New series of isoxazole derivatives targeting EGFR-TK: Synthesis, molecular modeling and antitumor evaluation

Article abstract of DOI:10.1016/j.bmc.2020.115674

New series of isoxazole derivatives were synthesized and evaluated for in vitro antitumor activity against HepG2, MCF-7 and HCT-116 cancer cells. Results showed that 4b and 25a are the most potent members against the three cancer cells (IC₅₀ = 6.38–9.96 μM). Further, 4a, 8a and 16b showed strong activity against the three cancer cells, whereas 6b, 10a, 10b and 16a exhibited moderate activity against the three cancer cells. Moreover, 25a showed low cytotoxicity against WISH and WI38 normal cells (IC₅₀ = 53.19 ± 3.1 and 38.64 ± 2.8 μM, respectively), and it might be used as a potent and safe antitumor agent. The nine active compounds 4a, 4b, 6b, 8a, 10a, 10b, 16a, 16b and 25a were studied for EGFR-TK inhibitory activity, where 10a, 10b and 25a showed the highest inhibitory activity (IC₅₀ = 0.064 ± 0.001, 0.066 ± 0.001 and 0.054 ± 0.001 μM, respectively). Compound 25a was also assessed against other four target proteins, and it showed promising inhibitory activities against VEGFR-2, CK2α and topoisomerase IIβ, and acceptable inhibitory activity against tubulin polymerization. Cell cycle analysis of cancer cells treated with 25a proved that it induces cell cycle arrest at G2/M and pre-G1 phases. Furthermore, it was confirmed that 25a induces cancer cell death through apoptosis, supported by increased caspases 3/9 levels and increased Bax/Bcl-2 ratio in the three cancer cells. In addition, docking studies proved the exact fit of 25a into the active site of EGFR-TK, VEGFR-2, CK2α, topoisomerase IIβ and tubulin. Lipinski's rule and Veber's standards were also analyzed, and results illustrated that 25a is expected to be well absorbed orally.

Full text of DOI:10.1016/j.bmc.2020.115674

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New series of isoxazole derivatives targeting EGFR-TK: Synthesis, molecular
modeling and antitumor evaluation
Eman T. Warda, Ihsan A. Shehata, Mahmoud B. El-Ashmawy, Nadia S. El-
Gohary
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BMC 115674
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Please cite this article as: Warda, E.T., Shehata, I.A., El-Ashmawy, M.B., El-Gohary, N.S., New series of
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New series of isoxazole derivatives targeting EGFR-TK: Synthesis,
molecular modeling and antitumor evaluation
a
a
a
Eman T. Warda , Ihsan A. Shehata , Mahmoud B. El-Ashmawy ,
Nadia S. El-Goharya,*
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Corresponding author: dr.nadiaelgohary@yahoo.com, nadiaelgohary2005@gmail.com
ABSTRACT: New series of isoxazole derivatives were synthesized and evaluated for in vitro
antitumor activity against HepG2, MCF-7 and HCT-116 cancer cells. Results showed that 4b and
2
5a are the most active members. Further, 4a, 8a and 16b showed strong activity against the three
cancer cells, whereas 6b, 10a, 10b and 16a exhibited moderate activity against the three cancer
cells. Moreover, 25a showed low cytotoxicity against WISH and WI38 normal cells (IC = 53.19
5
0
and 38.64 µM, respectively), and it might be used as a potent and safe antitumor agent. The nine
active compounds 4a, 4b, 6b, 8a, 10a, 10b, 16a, 16b and 25a were studied for EGFR-TK
inhibitory activity, where 10a, 10b and 25a showed the highest activity (IC = 0.064, 0.066 and
5
0
0
.054 µM, respectively). Compound 25a was also assessed against other four target proteins, and
it showed promising inhibitory activity against VEGFR-2, CK2 and topoisomerase II, and
acceptable inhibitory activity against tubulin polymerization. Cell cycle analysis of cancer cells
treated with 25a proved that it induces cell cycle arrest at G2/M and pre-G1 phases. Furthermore,
it was confirmed that 25a induces cancer cell death through apoptosis, supported by increased
caspases 3/9 levels and increased Bax/Bcl-2 ratio in the three cancer cells. In addition, docking
studies proved the exact fit of 25a into the active site of EGFR-TK, VEGFR-2, CK2,
topoisomerase II and tubulin. Lipinski’s rule and Veber’s standards were also analyzed, and
results illustrated that 25a is expected to be well absorbed orally.
Keywords: Isoxazoles; Antitumor; EGFR-TK inhibition; Cell cycle analysis; Apoptosis;
Computational studies
1

1
. Introduction
Epidermal growth factor receptor (EGFR) is a tyrosine kinase (TK) receptor of the ErbB
family [1]. It plays a key role in the regulation of various cellular functions that affect tumorous
growth and progression, including proliferation, differentiation, inhibition of apoptosis,
metastasis (through its effects on cell migration, invasiveness, and lack of adhesion dependence),
and angiogenesis [2]. Aberrant expression of EGFR-TK has been reported in various human
malignancies, including hepatocellular [3], breast [4] and colorectal [5] cancers, and in some
studies, EGFR expression was proved to be associated with poor prognosis and resistance to
conventional cancer therapies [6,7]. Therefore, inhibitors of EGFR-TK activity by competing
with its cognate ligands may potentially constitute an important class of effective drugs in cancer
therapy [8,9].
Pursing the different chemical libraries of reported potent antitumor and EGFR-TK inhibitors
(EGFR-TKIs) illustrated that several pyrimidine-based small molecules were developed as
antitumor EGFR-TKIs such as osimertinib [10], rociletinib [11] and WZ4002 [12] (Fig. 1). In
addition, other heterocycles such as isoxazole derivatives were reported as potent antitumor
EGFR-TKIs. For example, the isoxazole derivatives A exhibited excellent antitumor activity [13],
whereas derivative B was proved to have good antitumor and EGFR-TK inhibitory activities [14]
(Fig. 2). In addition, the isoindoline-1,3-diones C were reported to exert outstanding antitumor and
EGFR-TK inhibitory activities [15] (Fig. 2). Moreover, lots of bioactive compounds possessing
chloroacetamide and chloropropionamide moieties D were described as potent antitumor agents
with promising EGFR-TK inhibitory activity [16] (Fig. 2). On the other hand, the
piperazinylacetamide and piperazinylpropionamide derivatives E were established as promising
antitumor agents [17], whereas the piperazinylacetamide and piperazinylpropionamide derivatives
F were reported to have excellent antitumor and EGFR-TK inhibitory activities [16] (Fig. 2).
Besides, several thiazolone derivatives were established as antitumor agents and/or EGFR-TKIs.
For example, the thiazolone derivative G was described as antitumor agent and EGFR-TKI [18]
(Fig. 2). Additionally, lots of arylidenethiazolone derivatives H [18] and I [19] were reported to
possess outstanding antitumor and EGFR-TK inhibitory activities (Fig. 2).
On the other hand, various anthracene derivatives were proved to exert their anticancer
activity through targeting a variety of proteins such as EGFR-TK [20], VEGFR [21], CK2
2

[22,23], topoisomerase II [24] and tubulin [25,26] (Fig. 3). Further, the anthracene derivatives
were reported to trigger cell cycle arrest and apoptosis in human cancer cells through increasing
the levels of caspases 3/9 [27,28], and increasing Bax/Bcl-2 ratio [29,30].
1
.1. Rational design
The ATP-binding site of EGFR-TK (Fig. 4) has the following features: Adenine binding
region which is involved in two key hydrogen bonds formed by the interaction of N1 and N6 of
adenine ring with the corresponding amino acids. Many potent inhibitors utilize one of these
hydrogen bonds. Sugar pocket which is a hydrophilic region. Hydrophobic binding regions, though
not utilized by ATP but they have significant role in binding affinity and inhibitor selectivity.
Phosphate region which is largely solvent exposed, and can be utilized for enhancing inhibitor
selectivity [31]. The majority of EGFR-TKIs do not exploit the sugar pocket and phosphate region
[32]. The core structure of most inhibitors consists of (i) central heteroaromatic ring system that
contains at least one hydrogen bond acceptor, and it occupies the adenine binding region [33]. (ii)
Terminal hydrophobic head that interacts with the hydrophobic binding region I [34]. (iii) A spacer
corresponding to the linker region between the adenine binding region and the hydrophobic
binding region I [35]. (iv) Hydrophobic tail which is directly attached to the heteroaromatic ring
system, and it occupies the hydrophobic binding region II [36].
The main target of the current research was the synthesis of new isoxazole derivatives carrying
the essential pharmacophoric features of the reported and clinically available EGFR-TKIs as
osimertinib [10] and rociletinib [11] (Fig. 1) through applying various structural modifications at
the four different positions of EGFR-TKIs, including the central heteroaromatic ring system, the
hydrophobic head, the spacer and the hydrophobic tail (Fig. 5).
In addition, molecular hybridization is a commonly used strategy in medicinal chemistry to
design new effective compounds. It involves the combination of two or more critical entities with
relevant biological activities to attain new hybrids with improved potency, selectivity and safety
[37,38].
With regard to the reported EGFR-TKIs and adopting the molecular hybridization approach,
new series of 4,5,6,7-tetrachloro/tetrabromo-2-(isoxazol-3-yl)-2H-isoindoline-1,3-diones 2a,b
and 3a,b were designed and synthesized through incorporation of isoindoline-1,3-dione scaffold
3

as a hydrophobic head (occupies the hydrophobic binding region I) to the isoxazole nucleus as a
central heteroaromatic ring system (occupies the adenine binding region), in addition to the 5-
methyl/t-butyl substituent that acts as a hydrophobic tail (occupies the hydrophobic binding region
II) (Figs. 5 and 6). Additionally, our chemical diversity was extended to involve the combination
of morpholine or substituted piperazines to the N-(isoxazol-3-yl)acetamides 4a,b or
propionamides 5a,b to synthesize new morpholinylacetamide 6a,b, piperazinylacetamide 7a,b-
1
0a,b, morpholinylpropionamide 11a,b and piperazinylpropionamide derivatives 12a,b-15a,b,
whereas the acetamide or propionamide spacers are proposed to occupy the linker region, and the
morpholine or substituted piperazine moieties are proposed to occupy the hydrophobic binding
region I in the ATP-binding site of EGFR-TK (Figs. 5 and 7). Besides, we found it interesting to
incorporate different arylidene moieties to the (isoxazolyl)thiazolone scaffolds 16a,b to prepare
new series of 5-arylidene-2-((5-(methyl/t-butyl)isoxazol-3-yl)aminothiazol-4(5H)-ones 17a,b-
2
5a, whereas the aminothiazolone spacer is proposed to occupy the linker region, and the arylidene
moiety is proposed to occupy the hydrophobic binding region I in the ATP-binding site of EGFR-
TK (Figs. 5 and 7).
The designed new series of compounds were synthesized and evaluated for in vitro antitumor
and EGFR-TK inhibitory activities. However, just inhibiting EGFR-TK is not enough for
eradication of tumors, that is to say an anticancer agent that direct a single target is not sufficient
to improve the symptoms. Due to the limited anticancer activity of therapeutic agents that direct a
single target, researchers tried their best to explore other better protocols for cancer therapy, for
example, the multitarget-directed drug design strategy, by designing agents which would not only
direct a single target but direct multi-targets to trigger various changes in the cellular function, and
hence assure high curative effect and delayed resistance development [39]. Multitargeting
anticancer remedies exhibit synergistic or at least additive effects in comparison to single-targeted
ones, and they gained increased concern in fighting resistant cancer cells [40].
Accordingly, the most active member, [5-((anthracen-9-yl)methylidene)-2-((5-
methylisoxazol-3-yl)amino)thiazol-4(5H)-one (25a)] was further investigated to evaluate its
inhibitory activity against other target proteins, including VEGFR-2, CK2, topoisomerase II and
tubulin polymerization, as well as its ability to trigger cell cycle arrest and cell apoptosis in HepG2,
MCF-7 and HCT-116 cancer cells, and its effects on caspases 3/9 and Bax/Bcl-2 ratio. In addition,
the multi-targeting EGFR-TK, VEGFR-2, CK2, topoisomerase II and tubulin polymerization
4

