Synthesis of (Z)-(arylamino)-pyrazolyl/isoxazolyl-2-propenones as tubulin targeting anticancer agents and apoptotic inducers

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ARTICLE TYPE

Synthesis of (Z)ꢀ(arylamino)ꢀpyrazolyl/isoxazolylꢀ2ꢀpropenones as

Tubulin Targeting Anticancer Agents and Apoptotic Inducers

Ahmed Kamal,*^aVangala Santhosh Reddy,^aAnver Basha Shaik,^aG. Bharath Kumar,^aM V P S

Vishnuvardhan,^aSowjanya Polepalli,^bNishant Jain^b

5

A new class of pyrazole and isoxazole conjugates were synthesized and evaluated for their cytotoxic

activity against various human cancer cell lines. These compounds have shown significant cytotoxicity

with the lower IC₅₀values. FACS results revealed that A549 cells treated with these compounds arrested

10 cells at G2/M phase of the cell cycle apart from activating cyclinꢀB1 protein levels. Particularly,

compounds 9a and 9b demonstrated a remarkable inhibitory effect on tubulin polymerization and showed

pronounced inhibitory effect on the tubulin polymerization with IC₅₀values of 1.28 µM and 0.28 µM

respectively. Wereas, nocodazole a positive control have shown lower antitubulin activity with an IC₅₀

value 2.64 µM. Furthermore, these compounds induced apoptosis by loss of mitochondrial membrane

15 potential, propidium iodide (PI) staining and activation of caspaseꢀ3. A fluorescence based compititve

colchicine binding assay results suggest that these conjugates bind successfully at the colchicene binding

site of tubulin. These investigations reveal that such conjugates containing pyrazole with trimethoxy

phenyl ring and indole moieties have the potential in the development of newer chemotherapeutic agents.

20 Introduction

50

Combretastatins Aꢀ4 is a wellꢀknown natural product,

isolated from the bark of the South African tree Combretum

Microtubules are long, stiff filamentous, tubeꢀshaped

caﬀrum exhibit potent cytotoxicity and tubulin polymerization

polymers of protein; that are essential and ubiquitous in all

eukaryotic cells and are considered as an important target for

²⁵the development of anticancer agents.^1,2They are crucial in the

development and maintenance of cell shape, mitochondria, in

the transport of vesicles and other components throughout the

cells in cell signalling, cell division and mitosis.^3,4

Microtubules are polymers of α,βꢀtubulin heterodimer which

30 are in dynamic equilibrium with tubulin dimers. Microtubule

dynamics can be affected by treatment with some agents or

under influence of some conditions, which destabilize the

inhibition.^7ꢀ11Previously, a number of studies reported on

structure−activity relationships of CAꢀ4 indicating

55

OMe

MeO

OMe

MeO

OH

O

MeO

OMe

II

OMe

HN

O

I

O

N

equilibrium

between

tubulin

polymerization

and

O

H₃CO

OH

O

S

H

depolymerisation thereby effect cellular replication. This leads

35 to inhibition of the mitotic spindle formation thereby blocking

mitosis at the metaphase transition and inducing cell death.⁵

Thus, microtubules have become a significant target for

invention of new anticancer agents. Drugs that inhibit

polymerisation of microtubule are effective in the treatment of

⁴⁰breast, lung, colon, cervix and other cancers. Nevertheless,

occurrence of peripheral neuropathy is a most important

N

O

OMe

NH

N

OMe

IV

III

O

Cl

Ar

HN

R₁

O

HO

NH

complication

in

the

development

of

microtubule

R₂

A

depolymerising agents as drugs. For that reason, the discovery

of new molecules to defeat neuropathies is required. There has

45 been considerable interest in the invention and development of

small molecules that inhibit tubulin polymerisation. Some of

the important microtubule targeting agents are colchicines (I),

combretastatin Aꢀ4(II) and nocodazole (III) (Figure 1A).⁶

B

X

N

OH

N

R₃

NH

9a-q

V

Figure 1A Chemical Structures of some anticancer molecules:

Colchicines (I), combretastatin Aꢀ4 (II), nocodazole (III), isoxazole

derivative (IV), pyrazole derivative (V) and (Z)ꢀ1ꢀ(3ꢀarylꢀ1Hꢀpyrazolꢀ5ꢀ

60 yl)ꢀ3ꢀ(arylamino) propꢀ2ꢀenꢀ1ꢀone and (Z)ꢀ1ꢀ(3ꢀarylisoxazolꢀ5ꢀyl)ꢀ3ꢀ

(arylamino) propꢀ2ꢀenꢀ1ꢀone conjugates (9aꢀq).

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that cisꢀconfiguration at the olefinic bridge and 3,4,5ꢀ

trimethoxy unit on the Aꢀring of are essential for activity.^12ꢀ14

To retain the cisꢀconfiguration, alternative bridge groups like;

heteroaromatic rings (pyrazoles, thiazoles, isomeric triazoles,

tetrazoles, oxazoles, imidazoles, furans, furanones and

thiophenes) and nonꢀheterocyclic (ethers, olefins, ketones,

sulfonamides, sulfonates, amine, amide derivatives and

cyclopentanes) were introduced as substitutes to olefinic group

¹⁰without loss of potency and tubulin depolymerisation

activity.^15ꢀ19

Individually, the isoxazole (IV) core moieties have

been evaluated for antimicrobial activity, immunotropic activity,

analgesic activity, antihypertensive activity, antifungal activity

¹⁵and antitumor activity.^20ꢀ25Similarly, pyrazole (V)include

evaluated for their cytotoxicity against selected human cancer cell

lines and they also showed significant inhibition of tubulin

polymerization.

Results and Discussion

30 Chemistry

Synthesis of the (Z)ꢀ1ꢀ(3ꢀarylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ3ꢀ

(arylamino) propꢀ2ꢀenꢀ1ꢀone and (Z)ꢀ1ꢀ(3ꢀarylisoxazolꢀ5ꢀyl)ꢀ3ꢀ

(arylamino) propꢀ2ꢀenꢀ1ꢀone conjugates is carried out as shown

in Scheme 1. Diethyloxalate has been treated with substituted

³⁵acetophenones in the presence of base to obtain βꢀketoesters 3aꢀ

d. These intermediates (3aꢀd) were reacted with hydroxylamine

hydrochloride or hydrazine hydrochloride to provide aryl esters

4aꢀg. Reduction of 4aꢀg with LAH followed by oxidation with

IBX give substituted isoxazole/pyrazole aldehydes (6aꢀg). These

5

derivatives are reported for different biological activities like ⁴⁰intermediate aldehydes 6aꢀg were treated with ethynylmagnesium

antimicrobial,

antifungal,

antiꢀinflammatory

including

bromide in THF to obtain acetylene substituted arylꢀ

pyrazole/isoxazole alcohols 7aꢀg, further these alcohols (7aꢀg)

were oxidized to the desired precursors 8aꢀg with IBX in DMSO.

