Synthesis of new arylisoxazole–oxindole conjugates as potent antiproliferative agents

Article abstract of DOI:10.1111/cbdd.12884

A new series of arylisoxazole–oxindole derivatives (6a–r) were synthesized and evaluated for their antiproliferative activity against human cancer cell lines including non-small cell lung (A549), cervical (HeLa), breast (MCF-7), and prostate (DU-145) cancer cell lines. The synthesized compounds (6a–r) demonstrated excellent to moderate cytotoxicity with IC₅₀ values ranging from 0.82 to 3.69?μm. Some new compounds (6m–r) exhibited profound cytotoxicity better or similar to positive control. More particularly, the compound 6q possesses donating substituent like methoxy group presented at 5-position on D ring exhibited remarkable antiproliferative activity against A-549 (lung cancer) with an IC₅₀ value 0.82?μm. Further studies to determine the mechanistic aspects of these conjugates are under progress.

Full text of DOI:10.1111/cbdd.12884

Received Date : 19-Jul-2016
Revised Date : 09-Sep-2016
Accepted Date : 08-Oct-2016
Article type : Research Letter
Synthesis of New Arylisoxazole-Oxindole Conjugates as Potent
Antiproliferative agents
G. Bharath Kumar, Syed Nasir Abbas Bukhari*, Hua-Li Qin*,
Department of Pharmaceutical Engineering, School of chemistry, chemical engineering
and life science, Wuhan University of Technology, 205 Luoshi Road, Wuhan,430070,
P.R.China.
* Authors to whom correspondence should be addressed;
Hua-Li Qin: qinhuali@whut.edu.cn; Syed Nasir Abbas Bukhari: snab_hussaini@yahoo.com
Abstract
A new series of arylisoxazole-oxindole derivatives (6a-r) was synthesized and evaluated for their
antiproliferative activity against human cancer cell lines including non-small cell lung (A549),
cervical (HeLa), breast (MCF-7) and prostate (DU-145) cancer cell lines. The synthesized
compounds (6a-r) demonstrated excellent to moderate cytotoxicity with IC50 values ranging
from 0.82-3.69 µM. Some new compounds (6m-r) exhibited profound cytotoxicity better or
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similar to positive control. More particularly, the compound 6q possess donating substituent like
methoxy group presented at 5-position on D-ring exhibited remarkable antiproliferative activity
against A-549 (lung cancer) with an IC50 value 0.82 µM. Further studies to determine the
mechanistic aspects of these conjugates are under progress.
Keywords:. Arylisoxazole-oxindole, antiproliferative activity, nocadazole, cancer, drug discovery.
Cancer is considered to be one of the leading causes of morbidity and mortality worldwide. It is
estimated that 1,685,210 new cancer cases and 595,690 Americans will die from cancer this year,
corresponding to about 1,600 deaths per day more than HIV/AIDS, tuberculosis, and
malariambined (1). The most common causes of death are cancers of the lung, bronchus, prostate
and colorectum in men and lung, bronchus, breast, and colorectum in women. One-quarter (27%)
deaths occur due to lung cancer with uses of tobacco. Accelerating progress against cancer
requires both increased national investment in cancer research and the application of existing
cancer control knowledge across all segments of the population (2).
The discovery and development of new small compounds that demonstrate manifest
antiproliferative activity against human cancer cell lines remains an important goal in the search
for new cancer treatment agents and related therapeutic strategies. In this continuation, oxindoles
are small and important pharmacophores that are known to enhance anticancer activity. Some
established molecules such as sunitinib (1)(3), indirubin (2) (4, 5) and A-432411 (3, (Z)-3-((1H-
pyrrol-2-yl)methylene)-6-(4-hydroxy-3-methoxyphenyl)indolin-2-one) (6, 7) (Fig. 1) have
displayed excellent cytotoxic activity containing oxindole as the basic scaffold. In addition, these
natural conjugates are used in traditional medicine in china for treatment of leukaemia cancer.
Moreover, it has been observed that imidazopyridine-oxindole (8), pyrazole-oxindole (9),
imidazo[2,1b][1,3,4]thiadiazole-Linked oxindole (10) and oxindole-phenstatin/isocombretastatin
conjugates (11) exhibited profound growth inhibitory activity against various human cancer cell
lines.
The isoxazole moiety plays crucial role in several drugs such as leflunomide
(antirheumatic), valdecoxib (COX-2 inhibitor) and zonisamide (anti-convulsant). Besides,
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various isoxazole derivatives were reported to possess potent anticancer activity (12). In another
study the isoxazole heterocycle was incorporated in between the two aryl groups of CA-4 to
retain cis geometry without loss of cytotoxic activity in isoxazole liked combretastatin A-4
compounds (4) (Fig. 1) (13). Moreover, the isoxazole-containing 6-hydroxy coumarine (5) (Fig.