inhibitory activities of 25a was supported by molecular docking studies. Elaborate study of
structure-activity relationship will expand the future prospects for synthesis and evolution of new
more potent members.
2
. Results and discussion
2
.1. Chemistry
The 3-amino-5-(methyl/t-butyl)isoxazoles 1a,b were utilized as key starting materials for
synthesis of the new compounds 2-25 as delineated in Schemes 1-3. Pointing to Scheme 1, the
4
,5,6,7-tetrachloro/tetrabromo-2-(5-(methyl/t-butyl)isoxazol-3-yl)-2H-isoindoline-1,3-diones
a,b and 3a,b were synthesized through reaction of amines 1a,b with 4,5,6,7-
2
tetrachloro/tetrabromophthalic anhydride in refluxing glacial acetic acid for 20-30 min. Referring
to Scheme 2, the chloroacetamides 4a,b were synthesized through stirring amines 1a,b with
chloroacetyl chloride in chloroform in presence of triethylamine (TEA) as a catalyst, whereas the
chloropropionamides 5a,b were obtained through stirring amines 1a,b with chloropropionyl
chloride in toluene in presence of anhydrous potassium carbonate as a catalyst. Reaction of
chloroacetamides 4a,b or chloropropionamides 5a,b with morpholine or substituted piperazines in
refluxing toluene in presence of TEA as a catalyst produced the morpholinylacetamides 6a,b,
substituted piperazinylacetamides 7a,b-10a,b, morpholinylpropionamides 11a,b and substituted
piperazinylpropionamides 12a,b-15a,b, respectively. With regard to Scheme 3, the thiazolone
derivatives 16a,b were synthesized through heating chloroacetamides 4a,b with ammonium
thiocyanate in refluxing ethanol. Reaction of thiazolone derivatives 16a,b with aromatic aldehydes
in presence of ethanolic solution of sodium hydroxide gave the corresponding 5-
arylidenethiazolone derivatives 17a,b-25a. The new compounds were characterized by spectral
1
13
(
IR, MS, H NMR and C NMR) and elemental analyses.
5

2
.2. Biological screening
2
.2.1. Cytotoxicity testing
2
.2.1.1. In vitro antitumor evaluation
The new compounds 2-25 were evaluated for in vitro antitumor activity against hepatocellular
carcinoma (HepG2), breast carcinoma (MCF-7) and colorectal carcinoma (HCT-116) cells
following MTT assay [41-43]. Compounds’ concentrations that cause 50% inhibition of cell
viability (IC , µM) were calculated and compared to doxorubicin and erlotinib (standard drugs)
5
0
(
Table 1). The obtained data indicated that compounds 4b (IC = 9.74±0.8, 7.15±0.6 and 9.96±0.7
50
µM against HepG2, MCF-7 and HCT-116, respectively) and 25a (IC = 6.86±0.5, 6.38±0.4 and
5
0
7
.72±0.5 µM against HepG2, MCF-7 and HCT-116, respectively) have the highest potency against
all tested cancer cells. Further, compounds 4a (IC = 11.10±1.1, 10.39±0.9 and 13.07±1.3 µM
5
0
against HepG2, MCF-7 and HCT-116, respectively), 8a (IC50 = 19.49±1.6, 12.62±1.0 and
1
9
7.63±1.4 µM against HepG2, MCF-7 and HCT-116, respectively) and 16b (IC = 18.27±1.4,
50
.42±0.8 and 11.48±1.0 µM against HepG2, MCF-7 and HCT-116, respectively) displayed strong
activity against all tested cancer cells, whereas compounds 6b, 10a, 10b and 16a were evidenced
to be strongly active against MCF-7 cells (IC = 19.63±1.5, 16.70±1.3, 17.16±1.4 and 14.81±1.2
5
0
µM, respectively), and moderately active against HepG2 and HCT-116 cells (IC = 20.81-35.04
5
0
µM). On the other hand, compounds 8b (IC = 39.14±2.6, 27.05±2.1 and 38.38±2.7 µM against
5
0
HepG2, MCF-7 and HCT-116, respectively), 15b (IC = 37.15±2.6, 22.96±1.7 and 33.59±2.4 µM
5
0
against HepG2, MCF-7 and HCT-116, respectively) and 18a (IC = 32.83±2.3, 24.17±1.9 and
5
0
3
6.60±2.5 µM against HepG2, MCF-7 and HCT-116, respectively) were proved to be moderately
active against the three cancer cells, whereas 15a and 19b showed moderate activity against MCF-
cells (IC = 29.08±2.3 and 39.56±2.7 µM, respectively). The remaining compounds were found
7
5
0
to be of weakly active or inactive against the chosen cancer cells.
6

2
.2.1.1.1. Structure-activity relationship
Regarding the antitumor activity of compounds 2a,b and 3a,b
Presence of substituted 1,3-dioxoisoindoline moiety at 3-position of isoxazole ring resulted in
weak or no activity against the three tested cancer cells regardless of the type of substituent on
both the isoxazole and the isoindoline rings (compounds 2a,b and 3a,b).
Referring to the activity of chloroacetamides 4a,b and chloropropionamides 5a,b
The two chloroacetamides 4a,b showed higher activity than the corresponding
chloropropionamides 5a,b, and this indicates that increasing the distance between the amide
functionality and the halogen led to decreased activity. On the other hand, presence of 5-(t-
butyl)isoxazol-3-yl moiety in compound 4b led to mild increase in activity compared to the
corresponding 5-methylisoxazol-3-yl counterpart (compound 4a), the increased activity of 4b
compared to 4a might be attributed to the higher lipophilicity of 4b (LogP = 2.27) compared to 4a
(LogP = 0.55). On the opposite side, presence of 5-methylisoxazol-3-yl moiety in 5a led to higher
activity than the corresponding 5-(t-butyl)isoxazol-3-yl counterpart (compound 5b).
Taking into account the structures of morpholinylacetamides 6a,b and piperazinylacetamides
7
a,b-10a,b
The N-(5-(t-butyl)isoxazol-3-yl)-2-morpholinylacetamide (6b) showed strong activity against
MCF-7 cells, and moderate activity against HepG2 and HCT-116 cells. Replacing 5-(t-
butyl)isoxazol-3-yl moiety in 6b with 5-methylisoxazole counterpart led to weak activity against
MCF-7 cells, and no activity against HepG2 and HCT-116 cells, and this might be attributed to
decreased lipophilicity of 6a (LogP = 0.19) compared to 6b (LogP = 1.91). Further, the 2-(4-
ethylpiperazin-1-yl)-N-(5-methylisoxazol-3-yl)acetamide (8a) exhibited strong activity against
the three tested cancer cells, and it is considered to be the most active member amongst the tested
piperazinylacetamide derivatives. Replacing 5-methylisoxazol-3-yl moiety in 8a with 5-(t-
butyl)isoxazol-3-yl led to decreased activity against all tested cancer cells (compound 8b). On the
other hand, replacing 4-ethylpiperazine moiety in compounds 8a,b with 4-methylpiperazine led to
weak activity against the three tested cancer cells (compounds 7a,b), and this might be attributed
to decreased lipophilicity of 7a,b (LogP = 0.23 and 1.96, respectively) compared to 8a,b (LogP =
0
.61 and 2.33, respectively). From another point of view, replacing 4-ethylpiperazine moiety in 8a
with 4-phenylpiperazine led to decreased activity against the three tested cancer cells (compound
7

1
0a), whereas replacing 5-methylisoxazol-3-yl moiety in 10a with 5-(t-butyl)isoxazol-3-yl led to
mild increase in activity against HepG2 and HCT-116 cells, and mild decrease in activity against
MCF-7 cells (compound 10b). On contrary, replacing 4-phenylpiperazine moiety in compounds
1
0a,b with 4-benzylpiperazine led to decreased activity against the three tested cancer cells
(
compounds 9a,b versus 10a,b), and this might be attributed to decreased lipophilicity of 9a,b
LogP = 1.63 and 3.36, respectively) compared to 10a,b (LogP = 1.93 and 3.66, respectively).
(
Taking into account the structures of morpholinylpropionamides 11a,b and
piperazinylpropionamides 12a,b-15a,b
The N-(5-methylisoxazol-3-yl)-3-morpholinylpropionamide (11a) exhibited weak activity
against the three tested cancer cells. Replacing 5-methylisoxazol-3-yl moiety in 11a with 5-(t-
butyl)isoxazol-3-yl led to decreased activity against HepG2 and MCF-7 cells, and abolished
activity against HCT-116 cells (compound 11b). On the other hand, the 3-(4-ethylpiperazin-1-yl)-
5
-(methylisoxazol-3-yl)propionamide (12a) showed moderate activity against MCF-7 cells (IC50
44.16±3.0 µM) and weak activity against HepG2 and HCT-116 cells (IC = 56.72±3.5 and
=
5
0
7
5.81±4.2 µM, respectively). Replacing 5-methylisoxazol-3-yl moiety in 12a with 5-(t-
butyl)isoxazol-3-yl led to diminished activity against MCF-7 cells and abolished activity against
HepG2 and HCT-116 cells (compound 12b).
Replacing 4-methylpiperazine moiety in compounds 12a,b with 4-ethylpiperazine led to
increased activity against all tested cancer cells (compounds 13a,b), and this might be attributed
to increased lipophilicity of 13a,b (LogP = 0.51 and 2.24, respectively) compared to 12a,b (LogP
=
0.14 and 1.86, respectively). On the other hand, replacing 5-methylisoxazol-3-yl moiety in 13a
with 5-(t-butyl)isoxazol-3-yl led to decreased activity against the three tested cell lines (compound
3b). Additionally, replacing 4-ethylpiperazine moiety in 13a,b with 4-phenylpiperazine led to
1
apparent increase in activity against the three tested cell lines (compounds 15a,b), and this might
be attributed to increased lipophilicity of 15a,b (LogP = 1.83 and 3.56, respectively) compared to
1
3a,b (LogP = 0.51 and 2.24, respectively). On the other hand, replacing 5-methylisoxazol-3-yl
moiety in 15a with 5-(t-butyl)isoxazol-3-yl led to increased activity against the three tested cancer
cells (compound 15b). On contrary, replacing 4-phenylpiperazine moiety in 15a,b with 4-
benzylpiperazine led to weak or no activity against the three tested cancer cells (compounds
1
4a,b), and this might be attributed to decreased lipophilicity of 14a,b (LogP = 1.54 and 3.26,
respectively) compared to 15a,b (LogP = 1.83 and 3.56, respectively). Replacing 5-
8

methylisoxazol-3-yl moiety in 14a with 5-(t-butyl)isoxazol-3-yl led to mild increase in activity
against HepG2 and HCT-116 cells, and obvious increase in activity against MCF-7 cells
(
compound 14b). Comparing the activity of the piperazinylacetamides 7a,b-10a,b with that of the
piperazinylpropionamides 12a,b-15a,b, it is evident that the piperazinylacetamide derivatives
a,b-10a,b are more active against the three tested cell lines compared to their corresponding
7
piperazinylpropionamide derivatives 12a,b-15a,b, and this indicates that increasing the distance
between the amide functionality and the substituted piperazine moiety is not beneficial for the
activity.
Taking into consideration the structures of compounds 16a,b
The 2-((5-(t-butyl)isoxazol-3-yl)amino)thiazol-4(5H)-one (16b) showed strong activity
against the three tested cancer cells. Replacing 5-(t-butyl)isoxazol-3-yl moiety in 16b with 5-
methylisoxazol-3-yl led to mild decrease in activity (compound 16a), and this might be attributed
to decreased lipophilicity of 16a (LogP = 0.24) compared to 16b (LogP = 1.97).
With respect to the 5-arylidene-2-((5-(methyl/t-butyl)isoxazol-3-yl)amino)thiazol-4(5H)-ones
1
7a,b-25a
Presence of (anthracen-9-yl)methylidene moiety at 5-position of thiazole nucleus led to potent
activity against all tested cancer cells (compound 25a), and this might be attributed to the presence
of anthracene moiety that is proposed to be involved in hydrophobic interaction with different
target proteins (anthracene derivatives were previously reported to exert their antitumor activity
through targeting a variety of proteins [20-26]). Replacing anthracen-9-yl moiety in 25a with either
biphenyl-4-yl or naphthalen-2-yl counterparts led to abolished activity against the three tested
cancer cells (compounds 23a and 24a), and this might be attributed to decreased lipophilicity of
2
3a and 24a (LogP = 4.78 and 4.16, respectively) compared to 25a (LogP = 5.28). On the opposite
side, replacing 5-methylisoxazol-3-yl moiety in 23a and 24a with 5-(t-butyl)isoxazol-3-yl led to
increased activity against the three cancer cells (compounds 23b and 24b), and this might be
attributed to increased lipophilicity of 23b and 24b (LogP = 6.50 and 5.89, respectively) compared
to 23a and 24a (LogP = 4.78 and 4.16, respectively).
Additionally, replacing (anthracen-9-yl)methylidene moiety in 25a with (furan-2-yl/5-
bromothiophen-2-yl)methylidene or substituted benzylidene moieties led to obvious decrease in
activity against all tested cancer cells (compounds 17a-22a).
9