Finally, we achieved the target conjugates 9aꢀt by the reaction of

antiproliferative activity.^26ꢀ33However, several clinically useful

tubulin depolymerizing drugs have huge drawbacks. In the

20 present work, the antitubulin compounds developed have not

been proved in overcoming the problems of existing drugs. A ⁴⁵appropriate precursors (8aꢀg) with corresponding aryl amines in

new class of (Z)ꢀ(arylamino)ꢀisoxazolyl propꢀ2ꢀenꢀ1ꢀone and (Z)ꢀ

(arylamino)ꢀpyrazolyl propꢀ2ꢀenꢀ1ꢀones derivatives (9aꢀq) were

synthesized by keeping structural aspects like cisꢀdoble bond and

²⁵bridge groups (isoxazole and pyrazole) of combretastatin Aꢀ4 and

50

ethanol. The compound structures were confirmed by means of

¹H NMR, ¹³C NMR, HRMS and IR spectra. All the compounds

were obtained in pure Zꢀisomeric forms and Zꢀform of

compounds was confirmed based on their coupling constants³⁴.

Scheme:

O

R¹

R²

EtO

R¹

R²

(i)

(ii)

CH₃

1a-d

OEt

+

COOEt

55

60

65

O

3a-d

2

R³

COOEt

CHO

CH₂OH

X

R¹

R²

X

R¹

R²

(iii)

R¹

R²

X

(v)

(iv)

N

4a-g

5a-g

6a-g

R³

R¹

HO

O

Ar

HN

O

70

(vii)

(vi)

X

R¹

R²

R¹

R²

N

X

N

R³

9a-q

8a-g

7a-g

R³

75

Scheme: Reagents and conditions: (i) Na Metal, Ethanol, rt; 4ꢀ5 h; 85ꢀ90%; (ii) NH₂NH₂.2HCl for 4aꢀc, NH₂OH.HCl for 4dꢀg, EtOH, reflux, 2ꢀ3 h, 71ꢀ

o

81%; (iii) LAH, THF, rt, 2ꢀ3 h, (yield 70ꢀ80%); (iv) IBX, DMSO, 0 C, 2 h, 71ꢀ85%; (v) Ethynylmagnesium bromide, THF, 0^oC, rt, 4ꢀ5 h, 59ꢀ69%; (vi)

IBX, DMSO, 0^oC, 2 h, 73ꢀ86%; (vii) aryl amines, EtOH, rt, 4ꢀ5 h, 69ꢀ82%.

80

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Table 1. Structures of compounds 9a−q and their isolated yield.

Yield %

(mg)

Comp

Ar

X

R¹

R²

R³

9a

9b

9c

9d

9e

9f

5ꢀindolyl

6ꢀindolyl

NH

O

OCH₃

H

OCH₃

F

OCH₃

H

75 (120)

78 (125)

76 (131)

80 (126)

77 (139)

82 (137)

80 (163)

77 (127)

72 (116)

75 (118)

72 (130)

76 (143)

78 (135)

69 (130)

71 (120)

76 (151)

79 (140)

4ꢀtrifluoromethylphenyl

4ꢀmethoxyphenyl

3, 4, 5ꢀtrimethoxyphenyl

5ꢀindolyl

9g

9h

9i

3, 4, 5ꢀtrimethoxyphenyl

5ꢀindolyl

H

OCH₃

H

OCH₃

F

OCH₃

H

6ꢀindolyl

O

9j

4ꢀmethoxyphenyl

3, 4, 5ꢀtrimethoxyphenyl

6ꢀindolyl

O

9k

9l

O

9m

9n

9o

9p

9q

O

H

4ꢀtrifluoromethylphenyl

4ꢀmethoxyphenyl

3, 4, 5ꢀtrimethoxyphenyl

5ꢀindolyl

O

H

O

H

O

H

O

H

5

Biology

Antiproliferative activity

new class of (Z)ꢀ1ꢀ(3ꢀarylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ3ꢀ

compounds (9c and 9n) which are contain trifluorophenyl group

⁴⁵at aryl showed moderate cytotoxic activities.

A

To study the cytotoxic effect on normal cell lines,

10 (arylamino)propꢀ2ꢀenꢀ1ꢀone and (Z)ꢀ1ꢀ(3ꢀarylisoxazolꢀ5ꢀyl)ꢀ3ꢀ

(arylamino)propꢀ2ꢀenꢀ1ꢀone (9aꢀq) conjugates were synthesized

and tested for their cytotoxic activity against various human

cancer cell lines (MCFꢀ7 (breast), A549 (lung), HCT116 (colon)

and HeLa (cervix)) and the results are listed in Table 2.

compounds 9a, 9b, 9c and 9f were screened on HEKꢀ293 and the

results suggested that these conjugates (9a, 9b, 9c and 9f) were

80ꢀ100 fold less toxic to normal cell (IC₅₀values 98.5, 86.3,

50 101.7 and 102.5 µM, respectively) when it compare with cancer

cells (Table 2). Consequently, these results recommended that the

15

Most of the compounds have shown potential

cytotoxicity against tested cancer cell lines. Among the series

some compounds like 9a, 9b and 9f showed significant cytotoxic

potency with the IC₅₀values in the range of 0.36ꢀ 1.12 µM.

Structure activity relationship (SAR) (Figure 1B) studies revealed

presence of

a

pyrazole substitution on ring

B

with

trimethoxyphenyl substitution on ringꢀA and indolyl group at aryl

(Ar) resulted in promising molecules, which reveal effective

⁵⁵anticancer activity and inhibition of tubulin polymerization

activity.