1) demonstrated significant cell growth inhibition against pc-3 and A543 human cancer cell lines
(14). In this continuation, Kamal and coworkers synthesized 3, 5-diaryl isoxazoline/isoxazole
linked 2,3-dihydro quinazolinone hybrids (15) and (Z)-(arylamino)-pyrazolyl/isoxazolyl2-
propenones (16) with potent antiproliferative and tubulin depolymerisation activities.
In continuation of our search to discover new effective antimitotic agents, we have
developed an efficient and versatile convergent synthetic procedure for the preparation of a new
series of Arylisoxazole-Oxindole conjugates
(6a-r) (Fig. 1) employing Knoevenagel
condensation to generate a four ring scaffold (A: methoxy/methylene dioxy, B: isoxazole, C, D:
oxindole). The congeners (6a-r) were functionalized at A and D ring with various substituents
such as methoxy, methylene dioxy, chloro, fluoro and nitro groups. New synthesized compounds
were also evaluated for their effects on a panel of human cancer cell lines.
In present study, new arylisoxazole-oxindole conjugates (6a–r) were synthesized by employing
the Knoevenagel reaction between equimolar mixtures of 3-arylisoxazole-5-carbaldehyde (12a–
c) and oxindoles (13a–f). The key intermediates 3-arylisoxazole-5-carbaldehyde (12a–c) was
prepared in four sequential steps in good yields as presented in Table 1. Initially substituted
acetophenones (7a-c) were reacted with diethyl oxalate (8) in the presence of sodium ethanolate
in ethanol to yielde ethyl 2,4-dioxo-4-(substituted phenyl)butanoates (9a-c). It was further
cyclized by NH2-OH.2HCl in ethanol to produce ethyl 3- substituted phenylisoxazole-5-
carboxylate (10a-c) in good yields. The carboxylates were reduced to (3- substituted
phenylisoxazol-5-yl)methanol (11a-c) by LiAlH4 and obtained compounds were selectively
oxidized to 3-arylisoxazole-5-carbaldehyde (12a–c) by IBX in DMSO as outlined in Scheme 1.
Detailed synthesis procedure and characterization data of new compounds are provided in
supporting information.
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Table 1: Structures of arylisoxazole-oxindole conjugates (6a-r) melting points and their yields.
M.P (°C)
Compound
R1
R2
H
F
Cl
H
OMe
NO2
H
F
Cl
R3
H
H
H
Cl
H
H
H
H
H
Cl
H
H
%Yield (mg)
84% (132)
78% (129)
75% (131)
72% (126)
70% (120)
84% (150)
84% (126)
73% (115)
82% (136)
76% (125)
68% (110)
80% (135)
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
148-151
144-146
156-158
153-155
152-154
168-170
152-155
159-161
162-163
157-159
165-167
171-174
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
OMe
NO2
6j
6k
6l
3,4,5-trimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4,5-trimethoxyphenyl
H
F
Cl
H
OMe
NO2
H
H
H
Cl
H
H
148-150
162-163
165-168
151-153
172-174
179-181
83% (120)
82% (123)
84% (131)
77% (122)
76% (119)
90% (145)
6m
6n
6o
6p
6q
6r
To explore the structure activity relationship of arylisoxazole-oxindole conjugates (6a-r)
consisting A, B, C and D-rings, were derived as depicted in scheme 1 and biologically evaluated.
These eighteen new synthetic compounds differing in substitution on the A and D-rings, were
evaluated for cytotoxic activity against human cancer cell lines such as A549 (non-small cell
lung cancer), HeLa (cervical carcinoma), MCF-7 (breast cancer) and DU-145 (prostage
carcinoma) and the results are summarized in Table 2 and nocadazole was employed as a
positive control (17). Interestingly, all the tested conjugates exhibited wide spectrum of
anticancer effect towards all the cancer cell lines with IC50 value range of 0.82-31.95 µM. The
compounds like (6m-r) composed of 3,4,5-trimethoxy substituent’s in A-ring exhibited profound
cytotoxic activities with IC50 value range of 0.82-3.69 µM. Particularly, the compound 6q
possessing electron donating substituent like methoxy group presented at 5-position on D-ring
manifested excellent antiproliferative activity against human non-small cell lung cancer (A-549)
with an IC50 value 0.82 µM. In comparison, the compound 6r with strong electron withdrawing
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substituent like nitro group presented at 5-position on D-ring exhibited deleterious cytotoxicity
against prostate cancer cells (DU-145) with IC50 value 3.69 μM. Notably, presence of meta,
para-dimethoxy substituents in A-ring like compounds (6g-l) inhibited cell growth with an IC50
value range of 1.57-9.54 µM. The compound 6k with 5-methoxy substitution in D-ring inhibited
the growth of MCF-7 cells with IC50 value 1.57 µM. In contrast, 6l with 5-nitro group at D-ring
showed lower activity with IC50 value of 9.54 µM against HeLa cell line. The compounds like
(6a-f) composed of para-methoxy substituent at A-ring showed potent cytotoxic activity with
IC50 value range of 6.28-31.95 µM. In detail the compound 6e which possess an electron
donating group like methoxy on the D-ring inhibited the growth of breast cancer cells with IC50
value 6.28 µM. Taken together, the compounds with methoxy substituent on D-ring exhibited
better cytotoxic effect against all the cell lines than the compounds bearing nitro substituent in
the same ring.