On the other hand, the 5-((5-bromothiophen-2-yl)methylidene)-2-((5-methylisoxazol-3-
yl)amino)thiazol-4-(5H)-one (18a) exhibited moderate activity against the three tested cancer
cells. Replacing 5-methylisoxazol-3-yl moiety in 18a with 5-(t-butyl)isoxazol-3-yl led to
abolished activity against the three tested cancer cells (compound 18b). Replacing 5-
bromothiophen-2-yl moiety in 18a with furan-2-yl led to decreased activity against the three cancer
cell lines (compound 17a), whereas exchanging 5-bromothiophen-2-yl moiety in 18b with furan-
2
-yl led to increased activity against all tested cancer cells (compound 17b).
Taking into account the structures of 5-(substituted benzylidene)thiazol-4(5H)-ones 19a,b-
2a,b, it is evident that the activity of these compounds is dependent on the type of substituent on
2
the benzylidene moiety, whereas presence of electron-withdrawing substituents led to higher
activity than the electron-donating counterparts, and increasing the number of electron-
withdrawing groups led to higher activity, the order of activity is 21a and 22a (the benzylidene
carrying two electron-withdrawing groups) > 20a (the benzylidene carrying one electron-
withdrawing group) > 19a (the benzylidene carrying one electron-donating group).
On the other hand, replacing 5-methylisoxazol-3-yl in 19a and 20a with 5-(t-butyl)isoxazol-
3
-yl led to obvious increase in activity against the three tested cancer cells (compound 19b and
0b), and this might be attributed to increased lipophilicity of 19b and 20b (LogP = 5.15 and 5.51,
2
respectively) compared to 19a and 20a (LogP = 3.43 and 3.79, respectively). On the opposite side,
replacing 5-methylisoxazol-3-yl in 22a with 5-(t-butyl)isoxazol-3-yl led to weak or no activity
against the three tested cancer cells (compound 22b), whereas replacing 5-methylisoxazol-3-yl
moiety in 21a with 5-(t-butyl)isoxazol-3-yl led to mild increase in activity against HepG2 and
MCF-7 cells, and decreased activity against HCT-116 cells (compound 21b).
2
.2.1.2. In vitro cytotoxicity testing of 25a against normal cells
The potent antitumor compound 25a was assessed for its cytotoxicity against amnion
epithelial (WISH) and lung fibroblast (WI38) normal cells following MTT assay [41-43], and
using doxorubicin as a reference drug. IC50 values (µM) were set, and results revealed that
compound 25a has low cytotoxicity against WISH and WI38 normal cells (IC = 53.19±3.1 and
5
0
3
8.64±2.8 µM, respectively) compared to doxorubicin (IC50 = 3.18±0.2 and 6.72±0.5 µM,
respectively), and hence it was considered to be safe against the selected normal cells at their
cytotoxic concentrations against the selected cancer cells.
1
0

2
.2.2. Mechanistic studies
2
.2.2.1. Enzyme assays
The nine active antitumor compounds 4a, 4b, 6b, 8a, 10a, 10b, 16a, 16b and 25a were
subjected to in vitro EGFR-TK inhibition assay in order to explore their possible mode of action
44]. Results are presented as percentage inhibition at 10 µM (Fig. 8) and IC (µM) (Table 2), and
[
5
0
they are compared to erlotinib (standard drug). Compounds 10a, 10b and 25a displayed the highest
EGFR-TK inhibitory activity (IC50 = 0.064±0.001, 0.066±0.001 and 0.054±0.001 µM,
respectively). Compound 25a (with potent antitumor and EGFR-TK inhibitory activities) was
further assessed for its inhibitory activities against VEGFR-2 [44], CK2 [44], topoisomerase II
[45] and tubulin polymerization [46,47]. Results are expressed as % inhibition at 10 µM (Fig. 9)
and IC (µM) (Table 3), and compared to sorafenib (standard VEGFR-2 inhibitor), quinalizarin
5
0
(
standard CK2 inhibitor), doxorubicin (standard topoisomerase II inhibitor) and colchicine
standard tubulin polymerization inhibitor). Compound 25a displayed promising inhibitory
(
activity against VEGFR-2 (IC = 0.087±0.003 µM) compared to sorafenib (IC = 0.08±0.002
5
0
50
µM), CK2 (IC50 = 0.171±0.005 µM) compared to quinalizarin (IC50 = 0.11±0.003 µM) and
topoisomerase II (IC = 0.13±0.001 µM) compared to doxorubicin (IC = 0.727±0.005 µM).
5
0
50
On the other hand, its tubulin polymerization inhibitory activity (IC = 3.61±0.06 µM) was found
5
0
to be 3 times lower than that of colchicine (IC = 1.28±0.03 µM). These results referred that
5
0
EGFR-TK might be a good target for the new active isoxazole derivatives. Additionally, VEGFR-
2
, CK2 and topoisomerase II were proved to be other good targets for compound 25a.
2
.2.2.1.1. Structure-activity relationship
Referring to results of EGFR-TK inhibitory activity, the 5-((anthracen-9-yl)methylidene)-2-
((5-methylisoxazol-3-yl)amino)thiazol-4(5H)-one (25a) showed the highest EGFR-TK inhibitory
activity (IC = 0.054±0.001 M), the high inhibitory activity of 25a might be attributed to the
5
0
presence of the 5-((anthracen-9-yl)methylidene)thiazolone moiety that offers an additional site for
hydrophobic interaction, as well as two additional hydrogen bond acceptor sites. Replacing the 5-
((anthracen-9-yl)methylidene)thiazolone moiety in 25a with 5-unsubstituted thiazolone
counterpart led to decreased activity (compound 16a, IC = 0.081±0.002 M and this ensures
5
0
the importance of anthracene moiety for the enzymatic inhibitory activity. Replacing the 5-
1
1

methylisoxazolyl moiety in 16a with 5-t-butylisoxazolyl counterpart led to decreased activity
compound 16b, IC = 0.15±0.003 M, the decreased activity of 16b compared to 16a might be
(
5
0
attributed to the steric hindrance induced by the t-butyl substituent that hindered the exact fit of
6b with ATP-binding site of EGFR-TK. Interestingly, the N-(5-(methyl/t-butyl)isoxazol-3-yl)-2-
4-phenylpiperazin-1-yl)acetamides 10a and 10b displayed promising EGFR-TK inhibitory
activity (IC =0.064±0.001 and 0.066±0.001 M, respectively), the promising activity of 10a and
1
(
5
0
1
0b might be attributed to the presence of the phenylpiperazine moiety that offers an additional
site for hydrophobic interaction as well as two additional hydrogen bond acceptor sites. Replacing
the phenylpiperazine moiety in 10a with ethylpiperazine led to mild decrease in activity
(
compound 8a, IC = 0.081±0.002 M), the decreased activity of 8a might be attributed to its
5
0
decreased lipophilicity (LogP = 0.61) compared to 10a (LogP = 1.93). On the other hand, replacing
the phenylpiperazine moiety in 10b with morpholine led to decreased activity (compound 6b, IC50
=
0.144 ±0.003 M), the decreased activity of 6b compared to 10b might be attributed to the
absence of the phenyl moiety as a site for hydrophobic interaction in 6b. In addition, replacing the
-(4-phenypiperazinyl)acetamide moiety in 10a and 10b with chloroacetamide led to decreased
EGFR-TK inhibitory activity (compounds 4a and 4b, IC = 0.106±0.002 and 0.124±0.004 M,
2
5
0
respectively), the decreased activity of 4a and 4b compared to 10a and 10b might be attributed to
the absence of the phenyl moiety as a site for hydrophobic interaction as well as the absence of the
two additional hydrogen bond acceptor sites in 4a and 4b.
2
.2.2.2. Cell cycle analysis
Diverse studies proved that EGFR-TKIs trigger cell cycle arrest and cell apoptosis [48-50].
For further understanding of the mechanism of antitumor activity of the most potent compound
5a, its effects on cell cycle distribution in HepG2, MCF-7 and HCT-116 cancer cells were studied
2
following the flow cytometry assay [51-53]. Analysis of cell cycle arrest in the three cancer cells
treated with compound 25a (at the IC of the corresponding cell line) are illustrated in Figs. 10
5
0
and 11, and compared to that of the untreated cells. Results of cell cycle arrest in HepG2 revealed
that compound 25a decreased % cells at G0/G1 phase (from 51.88% to 38.11%) and S phase (from
3
4.03% to 24.96%), and increased % cells at G2/M phase (from 14.09% to 36.93%) and pre-G1
phase (from 1.44% to 13.29%) (Figs. 10 and 11). With regard to results of cell cycle arrest in
1
2

MCF-7, compound 25a decreased % cells at G0/G1 phase (from 56.14% to 44.29%) and S phase
from 32.91% to 26.41%), and increased % cells at G2/M phase (from 10.95% to 29.30%) and
(
pre-G1 phase (from 1.78% to 16.41%) (Figs. 10 and 11). Similarly, compound 25a decreased
HCT-116 cells in G0/G1 phase (from 48.52% to 29.67%) and S phase (from 35.61% to 27.44%),
and increased HCT-116 cells at G2/M phase (from 15.87% to 42.89%) and pre-G1 phase (from
1
.19% to 22.69%) (Figs. 10 and 11). The above results indicated that 25a exhibited significant
increase in cell population of the three cancer cells at G2/M and pre-G1 phases.
2
.2.2.3. Cell apoptosis
Compound 25a was studied for its ability to trigger cell apoptosis in the three tested cancer
cell lines applying the annexin V/PI double staining flow cytometry assay [51-53]. The dot plot
flow cytometry data of the pigmented cells with propidium iodide (PI) and annexin V-FITC are
displayed in Fig. 12. Results revealed that after 24 h of exposure, compound 25a (at the IC of
5
0
the corresponding cell line) triggered early and late apoptosis in the three cancer cells in
comparison to the control untreated cells. The proportion of total apoptosis in HCT-116 cells
treated with 10 µM of compound 25a was more than that of HepG2 and MCF-7 cells (Figs. 12 and
1
3). On the other hand, compound 25a triggered weak necrosis in the three cancer cells (Figs. 12
and 13) confirming that cancer cell death triggered by 25a occurs mostly through apoptosis.
2
.2.2.4. Effects on caspases 3/9
However multiple genes have been involved in apoptosis, caspases are the key mediators of
this process. Caspases are a family of cysteine-aspartic proteases that have fundamental role in
programmed cell death [54]. Caspases are divided into two main groups, initiator caspases like
caspase-9 that are involved in mitochondrial intrinsic pathway and effector caspases like caspase-3
that are included in extrinsic death receptor pathway as well as intrinsic mitochondrial pathway
[55]. The up-regulation of caspases in cancer cells is a prime determinant of the efficacy of
anticancer agents [56]; thus, drugs that up-regulate the levels of caspases are capable of inducing
apoptosis [56]. Caspase-3 is mostly expressed in HepG2 and HCT-116 cancer cells [57,58],
whereas MCF-7 cells do not express this caspase type [59,60], and instead they express caspase-9
[61,62]. Accordingly, compound 25a was screened for its capability to elevate the levels of
caspase-3 in HepG2 and HCT-116 [63], and caspase-9 in MCF-7 cells [63]. Results (Fig. 14A)
1
3