²⁰that among these compounds, 9a, 9b, 9f, 9h, 9i, 9m and 9q with

indole group at aryl position are more active than other

compounds. Moreover, 9a, 9b and 9f that contain indolyl

substitution at the aryl (Ar) and 3,4,5ꢀtrimethoxyphenyl

substitution on the Aꢀring have shown a remarkable cytotoxic

25 effect. Particularly, compound 9b with 6ꢀindolyl group is most

potent than compound 9a that possessing a 5ꢀindolyl group (6ꢀ

indolyl > 5ꢀindolyl) (Figure 1B). Subsequently, compounds 9a

and 9b exhibited notable inhibition effect on the tubulin

polymerization, which certainly correlated with observed

30 antiproliferative activity. In contrast, 9a (IC₅₀: 0.61 µM) and 9b

(IC₅₀: 0.36 µM) with a trimethoxyphenyl substitution on ring A

(similar to trimethoxy phenyl ring of colchicine) exhibited strong

activity, whereas, the presence of dimethoxyphenyl substitution

on ring A of 9f, (IC₅₀: 0.76 µM) decreased the activity in A549

35 cells (trimethoxyphenyl > dimethoxyphenyl). Furthermore, the

bridge rings (pyrazole and isoxazole) were played a key role in

cytotoxic activity. The presence of pyrazole substitution on ring

B of 9a and 9b are potent than the compounds 9h and 9i which

include isoxazole substitution on ring B and this result shows that

40 isoxazole substitution is decreasing activity and pyrazole

substitution is essential for better cytotoxic activity. In addition,

Structure Activity Relation

60

65

70

Figure 1B. Structure activity relationship of (Z)ꢀ(arylamino)ꢀ

pyrazolyl/isoxazolylꢀ2ꢀpropenones (9aꢀq). The compounds that

possessing 3,4,5ꢀtrimethoxyphenyl as Aꢀring and indolyl group showed

75 significant anticancer activity. Moreover, 3,4,5ꢀtrimethoxyphenyl ring is a

hall mark for tubulin binding.

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Table 2. In vitro cytotoxic effect (^[a]IC₅₀µM) of 40 under similar experimental conditions, nocodazole showed lower

pyrazole/isoxazole ꢀ arylpropenone (9aꢀq) conjugates.

antitubulin activity with an IC₅₀value 2.64 µM. Interestingly,

compounds 9a and 9b which demonstrated strong inhibition of

tubulin assembly, have the lowest IC₅₀values than the known

tubulin assembly inhibitor, nocodazole. These results support the

⁴⁵suggestion that compound 9b is a potent inhibitor of tubulin

assembly (Table 3).

entry

1

2

3

4

5

6

7

8

Compd

^[b]A549

0.61±0.4

0.36±0.1

1.40±0.8

2.52±0.2

4.13±0.3

0.76±0.8

2.43±0.6

2.06±0.9

1.68±1.1

3.39±0.6

2.93±0.6

3.27±0.1

2.46±1.1

1.89±0.9

2.89±0.6

3.08±0.5

2.19±0.9

0.9±0.2

^[c]HeLa

0.96±0.4

0.52±0.2

1.03±0.6

2.99±0.9

3.83±1.1

0.71±0.6

2.81±0.9

2.41±0.9

1.41±1.0

2.29±0.5

3.69±0.5

2.59±1.3

2.18±0.8

2.33±0.1

2.43±1.1

3.73±0.6

1.08±0.3

1.2±0.7

^[d]MCFꢀ7

0.68±0.1

0.57±0.1

1.54±0.7

1.38±1.5

3.96±0.9

0.87±0.1

3.77±0.6

1.79±1.1

1.55±0.8

3.86±0.3

2.80±0.4

2.58±1.4

3.03±1.1

2.06±0.3

2.62±0.6

4.03±0.6

2.51±0.2

1.25±0.9

^[e]HCT116

1.05±0.1

0.64±0.3

1.36±0.7

2.50±0.6

3.77±0.8

1.12±1.0

1.86±0.6

2.65±0.9

1.29±0.6

3.77±0.1

3.74±0.3

3.17±0.3

2.67±0.7

2.81±0.5

3.44±1.2

3.18±1.4

1.36±0.4

1.3±0.7

9a^[f]

9b^[f]

9c^[f]

9d

9e

9f^[f]

9g

Table 3. Effect of compounds 9a, 9b and 9f on Tubulin

Polymerization

9h

9i

9j

9k

9l

9m

9n

9o

9

10

Compound

Inhibition of tubulinpolymerization,^aIC₅₀(µM)

11

12

13

14

15

16

17

18

9a

9b

9f

1.28 ± 0.4

0.28 ± 0.2

7.34 ± 1.0

2.64 ± 0.4

Noc (III)

[a]

Compounds (final concentration: 3 µM and final volume: 10µl) were

preꢀincubated with tubulin at a final conc. of 10µM

9p

9q

Noc

^[a]Concentration required to inhibition 50% cell growth and the values represent

[b]

⁵⁰Effect on microtubule network

mean ± S.D. from three different experiments performed in triplicates. A549:

lung adenocarcinoma epithelial cell line. ^[c]HeLa: cervix cancer cell line ^[d]MCFꢀ

7: breast adenocarcinoma cell line. ^[e]HCT116: colon cell line. ^[f]9a, 9b, 9c and 9f

were inhibited normal cells (HEKꢀ293 cell line) with IC₅₀values 98.5, 86.3, 101.7

and 102.5 µM, respectively

Compounds 9a, 9b and 9f altered the cellꢀcycle

factors with preferential G2/M phase and also these exhibit

considerable effects on tubulin assembly. Furthermore, inhibition

of tubulin polymerization is strongly correlated with G2/M cellꢀ

⁵⁵cycle arrest.³⁵As 9a, 9b and 9f showed G2/M cellꢀcycle arrest, it

was considered of interest to study the microtubule inhibitory

function of these compounds. Thus, A549 cells were treated with

9a, 9b and 9f at 3 µM for 24 h and immunofluorescence analysis

reveals that the cells exhibited a rounded or irregular morphology

⁶⁰that is typical of mitotic arrest. Moreover, the chromatin was also

condensed in the nuclei, suggesting that it is metaphase cell

arrest. Whereas, cells treated with DMSO demonstrated a normal

and intact tubulin organization. The A549 cells were also

counterstained with DAPI to visualize the morphology of nuclei

⁶⁵(Figure 3).

Effect on cell cycle arrest

5

To elucidate whether the cytotoxicity induced by the

derivatives was due to cell cycle arrest, we performed flow

cytometry analysis for derivatives that exhibited potent

cytotoxicity. Thus, Aꢀ549 cells were treated with 9a, 9b and 9f at

3 µM for 24 h and it was found that major population of cells

¹⁰accumulated in the G2/M phase of the cell cycle with 75.21,

75.61 and 74.36 % respectively (Figure 2).

15

70

75

80

20

Figure 2. Antiꢀmitotic effect of 9a, 9b and 9f by FACS analysis:

25 Induction of cell cycle arrest at G2/M by compounds 9a, 9b and 9f. A549

cells were harvested after treatment at 3 µM for 24h. Untreated cells and

DMSO treated cells served as control. The percentage of cells in each

phase of cell cycle was quantified by flow cytometry.

