenyl)diazenyl)-3,5-dimethylisoxazole: Yield: 99%.
Recryst. from EtOH/H O 2:1. MP: 107-108 ºC. EA: for C H N O (Mι: 231.25 g/mole) Calc(%):
2
12 13
3
2
-5
C, 62.33; H, 5.67; N, 18.17; Found(%): C, 62.52; H, 5.82; N, 18.22. UV-vis in EtOH 4.58×10
-1
mole/L, λmax, nm(logε): 405sh; 346sh; 337(4.36); 242(4.15). IR, ν(cm ); ν(Ar-H): 3070w, 3039w;
ν(R-H): 2965w, 2934w, 2841w; ν(N=N) or ν(C=N): 1601s; ν(C=C): 1579s, 1497s; ν(N-O): 1407s.
1
H NMR (495 MHz in CDCl
3
) δ ppm: 2.52 (s, 3H, H
1
), 2.72 (s, 3H, H
5
), 3.88 (s, 3H, H
R
(CH O-)),
3
1
3
7
1
1
.02 – 6.94 (m, 2H, Ar-H) 7.86 – 7.72 (m, 2H, Ar-H). C NMR (495 MHz in CDCl ) δ ppm C :
3
1
2.20: C
2
: 154.09; C
3
: 132.40; C
4
: 168.30; C
5
: 11.79; C
6
, C
9
: 147.45, 161.96; C
7
, C
8
: 114.33,
24.10, C
R
: 55.73.
4
.6.5. Compound (5) (E)-3,5-dimethyl-4-(phenyldiazenyl)isoxazole: Yield: 65 %. Recryst. in
EtOH/H O 3:1.MP: 44-45 ºC. EA:for C H N O (Mι: 201.22 g/mole) Calc(%): C, 65.66; H, 5.51;
2
11 11
3
-5
N, 20.88; Found(%): C, 65.96; H, 5.59; N, 20.98. UV-vis in EtOH 5.19×10 mole/L, λmax
,
-1
nm(logε): 425(2.75); 317(4.23); 308sh(4.22); 233sh(3.98); 228(4.03); 222sh(4.00). IR, ν(cm ):

New Journal of Chemistry
Page 28 of 41
View Article Online
DOI: 10.1039/C4NJ02427C
ν(Ar-H): 3083w, 3042w; ν(R-H): 2988w, 2925m; ν(N=N) or ν(C=N): 1611f; ν(C=C): 1592m y
1
1
7
1
1
504w; ν(N-O): 1412s. H NMR (495 MHz in CDCl ) δ ppm: 2.54 (s, 3H, H ), 2.75 (s, 3H, H ),
3
1
5
13
.52-7.38 (m, 3H, Ar-H), 7.83-7.78 (m, 2H, Ar-H). C NMR (495 MHz in CDCl
3
1
) δ ppm: C :
2.15; C
2 3 4 5 6 9 7 8
: 153.84; C : 132.55; C : 169.42; C : 11.75; C , C : 130.79, 152.99; C , C : 122.27;
29.14.
4
.6.6. Compound (6) (E)-4-((4-chlorophenyl)diazenyl)-3,5-dimethylisoxazole: Yield: 98 %.
O 3:1. MP: 56-57 ºC. EA: for C11 O (Mι: 235.67 g/mole) Calc(%): C,
6.06; H, 4.28; N, 17.83; Found(%): C, 56.13; H, 4.54; N, 17.86. UV-vis in EtOH 5.13×10
Recryst. in EtOH/H
2
H10ClN
3
-5
5
-1
mole/L, λmax, nm(logε): 424(2.86); 324(4.35); 313sh; 239sh; 233(4.08); 227sh. I R, ν(cm ): ν(Ar-
H): 3068w; ν(R-H): 2982w, 2929w; ν(N=N) or ν(C=N): 1612s; ν(C=C): 1590m, 1576w; ν(N-O):
1
1
403s. H NMR (400 MHz in CDCl ) δ ppm: 2.52 (s, 3H, H ), 2.75 (s, 3H, H ), 7.56-7.35 (m,2H,
3
1
5
13
Ar-H), 7.94-7.6 (m, 2H, Ar-H). C NMR (400 MHz in CDCl
3
) δ ppm: C
1
2 3
: 12.25; C : 153.78; C :
1
4 5 6 9 7 8
32.60; C : 169.93; C : 11.84; C , C : 136.71, 151.40; C , C : 123.59, 129.46.
4
.6.7. Compound (7) (E)-4-((4-bromophenyl)diazenyl)-3,5-dimethylisoxazole: Yield: 91 %.