disclosed that 25a elevated the level of caspase-3 in HepG2 and HCT-116 cells by 5.4 and 14.2
folds, respectively in comparison to the corresponding untreated cells. On the other hand, 25a
elevated the level of caspase-9 in MCF-7 cells by 4.7 folds in comparison to the untreated cells
(Fig. 14B).
2
.2.2.5. Effects on Bax and Bcl-2 proteins
Bcl-2 is an example of antiapoptotic proteins that protect cells against apoptosis. This protein
inhibits the opening of mitochondrial permeability transition pores and significantly prevents
mitochondrial membrane permeability, and in turn prevents the liberation of the apoptogenic
factors to cytosol [64,65]. On contrary, Bax is an apoptotic protein, and elevated level of Bax
protein permits increased mitochondrial membrane permeability and allows liberation of the
apoptogenic factors to cytosol [66,67]. To ensure that compound 25a triggers apoptosis through
affecting the mitochondrial mediated pathway, we extended our investigation to measure the levels
of Bax and Bcl-2 proteins in HepG2, MCF-7 and HCT-116 cells [68]. Results (Fig. 15A) revealed
that compound 25a caused 4.1, 7.0 and 6.9 folds increase in the level of Bax protein in HepG2,
MCF-7 and HCT-116 cells, respectively in comparison to the control untreated cells. On contrary,
it decreased the level of Bcl-2 protein by 0.56, 0.62 and 0.64 folds in HepG2, MCF-7 and HCT-
1
16 cells, respectively in comparison to the control untreated cells (Fig. 15B). These results
confirmed that compound 25a might trigger apoptosis through increasing Bax/Bcl-2 ratio, and in
turn through affecting the mitochondrial mediated pathway.
3
. Computational studies
3
.1. Molecular docking
Molecular docking studies were conducted in order to better understand the binding
interactions of the potent antitumor agent 25a with EGFR-TK, VEGFR-2, CK2, topoisomerase
II and tubulin aiming to explain its promising inhibitory activities. Further, docking studies will
help to identify the key structural features essential for inhibition of each target, and hence it will
offer some insights into the extra structural modifications that will help in the development of new
more potent multi-targeting antitumor agents.
1
4

In the current research, molecular docking studies relied on the crystal structure of EGFR in
complex with erlotinib (PDB ID: 1M17) [69], crystal structure of VEGFR-2 in complex with
sorafenib (PDB ID: 4ASD) [70], crystal structure of human CK2α subunit in complex with
quinalizarin (PDB ID: 3FL5) [71], crystal structure of topoisomerase IIβ in complex with DNA and
etoposide (PDB ID: 3QX3) [72] and crystal structure of tubulin-(DAMA-colchicine) complex
(PDB code: 4O2B) [73] were implemented applying “molecular operating environment (MOE)
version 2015.10” Chemical Computing Group Inc. software [74] in order to attain an idea about
the binding mode of 25a to EGFR-TK, VEGFR-2, CK2, topoisomerase II and tubulin.
In order to explore the binding mode of 25a to EGFR-TK, first, the molecular docking setup
was validated by performing re-docking of erlotinib with the active site of EGFR-TK. The re-
docking validation step reproduced the experimental binding mode of the co-crystallized ligand
efficiently indicating the proportionality of the utilized setup for the purposed docking study, and
this is proved by the small root-mean-square deviation (RMSD) of 0.80 Å (<2 Å) between the
docked pose and the co-crystallized ligand, and by the capability of the docking poses to reproduce
the key interactions shown by the co-crystallized ligand with the hot spot amino acids in the active
site, including two H-bonding interactions, one formed between N1 atom of erlotinib and Met769
residue, and the second one is a water mediated H-bonding formed between N3 atom of erlotinib
and Thr766 residue, as well as arene-cation interaction between the phenyl ring of the anilino
moiety in erlotinib and Lys721 residue (Fig. 16).
Analysis of docking results illustrated that compound 25a can interact with the hot key amino
acids in the active site of EGFR-TK (docking energy score (ΔG) = -10.80 kcal/mol) in a manner
similar to the interactions of the co-crystalized ligand (erlotinib) (docking energy score (ΔG) = -
1
0.70 kcal/mol) (Fig. 17). Compound 25a displayed two key H-bonding interactions, one formed
between N atom of thiazolone and Met769 residue, and the second one is a water mediated H-
bonding formed between O atom of the carbonyl and Thr766 residue, as well as arene-cation
interaction between the anthracene moiety and Lys721 residue (Fig. 17).
Also, superimposition of the co-crystallized ligand (erlotinib) and the docking pose of
compound 25a into the active site of EGFR-TK showed the overlap of anthracene ring in 25a with
phenyl of anilino moiety in erlotinib, as well as the overlap of thiazolone ring in 25a with
quinazoline ring in erlotinib (Fig. 18). The obtained results proved that 25a can bind to the active
1
5

site of EGFR-TK to a good extent, and it occupied the active site similarly to erlotinib (Fig. 17);
thus, it might exert its anticancer activity via EGFR-TK inhibition.
In order to rationalize the promising VEGFR-2 inhibitory activity of compound 25a, and to
get an idea about its binding mode to VEGFR-2, molecular docking of 25a into the active site of
VEGFR-2 was carried out. The co-crystallized ligand (sorafenib) is involved in eight H-bonding
interactions with Glu885, Asp1046, Cys919, Cys1045 and Leu840 residues (Fig. 19 A).
Analysis of docking results of the binding interactions of 25a with VEGFR-2 illustrated that 25a
fits well into the active site of VEGFR-2 with docking energy score (ΔG) = -13.73 Kcal/mol close
to that of sorafenib with docking energy score (ΔG) = -15.10 Kcal/mol, and it is proved to be
involved in the formation of three key H-bonding interactions, one formed between N atom of
thiazolone and Asp1046 residue, the second one formed between S atom of thiazolone and Glu885
residue, and the third one is a water mediated H-bonding formed between NH and Glu885 residue
similarly to sorafenib. In addition, it displayed arene-H interaction between the anthracene moiety
and Ile892 residue (Fig. 19 B and C).
A molecular docking study based on the crystal structure of human CK2α subunit (PDB ID:
3
FL5) [71] was performed. The co-crystallized ligand (quinalizarin) is involved in two H-bonding
interactions formed between the two hydroxy groups at 1- & 2-positions of anthracene moiety and
Lys68 residue, as well as arene-H interaction between the anthracene moiety and Ile174 residue
(Fig. 20A). The structure model of 25a and CK2α complex showed that compound 25a fits well
into the active site of CK2α with docking energy score (ΔG) = -6.21 Kcal/mol close to that of
quinalizarin with docking energy score (ΔG) = -6.71 Kcal/mol, and it is involved in two key H-
bonding interactions, one formed between NH and Asn161 residue, and the second one formed
between O atom of the carbonyl and Lys68 residue, as well as arene-H interaction between the
anthracene moiety and Ile66 residue, and arene-arene interaction between the anthracene moiety
and Phe113 residue (Fig. 20B and C).
In addition, docking analysis was carried out to assess the docking energy score and binding
mode of 25a to topoisomerase IIβ using the crystal structure of topoisomerase IIβ in complex with
DNA and etoposide (PDB ID: 3QX3) [72]. Binding mode of doxorubicin to the active site of
topoisomerase IIβ revealed five key H-bonding interactions with Lys739, Asn786, Asn867 and
Lys814 residues (Fig. 21A). The detailed analysis of the binding interactions of 25a to
topoisomerase IIβ showed that it occupied the active site of topoisomerase IIβ with good docking
1
6

energy score (ΔG) = -5.86 Kcal/mol close to that of doxorubicin with docking energy score (ΔG)
-6.51 Kcal/mol, and it displayed three key H-bonding interactions, one formed between NH and
=
Met782 residue, the second one formed between S atom of thiazolone and Met782 residue, and
the third one formed between O atom of the carbonyl and Lys814 residue, as well as arene-cation
interaction between the anthracene moiety and Lys814 residue (Fig. 21B and C).
In order to explore the binding mode of compound 25a with tubulin, docking was done in the
colchicine-binding site of tubulin using the crystal structure of tubulin-(DAMA-colchicine)
complex (PDB ID: 4O2B) [73]. The co-crystallized ligand (DAMA-colchicine) is involved in two
H-bonding interactions with ValA181 and AlaA180 residues, as well as a hydrophobic interaction
with MetB259 (Fig. 22A). Binding interactions of 25a with the colchicine-binding site of tubulin
are displayed in Fig. 22B and C, and revealed that 25a showed a consistent binding mode (docking
energy score (ΔG) = -7.35 kcal/mol) close to that of the co-crystallized ligand (DAMA-colchicine)
(docking energy score (ΔG) = -8.09 kcal/mol), and it is involved in two key H-bonding interactions,
one formed between O atom of the carbonyl and LysB254 residue, and the second one formed
between S atom of thiazolone and GluA183 residue, as well as two arene-H interactions, one
between the anthracene moiety and SerA140 residue, and the second one between the isoxazole
moiety and Leu248 residue.
Summary of residues involved in arene-cation, arene-H and arene-arene interactions, number
of hydrogen bonds, residues involved in hydrogen bonding interaction and docking energy score
(kcal/mol) of compound 25a with EGFR-TK, VEGFR-2, CK2, topoisomerase II and tubulin is
presented in Table 4.
Generally speaking, docking results proved the exact fit of 25a into the active site of the five
target proteins (EGFR-TK, VEGFR-2, CK2, topoisomerase II and tubulin), and that the
anthracene and thiazolone moieties are the essential pharmacophoric features for binding
interactions with the five targets. Moreover, the NH group is an additional moiety essential for H-
bonding interaction with the active site of VEGFR-2, CK2 and topoisomerase II. On the other
hand, the isoxazole moiety is an additional pharmacophoric moiety essential for arene-H
interaction with the active site of tubulin. The identification of the pharmacophoric features of 25a
would enable the design of new more potent multi-targeting inhibitors as prospective antitumor
agents.
1
7