30 Effect on Inhibition of tubulin polymerization

Cell viability assays revealed that all compounds

showed potent cytotoxic activity against a majority of the cell

lines tested. To investigate whether the antiproliferative activities

of these conjugates are due to their antitubulin effects, we

35 employed in vitro tubulin polymerization assay (Cytoskeleton,

Inc.). For comparison, nocodazole was used as positive control in

all experiments. Results demonstrated that compounds 9a, 9b and

9f exhibited superior inhibition of tubulin assembly with IC₅₀

values as 1.28 µM, 0.28 µM and 7.34 µM respectively; however,

85 Figure 3. Effect of 9a, 9b and 9f on microtubules and nuclear

condensation: A549 cells were treated with 9a, 9b and 9f concentrations

of 3 ꢁM for 24 h, followed by fixation, permeabilization and indirect

immunofluorescent analysis with an antiꢀαꢀtubulinꢀFITC. Nuclei were

stained with DAPI. The merged images of cells stained for tubulin and

90 DAPI are represented.

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Analysis of free versus polymerized tubulin by western 60 control. Because the intrinsic fluorescence of colchicine increases

blotting

upon binding to tubulin, it was used as an index for 9a, 9b and 9f

competition with colchicine in tubulin binding. As shown in

figure 6, paclitaxel did not affect the binding to tubulin. However,

the fluorescence of colchicineꢀtubulin complex was reduced in

The

microtubules

continuously

undergo

polymerization and depolymerization which are mediated by αꢀ

and βꢀtubulin, and changing this dynamic balance is considered

5

as a potential target for cancer drug development (e.g., paclitaxel ⁶⁵the presence of compounds 9a, 9b, 9f and nocodazole in a doseꢀ

or colchicines). In order to extend the in vitro effects of the

compounds on tubulin polymerization to the cellular effects i.e.,

ratio of free vs polymerized tubulin, Western blot analysis of αꢀ

dependent manner. These observations indicate that the

compounds 9a, 9b and 9f inhibit the binding of colchicine to

tubulin, suggesting that the compounds 9a, 9band 9f binds at the

colchicine binding site of tubulin (Figure 6).

¹⁰tubulin in A549 cells was performed by treating the cells with

nocodazole (2 ꢁM), paclitaxel (1 ꢁM), 9a and 9b (2 µM) for 24

h. Immunoblot results show that the cells exposed to these

compounds 9a and 9b contain more tubulin in the soluble fraction

of cells, when compared to insoluble fraction. Hence, increased

¹⁵tubulin in soluble fraction of cells treated with these conjugates

supported well with the inhibition of tubulin polymerization

(Figure 4).

70

75

80

20

Figure 4. Distribution of tubulin in polymerized vs soluble fractions

as analyzed by immunobloting: A549 cells were treated with 2 µM of

²⁵9a, 9b and nocodazole (III) for 24 h. Paclitaxel was utilised at 1 µM

concentrations for 24h treatments. The fractions containing soluble and

polymerised tubulin were collected and separated by SDSꢀPAGE. Tubulin

was detected by immunoblot analysis.

Figure 6. Fluorescence based colchicine competitive binding assay of

conjugates 9a, 9b and 9f. It was carried out at various concentrations

0

containing 3 µM of tubulin and colchicine for 60 min at 37 C, whereas

⁸⁵nocodazole was used as a positive control and taxol was used as negative

control which binds at taxane site. Fluorescence values are normalized to

control.

30 Effect on cellular cyclinꢀB1 levels by immunoblot analysis

During a normal cell cycle, the progression of cells in

the G2 phase to M phase is triggered by the activation of the

cyclin B1 dependent kinase.^36ꢀ37So, we also investigated the

expression level of cyclinB1. The A549 cells were exposed to 9a,

³⁵9b and 9f and were then prepared for evaluation by western

blotting. As shown in Figure 9, there was a marked increase in

cyclin B1 protein levels for the compounds 9a, 9b and 9f as

compared with the control. These result showed that 9a, 9b and

9f arrest cell cycle in G2/M phase involving cellꢀcycle regulators

⁴⁰cyclin B1 (Figure 5).

Effect on mitochondrial depolarization

90

Mitochondria play an essential role in the propagation

of apoptosis. It is well established that at an early stage, apoptotic

stimuli alter the mitochondrial trans membrane potential (ꢂψ_mt).³⁹

This was monitored by the fluorescence of the dye 5,5′,6,6′ꢀ

tetrachloroꢀ1,1′,3,3′ꢀtetraethylbenzimidazolcarbocyanine (JCꢀ1).

⁹⁵Wherein, JCꢀ1 displays a red fluorescence (590 nm) with normal

cells (high ꢂψ_mt), which is caused by the spontaneous and local

formation of aggregates that are associated with a large shift in

the emission. In contrast, when the mitochondrial membrane is

depolarized (low ꢂψ_mt), JCꢀ1 forms monomers that emit at 530

100 nm.

45

105

Figure 5. Western blot analysis of CyclinꢀB1: Treatment of A549 cells

with 2 µM concentrations of 9a, 9b and 9f for 24 h resulted an increase

in Cyclin B1.

50

Competitive tubulinꢀbinding assay

110

To confirm the above observations, we have

investigated the effect of 9a, 9b and 9f on tubulin polymerization

using an in vitro tubulin polymerization assay. Compounds 9a,

55 9b and 9f inhibited the polymerization of tubulin similar to that

of nocodazole. Therefore we further assessed the ability of

compounds 9a, 9b and 9f to compete with the colchicine for

binding to tubulin via competitive binding assays.³⁸Nocodazole

was used as a positive control and paclitaxel as a negative

Figure 7. Assessment of ꢁψ_mt(mitochondrial depolarization) of

compounds 9a, 9b and 9h in A549 cells: Representative histograms of

control cells and cells incubated for 24 h in the presence of 9a, 9b and 9f

as indicated and stained with the fluorescent probe JCꢀ1 after treatment.

115 The horizontal axis shows fluorescence intensity of the JCꢀ1 monomer

and the vertical axis shows fluorescence of JCꢀ1 aggregates.

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In this study, A549 cells were treated with 9a, 9b and 35 Assessment of apoptosis by PI

9f at 2 µM that exhibited a remarkable shift increase to 54.8%,

54.9% and 53.8% of fluorescence respectively, whereas control

cells showed 13.1% and these result indicates depolarization of

the mitochondrial membrane potential by these conjugates that

results in the induction of mitochondrial apoptosis (Figure 7).

To determine the apoptotic effect of 9a, 9b and 9f, the

PIꢀstaining was studied by ﬂuorescence microscopy and these

induced morphological changes with characteristic of apoptosis

in A549 cells.^41,42Control cells displayed probable growth

⁴⁰characteristics, but compounds 9a, 9b and 9f showed typical

apoptotic features of cells such as membrane blebbing and cell

shrinkage using PI (Figure 9).