Recryst. in EtOH/H O 3:1 yields single crystals. MP: 89-90. EA: for C H BrN O (Mι: 280.12
2
11 10
3
g/mole): Calc(%): C, 47.16; H, 3.60; N, 15.00; Found(%): C, 47.20; H, 3.75; N, 14.94. UV-vis in
-5
EtOH 5.09×10 mole/L, λmax, nm(logε): 4.25(2.96); 325(4.35); 315sh; 240sh; 234(4.03); 227sh. IR,
-1
ν(cm ); ν(Ar-H): 2974w, 2932w; ν(N=N) or ν(C=N): 1605s; ν(C=C): 1584m y 1569m; ν(N-O):
1
1
407s. H NMR (495 MHz in CDCl
3
) δ ppm: 2.52 (s, 3H, H
1
), 2.75 (s, 3H, H
5
), 7.64-7.58 (m, 2H,
1
3
Ar-H), 7.71-7.65 (m, 2H, Ar-H). C NMR (495 MHz in CDCl ) δ ppm: C : 12.23; C : 153.77; C :
3
1
2
3
1
4 5 6 9 7 8
32.63; C : 169.98; C : 11.86; C , C : 125.12, 151.77; C , C : 123.83, 132.44.

Page 29 of 41
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ02427C
4
.6.8. Compound (8) (E)-4-((3,5-dimethylisoxazol-4-yl)diazenyl)benzoic acid: Yield: 89 %.
Recryst. from EtOH/H O 2:1. MP: 108-109 ºC. AE: for C H N O (Mι: 245.23 g/mole). Calc(%):
2
12 11
3
3
-5
C, 58.77; H, 4,52; N, 17.13; Found(%): C, 59.81; H, 4.92; N, 16.47. UV-vis in EtOH 4.79×10
-1
mole/L, λmax, nm(log
ε
): 432(2.88); 322(4.28); 233sh; 227(3.89); 221sh. IR, ν(cm ): ν(O-H): 3284-
1
2
175s, ν(C=O): 1674s; ν(C=C): 1597s, 1581m, 1505w; ν(N-O): 1410s. H NMR (495 MHz in
6
1 5
DMSO-d ) δ ppm 2.47 (s, 3H, H ), 2.78 (s, 3H, H ), 7.91-7.88 (m, 2H, Ar-H), 8.14-8.09 (m, 2H, Ar-
1
3
H), 13.16 (s, 1H, H (COOH)). C NMR (495 MHz in DMSO-d ) δ ppm: C : 11.71; C : 152.84;
R
6
1
2
C
3
: 132.17; C
4
: 171.26; C
5
: 11.43; C
6
, C
9
: 132.48, 154.66; C
7 8 R
, C :122.02, 130.58; C : 166.70
(>COOH).
4
.6.9. Compound (9) (E)-ethyl 4-((3,5-dimethylisoxazol-4-yl)diazenyl)benzoate: Yield: 67 %.
Recryst. from EtOH/H O 2:1. MP: 107-108 ºC. EA: for C H N O (Mι: 273.29 g/mole) Calc(%):
2
14 15
3
3
-
5
C, 61.53; H, 5.53; N, 15.38. Found(%): C, 61.93; H, 5.98; N, 15.18. UV-vis in EtOH 4.67×10
-1
mole/L, λmax, nm(log
ε
): 429(2.92); 332(4.32); 233sh; 227(3.94), 220sh. IR, ν(cm ): ν(R-H): 2986w,
1
2
954w; ν(C=O): 1715s; ν(N=N) or ν(C=N): 1611w; ν(C=C): 1598s; ν(N-O): 1409s. H NMR (400
MHz in DMSO-d
6
3
2 1 5
) δ ppm: 1.34 (t, 3H, (CH CH -)), 2.46 (s, 3H, H ), 2.77 (s, 3H, H ), 4.34 (q, 2H,
1
3
(
CH CH -)), 7.89 (d, J = 8.4 Hz, 2H, Ar-H), 8.11 (d, J = 8.5 Hz, 2H, Ar-H). C NMR (400 MHz in
3
2
DMSO-d
6
) δ ppm: C
1
: 11.68; C
2
: 154.75; C
3
: 132.17; C
4 5 6 9
: 171.36; C : 11.41; C , C : 131.41, 154.75,
C
7
, C : 122.11, 130.38; C
8
R
: 14.12 (CH
3
CH
2
-), 61.05, (CH
3
CH -), 165.09 (-COOR).
2
4
.6.10. Compound (10) (E)-1-(4-((3,5-dimethylisoxazol-4-yl)diazenyl)phenyl)ethanone: Yield: 99
. Recryst. from EtOH/H O 2:1. MP: 106-107 ºC. EA: for C H N O (Mι: 243.26 g/mole)
%
2
13 13
3
2
Calc(%): C, 64.19; H, 5.39; N, 17.27; Found(%): C, 62.79; H, 5.77; N, 19.54. UV-vis in EtOH
-5
-1
4
.77×10 mole/L, λmax, nm(logε): 427(3.02); 330(4.34); 229(4.01). IR, ν(cm ): ν(Ar-H): 3038w;