3
.2. Molinspiration calculations
In silico studies are considered to be valuable tools in drug discovery, since they are beneficial
in prediction of physicochemical properties, pharmacokinetics and toxicity of drugs [75]. The new
compounds were analyzed for prediction of Lipinski’s rule [76] and Veber’s standards [77]
applying Molinspiration software [78].
Lipinski’s rule is important in prognosis of oral absorption of drugs [76], and it is dependent
on the compound’s physicochemical properties. Similarly, topological polar surface area (TPSA)
and number of rotatable bonds (Nrotb) have great effect on the rate at which drugs are orally
absorbed [77]. Molinspiration software [78] was adopted for assessment of Lipinski’s rule and
Veber’s standards of the new members. Results (Table 5) suggested that most of the investigated
compounds are in harmony with the accepted standards of Lipinski’s rule, TPSA and Nrotb. It is
noted that all of the new compounds (except 3b) have zero or one violation of Lipinski’s rule, and
they are anticipated to be well absorbed orally. Molecules violating more than one might have poor
oral absorption [76].
4
. Conclusion
Results of in vitro antitumor screening revealed that 4b and 25a have the highest antitumor
activity against the three tested cancer cells (IC = 6.38-9.96 µM). In addition, 25a showed low
5
0
cytotoxicity against WISH and WI38 normal cells (IC = 53.19 and 38.64 µM, respectively), and
5
0
it might be used as a potent and safe antitumor agent. Attempts of studying the possible mode of
action of the active compounds in the current research revealed that they have potent EGFR-TK
inhibitory activity (IC = 0.054-0.15 µM), and it could be the possible target for the new active
5
0
compounds. Further, compound 25a (the potent antitumor EGFR-TKI) showed promising
VEGFR-2, CK2 and topoisomerase II inhibitory activities, and acceptable tubulin polymerization
inhibitory activity, hence it is considered to be a promising multi-targeting antitumor agent. Results
of cell cycle analysis disclosed that 25a triggers cell cycle arrest at G2/M and pre-G1 phases in the
three cancer cell lines. Additionally, cancer cell death was proved to occur mostly through
apoptosis (supported by increased levels of caspases 3/9, and increased Bax/Bcl-2 ratio). Further,
docking studies confirmed the exact binding interactions of 25a with the active site of EGFR-TK,
VEGFR-2, CK2, topoisomerase II and tubulin. Moreover, Molinspiration calculations assured
1
8

that 25a is expected to have good oral absorption. Taking together, compound 25a is considered
to be a promising lead for design and discovery of new more potent multi-targeting inhibitors as
prospective antitumor agents.
5
. Experimental
5
.1. Chemistry
Melting points (ºC) were measured on Stuart melting point (SMP30) apparatus. IR spectral
-1
analyses (KBr disc) were achieved on Mattson 5000 FT IR spectrometer (υ in cm ), Mansoura
University, Egypt. Brucker 400 (100) MHz and Joel 500 (125) MHz spectrometers were utilized
1
13
15
for analysis of H, C and N NMR spectra in CDCl or DMSO-d (chemical shifts in δ units,
3
6
ppm) using tetramethylsilane as a standard, Mansoura University, Egypt. Mass spectral analyses
were achieved on direct probe controller inlet part to single quadropole mass analyzer in (thermo
scientific GC-MS) model (ISQ LT) using thermo x-caliber software at the Regional Center of
Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Egypt. Elemental
microanalyses (% C, H, N) were carried out, and they agreed with the proposed structures within
±
0.4% of the calculated values, Microanalytical Center, Cairo University, Egypt. TLC sheets
(silica gel 60 F254) were used for the control of reaction times. Petroleum ether/ethyl acetate (8:2)
was used for elution, and UV (366 nm) was used for spot visualization. Compounds 1a,b were
purchased from Sigma Aldrich Chemicals Co., USA. Compounds 4a [79-81], 4b [82,83] and 16a
[81] were prepared according to the reported procedures. Compound 10a was previously
mentioned in literature [84]. Compounds 8a and 13a are commercially available but no available
data were reported for them in literature.
5
.1.1. Synthesis of 4,5,6,7-tetrachloro/tetrabromo-N-(5-(methyl/t-butyl)isoxazol-3-yl)-2H-
isoindoline-1,3-diones 2a,b and 3a,b
A mixture of 3-aminoisoxazole 1a-b (0.003 mol) and substituted phthalic anhydride (0.003
mol) in glacial acetic acid (10 mL) was heated at 100 °C for 20-30 min. The solution was
concentrated under vacuum, and the formed precipitate was filtered and crystallized from
ethanol/water (2:1) to give compounds 2a,b and 3a,b.
1
9

5
.1.1.1. 4,5,6,7-Tetrachloro-N-(5-methylisoxazol-3-yl)isoindoline-1,3-dione (2a)
1
Yield 65%, m.p. 215-217 °C. IR: 1706, 1619 (2C=O). H NMR (400 MHz, DMSO-d ): δ 2.43 (s,
6
13
3
H, CH ), 6.71 (s, 1H, Isoxazole-H). C NMR: δ 12.6, 96.8, 129.3, 133.9, 135.1, 157.9, 165.0,
3
+
+
+
1
70.8. MS m/z (%): 370 (1.82, M +4), 368 (62.01, M +2), 366 (100.00, M ). Anal. Calcd. (Found)
for C H Cl N O (365.98): C, 39.38 (39.21); H, 1.10 (1.30); N, 7.65 (7.82) %.
12
4
4
2
3
5
.1.1.2. N-(5-(t-Butyl)isoxazol-3-yl)-4,5,6,7-tetrachloroisoindoline-1,3-dione (2b)
1
Yield 62%, m.p. 245-247 °C. IR: 1793, 1737 (2C=O). H NMR (400 MHz, DMSO-d ): δ 1.38 (s,
6
13
9
H, C(CH ) ), 6.56 (s, 1H, Isoxazole -H). C NMR: δ 28.8, 33.3, 96.8, 128.7, 129.1, 139.3, 153.1,
3 3
+
+
+
1
61.1, 182.7. MS m/z (%): 412 (10.34, M +4), 410 (48.30, M +2), 408 (68.60, M ), 393 (100.00).
Anal. Calcd. (Found) for C H Cl N O (408.06): C, 44.15 (43.99); H, 2.47 (2.15); N, 6.87 (6.65)
15
10
4
2
3
%.
5
.1.1.3. 4,5,6,7-Tetrabromo-N-(5-methylisoxazol-3-yl)isoindoline-1,3-dione (3a)
1
Yield 42%, m.p. 249-251 °C. IR: 1782, 1726 (2C=O). H NMR (400 MHz, CDCl ): δ 2.53 (s, 3H,
3
13
CH ), 6.46 (s, 1H, Isoxazole-H). C NMR: δ 12.9, 98.2, 122.6, 129.8, 138.9, 147.7, 160.5, 171.5.
3
+
+
+
MS m/z (%): 548 (14.00, M +4), 546 (65.49, M +2), 544 (100.00, M ). Anal. Calcd. (Found) for
C H Br N O (543.79): C, 26.50 (26.23); H, 0.74 (0.57); N, 5.15 (4.85) %.
12
4
4
2
3
5
.1.1.4. 4,5,6,7-Tetrabromo-N-(5-(t-butyl)isoxazol-3-yl)isoindoline-1,3-dione (3b)
1
Yield 45%, m.p. 270-272 °C. IR: 1740, 1705 (2C=O). H NMR (400 MHz, DMSO-d ): δ 1.38 (s,
6
13
9
H, C(CH ) ), 6.55 (s, 1H, Isoxazole-H). C NMR: δ 28.8, 33.3, 96.9, 121.7, 131.4, 137.6, 153.3,
3 3
+
+
+
1
61.5, 182.6. MS m/z (%): 590 (5.72, M +4), 588 (34.23, M +2), 586 (57.66, M ), 571 (100.00).
Anal. Calcd. (Found) for C H Br N O (585.87): C, 30.75 (30.91); H, 1.72 (1.89); N, 4.78 (4.62)
15
10
4
2
3
%.
5
.1.2. Synthesis of 2-chloro-N-(5-(methyl/t-butyl)isoxazol-3-yl)acetamides 4a,b
A mixture of amine 1a,b (0.003 mol) and chloroacetyl chloride (0.67 g, 0.006 mol) in
chloroform (10 mL) in presence of TEA (0.1 mL) was stirred at room temperature for 2-4 h. The
solution was concentrated under vacuum, and then poured onto ice-water. The precipitated solid
was filtered and crystallized from chloroform to produce the desired compounds 4a,b.
2
0

5
.1.2.1. 2-Chloro-N-(5-methylisoxazol-3-yl)acetamide (4a) [79-81]
1
Yield 65%, m.p. 189-191 °C [79]. H NMR (500 MHz, DMSO-d ): δ 2.37 (s, 3H, CH ), 4.28 (s,
6
3
2
H, COCH ), 6.62 (s, 1H, Isoxazole-H), 11.27 (s, 1H, NH). Anal. Calcd. (Found) for C H ClN O
2
6
7
2
2
(174.58): C, 41.28 (41.00); H, 4.04 (3.87); N, 16.05 (15.75) %.
5
.1.2.2. N-(5-(t-Butyl)isoxazol-3-yl)-2-chloroacetamide (4b) [82,83]
1
Yield 60%, m.p. 93-95 °C. H NMR (400 MHz, CDCl ): δ 1.27 (s, 9H, C(CH ) ), 4.14 (s, 2H,
3
3 3
COCH ), 6.62 (s, 1H, Isoxazole-H), 9.63 (s, 1H, NH). Anal. Calcd. (Found) for C H ClN O
2
9
13
2
2
(216.07): C, 49.89 (50.17); H, 6.05 (6.21); N, 12.93 (13.15) %.
5
.1.3. Synthesis of 3-chloro-N-(5-(methyl/t-butyl)isoxazol-3-yl)propionamides 5a,b
A mixture of amine 1a,b (0.003 mol) and chloropropionyl chloride (0.76 g, 0.006 mol) in
toluene (10 mL) in presence of anhydrous potassium carbonate (0.82 g, 0.006 mol) was stirred at
room temperature for 2-4 h. The solution was concentrated under vacuum, and then poured onto
ice-water. The precipitated solid was filtered and crystallized from toluene to produce the desired
compounds 5a,b.
5
.1.3.1. 3-Chloro-N-(5-methylisoxazol-3-yl)propionamide (5a)
1
Yield 65%, m.p. 120-122 °C. IR: 3222 (NH), 1704 (C=O). H NMR (500 MHz, CDCl ): δ 2.43 (s,
3
3H, CH ), 2.93 (t, 2H, COCH CH , J =13.0 Hz), 3.88 (t, 2H, COCH CH , J = 13.0 Hz), 6.78 (s,
3 2 2 2 2
13
1
H, Isoxazole-H), 10.16 (s, 1H, NH). C NMR: δ 12.6, 39.1, 39.5, 96.7, 158.2, 168.2, 170.2. MS
+
+
+
m/z (%): 191 (1.85, M +2), 189 (5.28, M ), 188 (10.37, M -1), 63 (100.00). Anal. Calcd. (Found)
for C H ClN O (188.61): C, 44.58 (44.30); H, 4.81 (4.66); N, 14.85 (14.53) %.
7
9
2
2
5
.1.3.2. N-(5-(t-Butyl)isoxazol-3-yl)-3-chloropropionamide (5b)
1
Yield 60%, m.p. 84-86 °C. IR: 3257 (NH), 1716 (C=O). H NMR (400 MHz, CDCl ): δ 1.28 (s, 9H,
3
C(CH ) ), 2.87 (t, 2H, COCH CH , J =13.0 Hz), 3.81 (t, 2H, COCH CH , J = 13.0 Hz), 6.68 (s,
3
3
2
2
2
2
13
1
H, Isoxazole-H), 10.21 (s, 1H, NH). C NMR: δ 28.6, 33.1, 39.2, 39.8, 93.6, 157.8, 168.2, 182.0.
+
+
+
MS m/z (%): 233 (1.68, M +2), 232 (6.61, M +1), 231 (13.92, M ), 63 (100.00). Anal. Calcd.
Found) for C H ClN O (230.69): C, 44.58 (44.32); H, 4.81 (4.54); N, 14.85 (14.60) %.
(
10
15
2
2
2
1