5

Caspaseꢀ3 activation

There are some reports that the cell cycle arrest at

¹⁰G2/M phase takes place by the induction of cellular apoptosis.⁴⁰

Hence, it was considered of interest to understand the correlation

of cytotoxicity with that to apoptosis by 9a and 9b.

Conclusion

45

In conclusion, we have designed, synthesized a new

type of conjugates (9aꢀq) and tested for their in vitro anticancer

activities against a variety of different human cancer cell lines.

All the compounds showed moderate to good cytotoxic potency

against most of the tested cancer cell lines, however some of the

⁵⁰compounds 9a, 9b, 9c and 9f exhibited significant cytotoxic

potency with IC₅₀values in the range of 0.36 to 1.54 µM. FACS

results reveal that these conjugates 9a, 9b and 9f caused cell

cycle arrest and accumulate cells in the G2/M phase. They inhibit

tubulin assembly as shown by the tubilin polymerization assay.

⁵⁵Interestingly, it was observed that IC₅₀value of 9b (0.28 µM) was

less than nocodazole (2.64 µM),

a well known tubulin

polymerization inhibitor and activated the cyclinꢀB1 protein

levels and disrupted microtubule system. Moreover caspaseꢀ3

activation, loss of mitochondrial membrane potential and PI

⁶⁰staining results suggest that they cause cell death by inducing

apoptosis. The compounds 9a, 9b and 9f inhibit the binding of

colchicine to tubulin, thereby suggesting that the compounds 9a,

9band 9f binds at the colchicine binding site. Based on these

results it is evident that these conjugates have the potential to be

⁶⁵taken up for further detailed investigations either alone or in

combination with the existing therapies as potential tubulin

polymerization inhibitors.

15 Figure 8. Effect of compounds 9a and 9b on caspaseꢀ3 activity: Aꢀ549

cells were treated for 48 h with 2 µM concentrations of compounds 9a

and 9b.Values indicated are the mean _ SD of two different experiments

performed in triplicates.

Cysteine aspartase group, namely, caspases play a

20 crucial role in the induction of apoptosis and amongst them

caspaseꢀ3 happens to be one of the effecter caspase. Hence, we

treated Aꢀ549 cells with 9a and 9b examined the activation of

caspaseꢀ3. The results indicate that there is nearly 3ꢀ4 fold

induction in caspaseꢀ3 activity in cells treated with these

²⁵conjugates at 2 µM concentrations. Therefore activation of

caspaseꢀ3 by 9a and 9b indicate that they have the capacity to

induce apoptosis in Aꢀ549 (Figure 8).

Experimental protocols

70 General

All chemicals were purchased from Spectrochem Pvt.

Ltd (Mumbai, India), Aldrich (Sigma–Aldrich, St. Louis, MO,

USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward

Hill, MA, USA) and were used without further purification.

⁷⁵Reactions were monitored by TLC, performed on silica gel glass

plates containing 60 GFꢀ254, and visualization on TLC was

attained by UV light or iodine indicator. Column chromatography

1

was performed with Merck 60–120 mesh silica gel. H NMR and

¹³C NMR spectra were recorded on Bruker UXNMR/XWINꢀ

80 NMR (300 M Hz) instruments. Chemical shifts are reported in

parts per million (δ in ppm) relative to the peak for TMS

(tetramethylsilane) as an intrernal standard, coupling constants

are reported in hertz (Hz). ESI spectra were recorded on Micro

mass, Quattro LC using ESI⁺software with capillary voltage 3.98

85 kV and ESI mode positive ion trap detector. Highꢀresolution mass

spectra were recorded on a QSTAR XL Hybrid MS–MS mass

spectrometer. Melting points were determined with an Electro

thermal melting point apparatus, and are uncorrected. Purity was

evaluated by analytical HPLC using waters 515 pump coupled to

90 a waters 2487 dual λ absorbance UV detector. Mobile phase:

H₂OꢀCH₃CN (10:90 v/v). (buffer ammonium acetate pH 4.4) UV

detection at 254 nm. Column used was Luna 5u C18 with 5 ꢁm

30

Figure 9. Assessment of apoptosis by PI: Cells were treated with

compounds 9a, 9b and 9f at 2 µM concentration for 24 h. Cells were

examined under a ﬂuorescence microscope.

6

|

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particle size. Flow rate: 0.7 mL/min: injection volume: 5 ꢁL

retention times are given in minutes.

Chemistry

4.17ꢀ4.36 (q, 2H, J=7.5Hz, CH₂), 6.8 (s, 1H, ArH), 6.90 (d, 2H, J

60 = 2.2Hz, ArH), 7.62 (d, 2H, J = 9.0Hz, ArH) ppm; MS (ESI):

m/z 235 [M+H] ⁺.

Preparation

phenyl)butanoates (3aꢀd)

Diethyl oxalate (2) (1.0 mol) was added to freshly prepared

of

ethyl

2,4ꢀdioxoꢀ4ꢀ(substituted

Ethyl 3ꢀ(3,4,5ꢀtrimethoxyphenyl)isoxazoleꢀ5ꢀcarboxylate (4d)

was prepared using above method by the addition of ethyl 2,4ꢀ

5

sodium ethoxide at 0 °C and after 10 minutes substituted ⁶⁵dioxoꢀ4ꢀ(3,4,5ꢀtrimethoxyphenyl)butanoate (3a) (5.62 g, 18.2

acetophenones (1aꢀd) (1.0 mol) were added slowly in small

portions maintaining the temperature 0 °C. After completion of

¹⁰addition the stirring was continued at room temperature for 4ꢀ5 h.

the reaction mixture was neutralized by diluted H₂SO₄and

mmol) and hydroxylamine hydrochloride (1.87 g, 27.2 mmol) to

ethanol (50 mL) as yellow colored solid; (4.51 g, yield 81%); mp

137ꢀ139 ^oC; ¹H NMR (300 MHz, CDCl₃): δ 1.33ꢀ1.40 (t, 3H,

J=6.7 Hz, J=7.5 Hz, ꢀCH₃), 3.88 (s, 3H, ꢀOCH₃), 3.91 (s, 6H, ꢀ

extracted with ethyl acetate. It was recrystallized from methanol ⁷⁰OCH₃), 4.33ꢀ4.45 (q, 2H, J=6.7 Hz, J=7.5 Hz, CH₂), 7.01 (s, 2H,

to offered solid products βꢀdi ketoesters (3aꢀd) (yield 85ꢀ90%)

which were taken as such for the next step.