Table 2: Cytotoxic activity of arylisoxazole-oxindole conjugates (6a-r)
[a]IC50 (μM)
[c]HeLa
Compound
[b]A549
[e]DU145
[d]MCF-7
21.68 2.12
18.51 0.88
15.82 1.11
18.81 1.14
10.15 0.89
30.21 2.55
5.15 0.58
3.67 0.45
4.25 0.15
5.41 0.59
2.92 0.29
8.15 0.97
1.31 0.15*
0.91 0.09**
1.88 0.95*
1.57 0.16*
0.82 0.09**
1.92 0.18
1.08
20.26 1.99
19.25 1.22
22.23 3.25
21.21 1.85
11.54 1.12
28.26 2.85
4.53 0.45
4.46 0.75
3.72 0.27
6.58 0.65
1.91 0.22
9.54 1.11
1.97 0.18*
1.57 0.16*
1.67 0.66*
1.89 0.20*
0.91 0.01**
2.78 0.27
1.17
9.27 0.66
12.95 0.79
18.59 0.99
19.68 1.95
6.28 0.81
15.54 1.14
3.31 0.25
2.07 0.25
3.08 0.36
5.67 0.58
1.57 0.16*
6.55 0.77
1.62 0.11*
1.91 0.21*
2.02 0.12
2.39 0.22
1.02 0.90*
3.19 0.29
1.21
26.51 2.16
22.98 3.14
23.83 1.54
24.29 1.14
14.79 1.11
31.95 2.65
4.33 0.45
2.37 0.22
4.32 0.47
8.31 0.83
3.24 0.34
4.79 0.58
1.98 0.17*
2.59 0.22
2.48 0.48
2.95 0.27
1.52 0.44*
3.69 0.48
1.76
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
Nocadazole
[a]Concentration required to inhibition 50% cell growth following 48 h treatment with the tested drug and
the values represent mean S.D. from three different experiments performed in triplicates, [b]A549- non-
small cell lung cancer, [c]HeLa-cervix cancer, [d]MCF-7-brest cancer and [e]DU-145- prostate cancer cell
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line. Data is expressed as means
standard deviation (SD) of at least three independent
experiments. *p < 0.05 and **p < 0.01 are significant difference with respect to control.
In conclusion, a new series of arylisoxazole-oxindole derivatives (6a-r) have been synthesized
and evaluated for their cytotoxic potential against selected human cancer cell lines. All the
synthesized compounds showed potent cytotoxic activity against the tested cancer cell lines and
two of them (6n and 6q) showed IC50 values 0.91 and 0.82 µM, respectively against A549 cancer
cell line. Methoxy group presented at 5-position on D-ring manifested excellent antiproliferative
activity (6q).The results demonstrate that some compounds are promising leads and could be
taken up for further structural modifications in the development of improved anticancer agents.
Supporting Information
Additional Supporting Information related to synthesis procedures, characterization data of new
compounds and methods of biological evaluation can be found in the supporting information tab
for this article.
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Fig 1. Sunitinib (1), Indirubin (2, bis-indole alkaloid), A-432411 (3, (Z)-3-((1H-pyrrol-2-yl)methylene)-
6-(4-hydroxy-3-methoxyphenyl)indolin-2-one), cis-restricted isoxazole conjugate (4, 3,4-diarylisoxazole,
3,4-diary;isoxazole 5-amino isoxazole), Isoxazole-coumarin derivative (5, CBI-0997-[(5-(2,4-dimethoxy-
5-ethylphenyl)-4-(4-bromophenyl)isoxazole)], Arylisoxazole-oxindole conjugates (6a-r)
Scheme 1. Synthesis of arylisoxazole-oxindole conjugates (6a-r): reagents & conditions: (i) Na metal,
EtOH, rt; 4-5 H 85-90%; (ii) NH2OH.HCl, EtOH, reflux, 2-3 h, 71-81%; (iii) LAH, THF, rt, 2-3 h, 70-
80%; (iv) IBX, DMSO, 0 oC, 2-3 h, 71-85%; (v) substituted oxindoles 13a–f, piperidine, EtOH, reflux 3–
5 h, 50–70 %.
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