5
2
.1.4. Synthesis of 2-morpholinyl/2-(4-substituted piperazin-1-yl)acetamides 6a,b-10a,b and
-morpholinyl/2-(4-substituted piperazin-1-yl)propionamides 11a,b-15a,b
A mixture of acetamide 4a,b or propionamide 5a,b (0.002 mol), morpholine (0.17 g, 0.002
mol) or substituted piperazine (0.002 mol) and TEA (0.2 mL) in toluene (10 mL) was heated under
reflux for 15-24 h. The solvent was removed under vacuum, and the remained residue was taken
onto ice-water. The precipitated solid was filtered and crystallized from toluene to produce the
desired compounds 6a,b-10a,b and 11a,b-15a,b, respectively.
5
.1.4.1. N-(5-Methylisoxazol-3-yl)-2-morpholinylacetamide (6a)
1
Yield 50%, m.p. 99-101 °C. IR: 3483 (NH), 1704 (C=O). H NMR (400 MHz, CDCl ): δ 2.33 (s,
3
3
H, CH ), 2.60 (s, 4H, Morpholine-2CH ), 3.18 (s, 2H, COCH ), 3.71 (t, 4H, Morpholine-2CH , J
3 2 2 2
13
=
9.0 Hz), 6.63 (s, 1H, Isoxazole-H), 9.63 (s, 1H, NH). C NMR: δ 12.7, 53.7, 61.9, 66.6, 96.2,
+
1
57.3, 168.1, 170.2. MS m/z (%): 225 (7.38, M ), 42 (100.00). Anal. Calcd. (Found) for C H N O
10
15
3
3
(225.25): C, 53.32 (53.20); H, 6.71 (6.46); N, 18.66 (18.37) %.
5
.1.4.2. N-(5-(t-Butyl)isoxazol-3-yl)-2-morpholinylacetamide (6b)
1
Yield 55%, m.p. 94-96 °C. IR: 3449 (NH), 1690 (C=O). H NMR (500 MHz, CDCl ): δ 1.34 (s, 9H,
3
C(CH ) ), 2.60 (t, 4H, Morpholine-2CH , J = 9.0 Hz), 3.16 (s, 2H, COCH ), 3.76 (t, 4H,
3
3
2
2
13
Morpholine-2CH , J = 9.0 Hz), 6.69 (s, 1H, Isoxazole-H), 9.53 (s, 1H, NH). C NMR: δ 28.6, 33.0,
2
+
+
5
3.8, 62.2, 66.8, 92.9, 156.9, 168.3, 181.9. MS m/z (%): 268 (8.79, M +1), 267 (12.79, M ), 100
(
(
100.00). Anal. Calcd. (Found) for C H N O (267.33): C, 58.41 (58.69); H, 7.92 (8.08); N, 15.72
13
21
3
3
15.97) %.
5.1.4.3. N-(5-Methylisoxazol-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (7a)
1
Yield 49%, m.p. 76-78 °C. IR: 3424 (NH), 1704 (C=O). H NMR (400 MHz, CDCl ): δ 2.33 (s, 3H,
3
CH ), 2.41 (s, 3H, CH ), 2.52 (s, 4H, Piperazine-2CH ), 2.65 (s, 4H, Piperazine-2CH ), 3.16 (s,
3
3
2
2
1
3
2
9
H, COCH ), 6.73 (s, 1H, Isoxazole-H), 9.60 (s, 1H, NH). C NMR: δ 12.7, 45.8, 53.3, 54.9, 61.5,
2
+
+
6.1, 157.4, 168.7, 170.1. MS m/z (%): 239 (9.74, M +1), 238 (21.08, M ), 79 (100.00). Anal.
Calcd. (Found) for C H N O (238.29): C, 55.45 (55.17); H, 7.61 (7.44); N, 23.51 (23.19) %.
11
18
4
2
5.1.4.4. N-(5-(t-Butyl)isoxazol-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (7b)
1
Yield 46%, m.p. 92-94 °C. IR: 3450 (NH), 1656 (C=O). H NMR (500 MHz, CDCl ): δ 1.34 (s, 9H,
3
C(CH ) ), 2.33 (s, 3H, CH ), 2.52-2.64 (m, 8H, Piperazine-4CH ), 3.16 (s, 2H, COCH ), 6.69 (s,
3
3
3
2
2
13
1
H, Isoxazole-H), 9.57 (s, 1H, NH). C NMR: δ 28.6, 33.0, 45.0, 52.1, 54.4, 61.3, 92.9, 156.9, 168.1,
2
2

+
+
1
81.9. MS m/z (%): 281 (1.59, M +1), 280 (3.58, M ), 42 (100.00). Anal. Calcd. (Found) for
C H N O (280.37): C, 59.98 (59.86); H, 8.63 (8.38); N, 19.98 (19.64) %.
14
24
4
2
5.1.4.5. 2-(4-Ethylpiperazin-1-yl)-N-(5-methylisoxazol-3-yl)acetamide (8a)
1
Yield 40%, m.p. 53-55 °C. H NMR (400 MHz, CDCl ): δ 1.03 (t, 3H, CH CH , J =13.0 Hz), 2.33-
3
2
3
2.57 (m, 13H, CH , CH CH , Piperazine-4CH ), 3.09 (s, 2H, COCH ), 6.65 (s, 1H, Isoxazole-H),
3 2 3 2 2
13
9
.54 (s, 1H, NH). C NMR: δ 11.9, 12.7, 52.2, 52.6, 53.4, 61.6, 96.1, 157.4, 168.8, 170.1. Anal.
Calcd. (Found) for C H N O (252.32): C, 57.12 (56.86); H, 7.99 (7.67); N, 22.21 (21.99) %.
12
20
4
2
5.1.4.6. N-(5-(t-Butyl)isoxazol-3-yl)-2-(4-ethylpiperazin-1-yl)acetamide (8b)
1
Yield 45%, m.p. 75-77 °C. IR: 3234 (NH), 1701 (C=O). H NMR (500 MHz, CDCl ): δ 1.09 (t, 3H,
3
CH CH , J =15.0 Hz), 1.33 (s, 9H, C(CH ) ), 2.43-2.64 (m, 10H, CH CH , Piperazine-4CH ), 3.15
2
3
3 3
2
3
2
13
(
s, 2H, COCH ), 6.68 (s, 1H, Isoxazole-H), 9.59 (s, 1H, NH). C NMR: δ 11.5, 28.6, 32.9, 52.1,
2
+
5
2.4, 53.0, 61.5, 92.9, 156.9, 168.6, 181.8. MS m/z (%): 294 (20.22, M ), 46 (100.00). Anal. Calcd.
(
Found) for C H N O (294.40): C, 61.20 (61.04); H, 8.90 (8.63); N, 19.03 (18.78) %.
15 26 4 2
5.1.4.7. 2-(4-Benzylpiperazin-1-yl)-N-(5-methylisoxazol-3-yl)acetamide (9a)
1
Yield 70%, m.p. 110-112 °C. IR: 3453 (NH), 1719 (C=O). H NMR (400 MHz, CDCl ): δ 2.41 (s,
3
3H, CH ), 2.53 (s, 4H, Piperazine-2CH ), 2.63 (s, 4H, Piperazine-2CH ), 3.15 (s, 2H, COCH ),
3 2 2 2
3
.54 (s, 2H, Phenyl-CH ), 6.73 (s, 1H, Isoxazole-H), 7.27-7.33 (m, 5H, Ar-H), 9.62 (s, 1H, NH).
2
13C NMR: δ 12.6, 52.7, 53.2, 61.5, 62.6, 96.1, 127.4, 128.3, 129.3, 136.9, 157.3, 168.7, 170.1. MS
+
+
+
m/z (%): 315 (100.00, M +1), 314 (57.68, M ), 313 (28.54, M -1). Anal. Calcd. (Found) for
C H N O (314.39): C, 64.95 (65.23); H, 7.05 (7.22); N, 17.82 (18.12) %.
17
22
4
2
5.1.4.8. 2-(4-Benzylpiperazin-1-yl)-N-(5-(t-butyl)isoxazol-3-yl)acetamide (9b)
1
Yield 75%, m.p. 64-66 °C. IR: 3390 (NH), 1705 (C=O). H NMR (500 MHz, CDCl ): δ 1.34 (s, 9H,
3
C(CH ) ), 2.52 (s, 4H, Piperazine-2CH ), 2.62 (s, 4H, Piperazine-2CH ), 3.14 (s, 2H, COCH ), 3.53
3
3
2
2
2
13
(
s, 2H, Phenyl-CH ), 6.69 (s, 1H, Isoxazole-H), 7.26-7.32 (m, 5H, Ar-H), 9.61 (s, 1H, NH). C
2
NMR: δ 28.6, 32.9, 52.8, 53.4, 61.6, 62.8, 92.8, 127.1, 128.2, 129.1, 137.6, 156.9, 168.8, 181.8. MS
+
+
m/z (%): 357 (54.82, M +1), 356 (100.00, M ). Anal. Calcd. (Found) for C H N O (356.47): C,
20
28
4
2
67.39 (67.55); H, 7.92 (8.19); N, 15.72 (15.97) %.
5.1.4.9. N-(5-Methylisoxazol-3-yl)-2-(4-phenylpiperazin-1-yl)acetamide (10a) [84]
1
Yield 60%, m.p. 118-120 °C. H NMR (400 MHz, CDCl ): δ 2.34 (s, 3H, CH ), 2.69 (t, 4H,
3
3
Piperazine-2CH , J = 9.6 Hz), 3.15 (s, 2H, COCH ), 3.18 (t, 4H, Piperazine-2CH , J = 9.6 Hz),
2
2
2
6.66 (s, 1H, Isoxazole-H), 6.80-6.87 (m, 3H, Ar-H), 7.20 (t, 2H, Ar-H, J = 16.0 Hz ), 9.55 (s, 1H,
2
3

13
NH). C NMR: δ 12.7, 49.4, 53.6, 61.8, 96.2, 116.4, 120.2, 129.3, 150.9, 157.4, 168.6, 170.2. Anal.
Calcd. (Found) for C H N O (300.36): C, 63.98 (63.73); H, 6.71 (6.42); N, 18.65 (18.31) %.
16
20
4
2
5.1.4.10. N-(5-(t-Butyl)isoxazol-3-yl)-2-(4-phenylpiperazin-1-yl)acetamide (10b)
1
Yield 65%, m.p. 136-138 °C. IR: 3267 (NH), 1713 (C=O). H NMR (400 MHz, CDCl ): δ 1.35 (s,
3
9
2
1
1
H, C(CH ) ), 2.76 (t, 4H, Piperazine-2CH , J = 9.6 Hz), 3.23-3.26 (m, 6H, COCH , Piperazine-
3 3 2 2
CH ), 6.71 (s, 1H, Isoxazole-H), 6.87-6.94 (m, 4H, Ar-H), 7.28 (t, 1H, Ar-H, J = 14.8 Hz), 9.62 (s,
2
13
H, NH). C NMR: δ 28.7, 33.1, 49.3, 53.6, 61.6, 92.9, 116.4, 120.2, 129.3, 150.9, 157.1, 168.6,
+
+
81.9. MS m/z (%): 243 (45.37, M +1), 342 (100.00, M ). Anal. Calcd. (Found) for C H N O
2
19
26
4
(342.44): C, 66.64 (66.51); H, 7.65 (7.48); N, 16.36 (16.10) %.
5.1.4.11. N-(5-Methylisoxazol-3-yl)-3-morpholinylpropionamide (11a)
1
Yield 50%, m.p. 94-96 °C. IR: 3216 (NH), 1705 (C=O). H NMR (500 MHz, CDCl ): δ 2.39 (s, 3H,
3
CH ), 2.55 (t, 2H, COCH CH , J = 12.0 Hz), 2.60 (s, 4H, Morpholine-2CH ), 2.72 (t, 2H,
3
2
2
2
COCH CH , J = 12.0 Hz), 3.82 (t, 4H, Morpholine-2CH , J = 9.0 Hz), 6.65 (s, 1H, Isoxazole-H),
2
2
2
1
3
1
2
7
5
1.45 (s, 1H, NH). C NMR: δ 12.6, 31.8, 52.7, 53.6, 66.8, 96.5, 157.9, 169.7, 170.3. MS m/z (%):
+
39 (5.60, M ), 43 (100.00). Anal. Calcd. (Found) for C H N O (239.27): C, 55.22 (55.08); H,
11
17
3
3
.16 (6.97); N, 17.56 (17.33) %.
.1.4.12. N-(5-(t-Butyl)isoxazol-3-yl)-3-morpholinylpropionamide (11b)
1
Yield 55%, m.p. 102-104 °C. IR: 3256 (NH), 1709 (C=O). H NMR (400 MHz, CDCl ): δ 1.27 (s,
3
9
H, C(CH ) ), 2.51 (t, 2H, COCH CH , J = 11.6 Hz), 2.56 (s, 4H, Morpholine-2CH ), 2.68 (t, 2H,
3 3 2 2 2
COCH CH , J = 11.6 Hz), 3.76 (t, 4H, Morpholine-2CH , J = 8.8 Hz), 6.56 (s, 1H, Isoxazole-H),
2
2
2
13
1
1.48 (s, 1H, NH). C NMR: δ 28.7, 31.9, 33.0, 52.8, 53.7, 66.8, 93.3, 157.8, 170.3, 181.6. MS m/z
+
+
(
%): 282 (100.00, M +1), 281 (14.37, M ). Anal. Calcd. (Found) for C H N O (281.36): C, 59.77
14 23 3 3
(59.62); H, 8.24 (8.11); N, 14.94 (14.77) %.
5.1.4.13. N-(5-Methylisoxazol-3-yl)-3-(4-methylpiperazin-1-yl)propionamide (12a)
1
Yield 45%, m.p. 82-84 °C. IR: 3194 (NH), 1699 (C=O). H NMR (500 MHz, CDCl ): δ 2.33 (s, 3H,
3
CH ), 2.37 (s, 3H, CH ), 2.50-2.72 (m, 12H, COCH CH , Piperazine-4CH ), 6.64 (s, 1H, Isoxazole-
3
3
2
2
2
13
15
H), 11.63 (s, 1H, NH). C NMR: δ 12.6, 32.1, 45.5, 51.9, 53.0, 54.7, 96.5, 158.0, 169.6, 170.5. N
+
NMR: δ -6.9, 213.6, 336.1. MS m/z (%): 252 (5.74, M ), 56 (100.00). Anal. Calcd. (Found) for
C H N O (252.32): C, 57.12 (57.37); H, 7.99 (8.22); N, 22.21 (22.48) %.
12
20
4
2
2
4