¹⁵Preparation of ethyl 3ꢀsubstituted phenylꢀ1Hꢀpyrazoleꢀ5ꢀ

carboxylates (4aꢀc) and ethyl 3ꢀ(substituted phenyl)isoxazoleꢀ

5ꢀcarboxylate (4dꢀg)

To each ethyl 2,4ꢀdioxoꢀ4ꢀ(substituted phenyl)butanoates (3aꢀd)

obtained in the earlier step was added to hydrazine

²⁰dihydrochloride (NH₂ꢀNH₂.2HCl) for (4aꢀc) and hydroxylamine

hydrochloride (NH₂OH.HCl) for (4dꢀg) in ethanol and heated to

ArH), 7.05 (s, 1H, ArH) ppm; MS (ESI): m/z 308 [M+H] ⁺.

Ethyl 3ꢀ(3,4ꢀdimethoxyphenyl)isoxazoleꢀ5ꢀcarboxylate (4e)

Ethyl 3ꢀ(3,4ꢀdimethoxyphenyl)isoxazoleꢀ5ꢀcarboxylate (4e) was

prepared using above method by the addition of ethyl 2,4ꢀdioxoꢀ

⁷⁵4ꢀ(3,4ꢀdimethoxyphenyl)butanoate (3b) (6.12g, 21.8 mmol) and

hydroxylamine hydrochloride (2.26g, 32.7 mmol) to ethanol (60

mL) as yellow colored solid; (4.72g, yield 78%); mp 131ꢀ132 ^oC;

¹H NMR (300 MHz, CDCl₃): δ 1.40 (t, 3H, J=7.4 Hz, CH₃), 3.81

(s, 6H, OCH₃), 4.39ꢀ4.46 (q, 2H, J=7.4 Hz, CH₂), 6.76 (s, 1H,

reflux for 2ꢀ3 h. The solvent was removed under vacuum then 80 ArH), 7.18 (d, 1H, J=7.8 Hz, ArH), 7.31 (s, 1H, ArH), 7.42 (dd,

added water to the residue followed by extracted with ethyl

acetate (50 ml x 4). The organic layer was dried on anhydrous

²⁵Na₂SO₄and evaporated the solvent to obtain crude product that

was further purified by column chromatography using ethyl

1H, J=5.9 Hz, J=1.6 Hz, ArH) ppm; MS (ESI): m/z 278 [M+H] ⁺.

Ethyl 3ꢀ(4ꢀmethoxyphenyl)isoxazoleꢀ5ꢀcarboxylate (4f)

Ethyl 3ꢀ(4ꢀmethoxyphenyl)isoxazoleꢀ5ꢀcarboxylate (4f) was

prepared using above described method by the addition of ethyl

acetate and hexane. The pure aryl esters (4aꢀg) were eluted at 30ꢀ ⁸⁵2,4ꢀdioxoꢀ4ꢀ(4ꢀmethoxyphenyl)butanoate (3d) (5.89 g, 23.5

40% of ethyl acetate with good yields.

Ethyl 3ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylate

³⁰(4a):

Ethyl 3ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylate (4a)

mmol) and hydroxylamine hydrochloride (2.44 g, 35.3 mmol) to

ethanol (55 mL) as yellow colored solid; (4.36 g, yield 75%); mp

126ꢀ127 C; H NMR (400 MHz, CDCl₃): δ 1.44 (t, 1H, J=7.2

Hz, CH₃), 3.87 (s, 3H, OCH₃), 4.42ꢀ4.52 (q, 2H, J=7.2 Hz, CH₂),

o

1

was prepared using above method by the addition of ethyl 2,4ꢀ ⁹⁰6.81 (s, 1H, ArH), 7.00 (d, 2H, J=8.9 Hz, ArH), 7.75 (d, 2H,

dioxoꢀ4ꢀ(3,4,5ꢀtrimethoxyphenyl)butanoate (3a) (5.59 g, 18

mmol) and hydrazine dihydrochloride (2.81 g, 27 mmol) to

³⁵ethanol (50 mL) as yellow colored solid; (4.14 g, yield 75%); mp

J=8.9 Hz, ArH) ppm; MS (ESI): m/z 248 [M+H] ⁺.

Ethyl 3ꢀ(4ꢀfluorophenyl)isoxazoleꢀ5ꢀcarboxylate (4g)

Ethyl 3ꢀ(4ꢀfluorophenyl)isoxazoleꢀ5ꢀcarboxylate (4g) was

prepared using above explained method by the addition of ethyl

o

1

129ꢀ130 C; H NMR (300 MHz, CDCl₃):

δ 1.45 (t, 3H, J= 7.2

Hz, ꢀCH₃), 3.91 (s, 6H, ꢀOCH₃), 3.95 (s, 3H, ꢀOCH₃), 4.43ꢀ4.53 ⁹⁵2,4ꢀdioxoꢀ4ꢀ(4ꢀfluorophenyl)butanoate (3c) (5.29 g, 22.2mmol)

(q, 2H, J=7.2 Hz, CH₂), 6.87 (s, 1H, ArH), 7.02 (s, 2H, ArH)

ppm; MS (ESI): m/z 307 [M+H]⁺.

and hydroxylamine hydrochloride (2.30 g 33.3 mmol) to ethanol

(50 mL) as yellow colored solid; (3.81g, yield 73%); mp 115−116

1

40 Ethyl

3ꢀ(3,4ꢀdimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylate

^oC; H NMR (300 MHz, CDCl₃): δ 1.39 (t, 3H, J=6.9 Hz, CH₃),

(4b):

4.47ꢀ4.56 (m, 2H, CH₂), 6.69 (s, 1H, ArH), 7.21 (d, 2H, J=7.6

Ethyl 3ꢀ(3,4ꢀdimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylate 4b 100 Hz, ArH), 7.47 (d, 2H, J=7.6 Hz, ArH) ppm; MS (ESI): m/z 236

was prepared using above method by the addition of ethyl 2,4ꢀ

dioxoꢀ4ꢀ(3,4ꢀtrimethoxyphenyl)butanoate (3b) (4.95 g, 17.7

45 mmol) and hydrazine dihydrochloride (2.76 g, 26.5 mmol) to

ethanol (45 mL) as yellow colored solid (3.90g, yield 80%); mp

[M+H] ⁺.

Preparation

of

(3ꢀsubstitutedphenylꢀ1Hꢀpyrazolꢀ5ꢀ

yl)methanols (5aꢀc) and (3ꢀ(substitutedphenyl)isoxazolꢀ5ꢀ

yl)methanol (5dꢀg)

o

1

116ꢀ117 C; H NMR (300 MHz, CDCl₃):

δ

1.23ꢀ1.31 (t, 3H, 105 To the ethyl 3ꢀsubstituted phenylꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylates/

J=7.1 Hz, ꢀCH₃), 3.85 (s, 3H, ꢀOCH₃), 3.90 (s, 3H, ꢀOCH₃) 4.19ꢀ

4.32 (q, 2H, J=7.1 Hz, ꢀCH₂), 6.88 (d, 1H, J=7.1 Hz, ꢀArH), 6.94

50 (s, 1H, ArH), 7.21ꢀ7.28 (m, 1H, ArH) 7.29ꢀ7.34 (m, 1H, ArH)

9.67 (brs, 1H, ꢀNH) ppm; MS (ESI): m/z 277 [M+H] ⁺.

Ethyl 3ꢀ(4ꢀfluorophenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylate (4c)

Ethyl 3ꢀ(4ꢀfluorophenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylate (4c) was

prepared using above method by the addition of ethyl 2,4ꢀdioxoꢀ

ethyl 3ꢀsubstituted phenylꢀisoxazoleꢀ5ꢀcarboxylates (4aꢀg),

obtained in the above step was added to LiAH₄(0.5 mol) in dry

THF at 0 °C and stirred for 2ꢀ3 h at room temperature. Added

saturated NH₄Cl solution drop wise to quench the unreacted

¹¹⁰LiAlH₄and removed the THF under vacuum then extracted with

ethyl acetate (100 ml X 4). The organic layer was dried on

anhydrous Na₂SO₄and evaporated ethyl acetate to obtain color

less solid products of (3ꢀsubstitutedphenylꢀ1Hꢀpyrazolꢀ5ꢀ

yl)methanols/(3ꢀsubstitutedphenylꢀisoxazoleꢀ5ꢀyl)methanols (5aꢀ

55 4ꢀ(4ꢀfluorophenyl)butanoate (3c) (5.16g,

21.6 mmol) and

hydrazine dihydrochloride (3.38g, 32.5 mmol) to ethanol (50 mL)

o

1

as yellow colored solid (3.60g, yield 71%); mp 121ꢀ122 C; H 115 g) (yield 70ꢀ80%). The alcohols produced in this step were pure,

NMR (400 MHz, CDCl₃): 1.23ꢀ1.37 (t, 3H, J=7.5Hz, ꢀCH₃),

δ

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and no further purification was required. These compounds were

taken as such for the next step.

MHz, CDCl₃): δ 3.96 (s, 6H, OCH₃), 6.79 (s, 1H, ArH), 6.97 (d,

60 1H, J=8.3 Hz, ArH), 7.32 (s, 1H, ArH), 7.41 (dd, 1H, J=6.4 Hz,

J=1.9 Hz, ArH), 10.18 (s, 1H, CHO) MS (ESI) m/z 234 [M+H]⁺.

3ꢀ(4ꢀMethoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6f)

3ꢀ(4ꢀMethoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6f) was prepared

using method by the addition of (3ꢀ(4ꢀmethoxyphenyl)isoxazolꢀ5ꢀ

Preparation

of

3ꢀsubtitutedphenylꢀ1Hꢀpyrazoleꢀ5ꢀ

carbaldehydes (6aꢀc) and 3ꢀ(substitutedphenyl)isoxazoleꢀ5ꢀ

carbaldehyde (6dꢀg)

5

To

the

(5ꢀsubstitutedphenylꢀ1Hꢀpyrazolꢀ3ꢀyl)methanols/(5ꢀ

substitutedphenylꢀisoxazoleꢀ3ꢀyl)methanols (5aꢀg) (1 eq) ⁶⁵yl)methanol (5f) (2.32g, 11.3mmol) and IBX (4.75 g, 16.9 mmol)

produced in the above step was added IBX (1.5 eq) in DMSO and

stirred for 2 h at room temperature. After completion reaction

to DMSO (10 mL) as white colored solid (1.86 g, yield 81%); mp

114ꢀ115 ^oC; ¹H NMR (300 MHz, CDCl₃): δ 3.88 (s, 3H, OCH₃),

6.76 (s, 1H, ArH), 7.01 (d, 2H, J=8.9 Hz, ArH), 7.76 (d, 2H,

J=8.9 Hz, ArH), 10.18 (s, 1H, CHO) ppm; MS (ESI) m/z 204

¹⁰added ice cold water to the reaction mixture and extracted with

ethyl acetate (50 ml X 4). The organic layer was dried on

anhydrous Na₂SO₄and evaporated the ethyl acetate. It was ⁷⁰[M+H]⁺.

recrystallized from methanol to obtain pure corresponding 5ꢀ

subtitutedphenylꢀ1Hꢀpyrazoleꢀ3ꢀcarbaldehydes/5ꢀ

¹⁵subtitutedphenylꢀisoxazoleꢀ3ꢀcarbaldehydes (6aꢀg) in good yields

(71ꢀ85%).

3ꢀ(4ꢀFluorophenyl)isoxazoleꢀ5ꢀcarbaldehyde (6g)

3ꢀ(4ꢀFluorophenyl)isoxazoleꢀ5ꢀcarbaldehyde (6g) was prepared

using method by the addition of (3ꢀ(4ꢀfluorophenyl)isoxazolꢀ5ꢀ

yl)methanol (5g) (2.40 g, 12.4 mmol) and IBX (5.20 g, 18.6

⁷⁵mmol) to DMSO (12 mL) as white colored solid (1.88 g, yield

3ꢀ(3,4,5ꢀTrimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde (6a)

3ꢀ(3,4,5ꢀTrimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde 6a was

prepared using above method by the addition of (3ꢀ(3,4,5ꢀ

²⁰trimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)methanol 5a (2.64 g, 10

mmol) and IBX (4.15 g, 15 mmol) to DMSO (20 mL) as brown

o

1

79%); mp 119ꢀ120 C; H NMR (300 MHz, CDCl₃): δ 6.71 (s,

1H, ArH), 7.10 (d, 2H, J=6.7 Hz, ArH), 7.53 (d, 2H, J=6.7 Hz,

ArH), 10.12 (s, 1H, CHO) ppm; MS (ESI) m/z 192 [M+H] ⁺.