5.1.4.14. N-(5-(t-Butyl)isoxazol-3-yl)-3-(4-methylpiperazin-1-yl)propionamide (12b)
1
Yield 49%, m.p. 72-74 °C. IR: 3181 (NH), 1698 (C=O). H NMR (500 MHz, CDCl ): δ 1.33 (s, 9H,
3
C(CH ) ), 2.36 (s, 3H, CH ), 2.51-2.74 (m, 12H, COCH CH , Piperazine-4CH ), 6.63 (s, 1H,
3
3
3
2
2
2
13
Isoxazole-H), 11.70 (s, 1H, NH). C NMR: δ 28.6, 32.1, 32.9, 45.5, 51.8, 53.0, 54.7, 93.3, 157.8,
1
5
+
1
70.4, 181.4. N NMR: δ -12.8, 199.9, 406.9. MS m/z (%): 294 (19.32, M ), 70 (100.00). Anal.
Calcd. (Found) for C H N O (294.40): C, 61.20 (61.08); H, 8.90 (8.66); N, 19.03 (18.76) %.
15
26
4
2
5.1.4.15. 3-(4-Ethylpiperazin-1-yl)-N-(5-methylisoxazol-3-yl)propionamide (13a)
1
Yield 50%, m.p. 74-76 °C. H NMR (400 MHz, CDCl ): δ 1.11 (t, 3H, CH CH , J = 14.40 Hz), 2.39-
3
2
3
2
.73 (m, 17H, CH , CH CH , COCH CH ,Piperazine-4CH ), 6.66 (s, 1H, Isoxazole-H), 11.71 (s, 1H,
3 2 3 2 2 2
13
NH). C NMR: δ 11.9, 12.6, 32.1, 52.1, 52.2, 52.7, 53.1, 96.6, 158.1, 169.6, 170.6. Anal. Calcd.
(
Found) for C H N O (266.35): C, 58.62 (58.90); H, 8.33 (8.59); N, 21.04 (21.26) %.
13 22 4 2
5.1.4.16. N-(5-(t-Butyl)isoxazol-3-yl)-3-(4-ethylpiperazin-1-yl)propionamide (13b)
1
Yield 55%, m.p. 89-91 °C. IR: 3182 (NH), 1697 (C=O). H NMR (400 MHz, CDCl ): δ 1.13 (t, 3H,
3
CH CH , J = 14.40 Hz), 1.34 (s, 9H, C(CH ) ), 2.48-2.75 (m, 14H, CH CH , COCH CH ,
2
3
3 3
2
3
2
2
13
Piperazine-4CH ,), 6.63 (s, 1H, Isoxazole-H), 11.79 (s, 1H, NH). C NMR: δ 11.9, 28.6, 32.1, 32.9,
2
1
5
5
2.1, 52.2, 52.6, 53.1, 93.3, 157.8, 170.6, 181.4. N NMR: δ 34.2, 89.7, 199.9. MS m/z (%): 309
+
+
(
5.23, M +1), 308 (8.60, M ), 57 (100.00). Anal. Calcd. (Found) for C H N O (308.43): C, 62.31
16 28 4 2
(62.20); H, 9.15 (9.03); N, 18.17 (17.89) %.
5.1.4.17. 3-(4-Benzylpiperazin-1-yl)-N-(5-methylisoxazol-3-yl)propionamide (14a)
1
Yield 60%, m.p. 74-76 °C. IR: 3194 (NH), 1698 (C=O). H NMR (500 MHz, CDCl ): δ 2.39 (s, 3H,
3
CH ), 2.51-2.73 (m, 12H, COCH CH , Piperazine-4CH ), 3.55 (s, 2H, Phenyl-CH ), 6.65 (s, 1H,
3
2
2
2
2
13
Isoxazole-H), 7.26-7.33 (m, 5H, Ar-H), 11.72 (s, 1H, NH). C NMR: δ 12.6, 32.0, 52.1, 52.8, 53.0,
+
6
2.6, 96.5, 127.2, 128.2, 129.2, 137.6, 158.0, 169.6, 170.5. MS m/z (%): 329 (100.00, M +1), 328
+
+
(
73.38, M ), 327 (29.90, M -1). Anal. Calcd. (Found) for C H N O (328.42): C, 65.83 (65.67);
18 24 4 2
H, 7.37 (7.05); N, 17.06 (16.84) %.
5.1.4.18. 3-(4-Benzylpiperazin-1-yl)-N-(5-(t-butyl)isoxazol-3-yl)propionamide (14b)
1
Yield 65%, m.p. 113-115 °C. IR: 3448 (NH), 1695 (C=O). H NMR (500 MHz, CDCl ): δ 1.33 (s,
3
9
H, C(CH ) ), 2.56-2.76 (m, 12H, COCH CH , Piperazine-4CH ), 3.59 (s, 2H, Phenyl-CH ), 6.63
3 3 2 2 2 2
13
(
s, 1H, Isoxazole-H), 7.26-7.35 (m, 5H, Ar-H), 11.69 (s, 1H, NH). C NMR: δ 28.6, 32.0, 32.9,
51.9, 52.7, 52.9, 62.5, 93.3, 127.4, 128.3, 129.3, 137.0, 157.8, 170.4, 181.4. MS m/z (%): 371
2
5

+
+
+
(
100.00, M +1), 370 (68.54, M ), 369 (10.26, M -1). Anal. Calcd. (Found) for C H N O
21 30 4 2
(370.50): C, 68.08 (67.77); H, 8.16 (7.94); N, 15.12 (14.83) %.
5.1.4.19. N-(5-Methylisoxazol-3-yl)-3-(4-phenylpiperazin-1-yl)propionamide (15a)
1
Yield 52%, m.p. 134-136 °C. IR: 3223 (NH), 1698 (C=O). H NMR (500 MHz, CDCl ): δ 2.39 (s,
3
3
H, CH ), 2.60 (t, 2H, COCH CH , J = 11.5 Hz ), 2.82 (s, 6H, Piperazine-3CH ), 3.32-3.34 (m, 4H,
3 2 2 2
COCH2CH , Piperazine-CH ), 6.65 (s, 1H, Isoxazole-H), 6.87-7.29 (m, 5H, Ar-H), 11.50 (s, 1H,
2
2
13
NH). C NMR: δ 12.6, 32.1, 49.2, 52.4, 53.2, 96.5, 116.4, 120.2, 129.1, 150.8, 157.9, 169.7, 170.4.
+
+
MS m/z (%): 315 (34.80, M +1), 314 (100.00, M ). Anal. Calcd. (Found) for C H N O (314.39):
17
22
4
2
C, 64.95 (64.72); H, 7.05 (6.86); N, 17.82 (17.53) %.
5.1.4.20. N-(5-(t-Butyl)isoxazol-3-yl)-3-(4-phenylpiperazin-1-yl)propionamide (15b)
1
Yield 58%, m.p. 123-125 °C. IR: 3240 (NH), 1704 (C=O). H NMR (500 MHz, CDCl ): δ 1.33 (s,
3
9H, C(CH ) ), 2.59 (t, 2H, COCH CH , J = 11.5 Hz ), 2.81 (s, 6H, Piperazine-3CH ), 3.32-3.34 (m,
3 3 2 2 2
4
H, COCH CH , Piperazine-CH ), 6.62 (s, 1H, Isoxazole-H), 6.87-7.27 (m, 5H, Ar-H), 11.62 (s, 1H,
2
2
2
13
NH). C NMR: δ 28.6, 32.1, 32.9, 49.2, 52.3, 53.2, 93.3, 116.4, 120.2, 129.1, 150.9, 157.7, 170.7,
+
+
1
81.5. MS m/z (%): 357 (18.84, M +1), 356 (100.00, M ). Anal. Calcd. (Found) for C H N O
20
28
4
2
(356.47): C, 67.39 (67.50); H, 7.92 (8.14); N, 15.72 (15.83) %.
5
.1.5. Synthesis of 2-((5-(methyl/t-butyl)isoxazol-3-yl)amino)thiazol-4(5H)-ones 16a,b
A mixture of 2-chloroacetamide 4a,b (0.002 mol) and ammonium thiocyanate (0.30 g, 0.004
mol) in absolute ethanol (10 mL) was heated under reflux for 3-5 h. The reaction mixture was
concentrated under vacuum, and the precipitate formed was filtered, washed with water and
crystallized from ethanol to produce the desired compounds 16a,b.
5.1.5.1. 2-((5-Methylisoxazol-3-yl)amino)thiazol-4(5H)-one (16a) [81]
1
Yield 60%, m.p. 204-206 °C [81]. H NMR (400 MHz, DMSO-d ): δ 2.49 (s, 3H, CH ), 4.13 (s,
6
3
13
2
H, Thiazole-H), 6.64 (s, 1H, Isoxazole-H), 11.38 (s, 1H, NH). C NMR: δ 12.6, 37.1, 96.6, 158.1,
1
65.1, 170.6, 174.7. Anal. Calcd. (Found) for C H N O S (197.21): C, 42.63 (42.37); H, 3.58 (3.35);
7
7
3
2
N, 21.31 (21.02) %.
5.1.5.2. 2-((5-(t-Butyl)isoxazol-3-yl)amino)thiazol-4(5H)-one (16b)
1
Yield 75%, m.p. 240-242 °C. IR: 3447 (NH), 1732 (C=O). H NMR (400 MHz, DMSO-d ): δ 1.30
6
13
(
s, 9H, C(CH ) ), 4.04 (s, 2H, Thiazole-H), 6.08 (s, 1H, Isoxazole-H). C NMR: δ 28.9, 32.9, 35.9,
3 3
2
6

+
9
6.5, 163.6, 165.2, 175.0, 181.6. MS m/z (%): 239 (19.71, M ), 102 (100.00). Anal. Calcd. (Found)
for C H N O S (239.29): C, 50.19 (50.04); H, 5.48 (5.19); N, 17.56 (17.35) %.
10
13
3
2
5
1
.1.6. Synthesis of 5-arylidene-2-((5-(methyl/t-butyl)isoxazol-3-yl)amino)thiazol-4(5H)-ones
7a,b-25a
A mixture of compound 16a,b (0.001 mol) and the appropriate araldehyde (0.001 mol) in
absolute ethanol (10 mL) was stirred at room temperature for 10 min, then an aqueous solution of
0% NaOH (10 mL) was added dropwise. The mixture was stirred at room temperature for 24 h,
1
then neutralized with 1N HCl. The precipitated solid was filtered, washed with water and
crystallized from ethanol to give the desired compounds 17a,b-25a.
5
.1.6.1. 5-((Furan-2-yl)methylidene)-2-((5-methylisoxazol-3-yl)amino)thiazol-4(5H)-one (17a)
1
Yield 50%, m.p. 80-82 °C. IR: 3425 (NH), 1699 (C=O). H NMR (500 MHz, DMSO-d ): δ 2.29 (s,
6
3
H, CH ), 6.55-6.60 (m, 3H, Isoxazole-H, Furan-2H), 7.06 (s, 1H, Furan-H), 7.82 (s, 1H, C=CH).
3
13C NMR: δ 12.3, 99.3, 109.9, 112.6, 132.9, 144.0, 151.7, 166.8, 167.6, 168.4, 172.4, 180.2. MS m/z
+
(
%): 275 (32.33, M ), 168 (100.00). Anal. Calcd. (Found) for C H N O S (275.28): C, 52.36
12
9
3
3
(52.11); H, 3.30 (3.12); N, 15.26 (15.04) %.
5
.1.6.2. 2-((5-(t-Butyl)isoxazol-3-yl)amino)-5-((furan-2-yl)methylidene)thiazol-4(5H)-one (17b)
1
Yield 55%, m.p. 216-218 °C. IR: 3450 (NH), 1708 (C=O). H NMR (500 MHz, DMSO-d ): δ 1.30
6
(
s, 9H, C(CH ) ), 6.17 (s, 1H, Isoxazole-H), 6.74 (t, 1H, Furan-H, J = 3.5 Hz), 7.05 (d, 1H, Furan-
3 3
13
H), 7.49 (s, 1H, Furan-H), 8.07 (s, 1H, C=CH), 12.50 (s, 1H, NH). C NMR: δ 28.4, 32.6, 96.7,
13.5, 117.5, 132.0, 147.3, 149.6, 164.6, 164.7, 167.5, 167.6, 181.5. MS m/z (%): 318 (22.23,
1
+
+
M +1), 317 (100.00, M ). Anal. Calcd. (Found) for C H N O S (317.36): C, 56.77 (56.92); H,
15
15
3
3
4.76 (4.95); N, 13.24 (13.45) %.
5.1.6.3. 5-((5-Bromothiophen-2-yl)methylidene)-2-((5-methylisoxazol-3-yl)amino)thiazol-
4(5H)-one (18a)
1
Yield 65%, m.p. 259-261 °C. IR: 3449 (NH), 1710 (C=O). H NMR (500 MHz, DMSO-d ): δ 2.29
6
(
s, 3H, CH ), 6.54 (s, 1H, Isoxazole-H), 7.18 (d, 1H, Thiophene-H, J = 3.5 Hz), 7.27 (d, 1H,
3
13
Thiophene-H, J = 3.5 Hz), 7.41 (s, 1H, C=CH). C NMR: δ 12.8, 99.6, 115.4, 130.8, 132.0, 134.9,
+
+
1
43.6, 167.5, 167.9, 169.1, 171.6, 180.4. MS m/z (%): 372 (1.28, M +2), 370 (4.98, M ), 95
100.00). Anal. Calcd. (Found) for C H BrN O S (370.24): C, 38.93 (38.71); H, 2.18 (2.43); N,
(
12
8
3
2 2
11.35 (11.12) %.
2
7

5
4
.1.6.4.
5-((5-Bromothiophen-2-yl)methylidene)-2-((5-(t-butyl)isoxazol-3-yl)amino)thiazol-
(5H)-one (18b)
1
Yield 68%, m.p. 243-245 °C. IR: 3451 (NH), 1706 (C=O). H NMR (400 MHz, DMSO-d ): δ 1.33
6
(
s, 9H, C(CH ) ), 6.24 (s, 1H, Isoxazole-H), 7.45 (d, 1H, Thiophene-H, J = 4.0 Hz), 7.52 (d, 1H,
3 3
13
Thiophene-H, J = 4.0 Hz), 7.92 (s, 1H, C=CH), 12.82 (s, 1H, NH). C NMR: δ 28.9, 33.1, 97.2,
+
1
18.9, 132.8, 134.4, 135.2, 139.6, 162.2, 164.8, 167.5, 167.7, 182.2. MS m/z (%): 414 (3.38, M +2),
+
4
12 (10.15, M ), 57 (100.00). Anal. Calcd. (Found) for C H BrN O S (412.32): C, 43.70 (43.91);
15
14
3
2 2
H, 3.42 (3.67); N, 10.19 (10.37) %.
5
.1.6.5. 2-((5-Methylisoxazol-3-yl)amino)-5-((4-methylphenyl)methylidene)thiazol-4(5H)-one
19a)
Yield 50%, m.p. 242-244 °C. IR: 3450 (NH), 1711 (C=O). H NMR (400 MHz, DMSO-d ): δ 2.37
(
1
6
(
s, 3H, CH ), 2.42 (s, 3H, CH ), 6.24 (s, 1H, Isoxazole-H), 7.38 (d, 2H, Ar-H, J = 8.0 Hz), 7.52 (d,
3 3
13
2
H, Ar-H, J = 8.0 Hz), 7.69 (s, 1H, C=CH), 12.68 (s, 1H, NH). C NMR: δ 12.7, 21.6, 99.8, 129.6,
+
1
29.8, 130.5, 130.9, 141.0, 143.3, 165.5, 167.8, 167.9, 171.2. MS m/z (%): 300 (10.21, M +1), 299
+
(
47.98, M ), 43 (100.00). Anal. Calcd. (Found) for C H N O S (299.35): C, 60.18 (60.05); H, 4.38
15
13
3
2
(
4.21); N, 14.04 (13.83) %.
.1.6.6. 2-((5-(t-Butyl)isoxazol-3-yl)amino)-5-((4-methylphenyl)methylidene)thiazol-4(5H)-one
19b)
Yield 53%, m.p. 246-248 °C. IR: 3451 (NH), 1711 (C=O). H NMR (500 MHz, DMSO-d ): δ 1.30
5
(
1
6
(s, 9H, C(CH ) ), 2.35 (s, 3H, CH ), 6.19 (s, 1H, Isoxazole-H), 7.36 (d, 2H, Ar-H, J = 8.0 Hz), 7.51
3 3 3
13
(
d, 2H, Ar-H, J = 8.0 Hz), 7.66 (s, 1H, C=CH), 12.62 (s, 1H, NH). C NMR: δ 21.6, 28.9, 33.1,
7.2, 130.6, 130.9, 131.7, 137.8, 140.9, 164.9, 166.6, 168.0, 168.2, 182.1. MS m/z (%): 342 (23.08,
9
+
+
M +1), 341 (92.93, M ), 148 (100.00). Anal. Calcd. (Found) for C H N O S (341.43): C, 63.32
18
19
3
2
(63.13); H, 5.61 (5.30); N, 12.31 (12.09) %.
5
.1.6.7. 5-((4-Bromophenyl)methylidene)-2-((5-methylisoxazol-3-yl)amino)thiazol-4(5H)-one
(20a)
1
Yield 70%, m.p. 248-250 °C. IR: 3451 (NH), 1715 (C=O). H NMR (500 MHz, DMSO-d ): δ 2.29
6
(
s, 3H, CH ), 6.57 (s, 1H, Isoxazole-H), 7.25 (s, 1H, C=CH), 7.46 (d, 2H, Ar-H, J = 8.0 Hz), 7.64
3
13
(
d, 2H, Ar-H, J = 8.0 Hz). C NMR: δ 12.8, 99.7, 121.2, 131.3, 132.3, 135.6, 135.9, 167.5, 167.9,
+
+
1
69.0, 172.5, 180.9. MS m/z (%): 366 (2.18, M +2), 364 (4.52, M ), 89 (100.00). Anal. Calcd.
(
Found) for C H BrN O S (364.22): C, 46.17 (46.43); H, 2.77 (2.56); N, 11.54 (11.24) %.
14 10 3 2
2
8

5
.1.6.8. 5-((4-Bromophenyl)methylidene)-2-((5-(t-butyl)isoxazol-3-yl)amino)thiazol-4(5H)-one
20b)
Yield 76%, m.p. 258-260 °C. IR: 3450 (NH), 1718 (C=O). H NMR (400 MHz, DMSO-d ): δ 1.33
(
1
6
(
s, 9H, C(CH ) ), 6.23 (s, 1H, Isoxazole-H), 7.58 (d, 2H, Ar-H, J = 8.0 Hz), 7.69 (s, 1H, C=CH),
3 3
13
7
.78 (d, 2H, Ar-H, J = 8.0 Hz), 12.75 (s, 1H, NH). C NMR: δ 28.9, 33.1, 97.2, 124.2, 125.4, 130.4,
+
+
1
32.3, 132.8, 132.9, 156.9, 164.9, 167.6, 182.2. MS m/z (%): 408 (35.17, M +2), 406 (100.00, M ).
Anal. Calcd. (Found) for C H BrN O S (406.30): C, 50.26 (50.14); H, 3.97 (3.73); N, 10.34 (10.11)
17
16
3
2
%.
5
.1.6.9. 5-((3,4-Dichlorophenyl)methylidene)-2-((5-methylisoxazol-3-yl)amino)thiazol-4(5H)-
one (21a)
Yield 80%, m.p. 223-225 °C. IR: 3451 (NH), 1721 (C=O). H NMR (500 MHz, DMSO-d ): δ 2.29
1
6
(
s, 3H, CH ), 6.57 (s, 1H, Isoxazole-H), 7.26 (s, 1H, C=CH), 7.50 (s, 1H, NH), 7.68-7.77 (m, 3H,
3
13
Ar-H). C NMR: δ 12.8, 99.7, 128.7, 129.5, 130.2, 131.5, 131.9, 135.0, 137.3, 167.3, 167.9, 169.0,
+
+
+
1
69.3, 172.0. MS m/z (%): 358 (1.10, M +4), 356 (4.87, M +2), 354 (14.06, M ), 124 (100.00).
Anal. Calcd. (Found) for C H Cl N O S (354.20): C, 47.47 (47.31); H, 2.56 (2.24); N, 11.86 (11.65)
14
9
2
3
2
%.
5
4
.1.6.10.
2-((5-(t-Butyl)isoxazol-3-yl)amino)-5-((3,4-dichlorophenyl)methylidene)thiazol-
(5H)-one (21b)
1
Yield 85%, m.p. 226-228 °C. IR: 3449 (NH), 1719 (C=O). H NMR (400 MHz, DMSO-d ): δ 1.32
6
(
s, 9H, C(CH ) ), 6.24 (s, 1H, Isoxazole-H), 7.58 (d, 1H, Ar-H, J = 8.0 Hz), 7.69 (s, 1H, C=CH),
3 3
13
7
.83 (d, 1H, Ar-H, J = 8.0 Hz), 7.92 (s, 1H, Ar-H), 12.87 (s, 1H, NH). C NMR: δ 28.8, 33.1, 97.2,
28.9, 129.4, 131.5, 132.5, 132.9, 134.5, 136.3, 164.7, 165.9, 167.6, 167.9, 182.2. MS m/z (%): 400
1
+
+
+
+
(
5.23, M +4), 398 (15.65, M +2), 397 (100.00, M +1), 396 (20.71, M ). Anal. Calcd. (Found) for
C H Cl N O S (396.29): C, 51.53 (51.65); H, 3.82 (3.96); N, 10.60 (10.83) %.
17
15
2
3
2
5
4
.1.6.11. 5-(2-Chloro-6-fluorophenyl)methylidene)-2-((5-methylisoxazol-3-yl)amino)thiazol-
(5H)-one (22a)
1
Yield 82%, m.p. 245-247 °C. IR: 3450 (NH), 1722 (C=O). H NMR (400 MHz, DMSO-d ): δ 2.39
6
(
s, 3H, CH ), 6.22 (s, 1H, Isoxazole-H), 7.42 (t, 1H, Ar-H, J = 18.0 Hz), 7.51 (d, 1H, Ar-H, J = 8.0
3
13
Hz), 7.57 (t, 1H, Ar-H, J = 14.0 Hz), 7.61 (s, 1H, C=CH), 12.84 (s, 1H, NH). C NMR: δ 12.7, 99.8,
15.8, 122.8, 122.9, 125.8, 126.6, 132.4, 132.9, 134.3, 159.8, 160.6, 160.9, 164.8, 165.2, 171.4. MS
1
2
9