General procedure for the Synthesis of 1ꢀ(3ꢀarylꢀ1Hꢀpyrazolꢀ

colored solid (1.84 g, yield 71%); mp 128ꢀ129 ^oC; ¹H NMR (400 80 5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7aꢀc) and 1ꢀ(3ꢀaryl isoxazolꢀ5ꢀyl)propꢀ2ꢀ

MHz, CDCl₃): δ 3.90 (s, 3H, ꢀOCH₃), 3.94 (s, 6H, ꢀOCH₃), 6.86

(s, 1H, ArH), 7.06 (S, 2H, ArH), 10.19 (s, 1H, CHO) ppm; MS

25 (ESI): m/z 263 [M+H] ⁺.

ynꢀ1ꢀol (7dꢀg)

A solution of 5ꢀsubtitutedphenylꢀ1Hꢀpyrazoleꢀ3ꢀcarbaldehydes/5ꢀ

subtitutedphenylꢀisoxazoleꢀ3ꢀcarbaldehydes (6aꢀg) in dry

3ꢀ(3,4ꢀDimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde (6b)

3ꢀ(3,4ꢀDimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde (6b) was ⁸⁵magnesium bromide in tetrahydrofuran (0.5 M) at 0 C and then

tetrahydrofuran (THF) was added to stirred solution of ethynyl

o

prepared using above method by the addition of (3ꢀ(3,4ꢀ

dimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)methanol (5b) (2.39g, 10.2

³⁰mmol) and IBX (4.28g 15.3 mmol) to DMSO (20 mL) as brown

colored solid (1.94 g, yield 82%); mp 116ꢀ117 ^oC; ¹H NMR (300

stirred at room temperature for 4ꢀ5 h. After completion of

reaction saturated aqueous ammonium chloride solution (5ꢀ10

ml) was added, and the THF was removed in vacuum followed by

ethyl acetate was added. The organic layer was extracted and

MHz, CDCl₃): δ 3.87 (s, 6H, OCH₃), 6.86 (s, 1H, ArH), 7.02 (d, ⁹⁰washed with brine solution, dried over anhydrous Na₂SO₄and

1H, J=8.7 Hz, ArH), 7.34ꢀ7.39 (m, 2H, ArH), 10.02 (s, 1H,

evaporated in vacuum. It was recrystallized from methanol to

obtain pure acetylene substituted arylꢀpyrazole/isoxazole alcohols

(7aꢀg) and these were used for next step.

CHO) ppm; MS (ESI): m/z 233 [M+H] ⁺.

³⁵3ꢀ(4ꢀFluoroyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde (6c)

3ꢀ(4ꢀFluorophenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde

(6c)

was

1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀ

prepared using above method by the addition of (3ꢀ(4ꢀ ⁹⁵ol (7a)

fluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)methanol (5c) (2.04 g. 10.6

mmol) and IBX (4.40 g, 15.8 mmol) to DMSO (20 mL) as brown

⁴⁰colored solid (1.61 g, yield 80%); mp 121ꢀ122 ^oC; ¹H NMR (500

1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol

(7a) was obtained using above described method by adding 3ꢀ

(3,4,5ꢀtrimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde (6a) (1.65

g, 6.3 mmol) to the ethynyl magnesium bromide solution(18.8

MHz, CDCl₃)

δ 7.78ꢀ7.82 (m, 1H, ArH), 7.86ꢀ7.93 (m, 2H,

ArH), 7.95ꢀ8.03 (m, 2H, ArH) 9.95 (s, 1H, CHO) ppm; MS ¹⁰⁰ml, 9.5mmol) as brown solid (1.25 g, yield 69%); mp 131−133

(ESI): m/z 191 [M+H] ⁺.

^oC; H NMR (400 MHz, CDCl₃+ DMSOꢀd₆): δ 2.85 (m, 1H,

H), 3.88 (s, 3H, OCH₃), 3.90 (s, 6H, OCH₃), 5.63 (d, 1H,

J=2.1 Hz, HꢀCꢀOH), 6.62 (s, 1H, ArH), 6.86 (s, 2H, ArH) ppm;

MS (ESI): m/z 289 [M+H] ⁺.

1

3ꢀ(3,4,5ꢀTrimethoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6d)

45 3ꢀ(3,4,5ꢀTrimethoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6d) was

prepared using method by the addition of (3ꢀ(3,4,5ꢀ

trimethoxyphenyl)isoxazolꢀ5ꢀyl)methanol (5d) (2.44 g, 9.2 105 1ꢀ(3ꢀ(3,4ꢀDimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol

mmol) and IBX (3.87 g, 13.8 mmol) to DMSO (20 mL) as white

colored solid (2.06 g, yield 85%); mp 133ꢀ134 ^oC; ¹H NMR (400

(7b)

1ꢀ(3ꢀ(3,4ꢀDimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol

(7b) was obtained using above described method by adding 3ꢀ

(3,4ꢀdimethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde (6b) (1.72 g,

¹¹⁰7.4 mmol) to the ethynyl magnesium bromide solution (22.2 ml,

11 mmol) as brown solid (1.13 g, yield 59%); mp 127ꢀ128 ^oC ¹H

50 MHz, CDCl₃):

δ 3.86 (s, 3H, OCH₃), 3.89 (s, 6H, OCH₃), 6.86ꢀ

7.24 (m, 3H, ArH), 9.98 (s, 1H, CHO); MS (ESI) m/z 264

[M+H] ⁺.

3ꢀ(3,4ꢀDimethoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6e)

3ꢀ(3, 4ꢀDimethoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6e) was

55 prepared using above method by the addition of (3ꢀ(3,4ꢀ

dimethoxyphenyl)isoxazolꢀ5ꢀyl)methanol (5e) (2.65 g, 11.3

NMR (300 MHz, CDCl₃): δ 2.73 (d, 1H, J=2.8 Hz,

3H, –OCH₃), 3.93 (s, 3H, –OCH₃), 5.66 (d, 1H, J=2.8 Hz, HꢀCꢀ

OH), 6.89 (s, 1H, ArH), 6.96 (d, 1H, J=6.9 Hz, ArH), 7.19ꢀ7.25

), 3.96 (s,

mmol) and IBX (4.73 g, 16.9 mmol) to DMSO (20 mL) as white 115 (m, 2H, ArH) ppm; MS (ESI): m/z 259 [M+H] ⁺.

colored solid (1.99 g, yield 76%); mp 129ꢀ130 ^oC; ¹H NMR (300

1ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7c)

8

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1ꢀ(3ꢀ(4ꢀfluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7c) was

was stirred for 2 h. After completion of reaction, ice water was

obtained using above described method by adding 3ꢀ(4ꢀ 60 added to reaction mixture and the mixture was stirred for another

fluorophenyl)ꢀ1Hꢀpyrazoleꢀ5ꢀcarbaldehyde (6c) (1.44 g, 7.6

10 min. To this mixture ethyl acetate was added and filtered

through celite. The organic layer was separated and washed with

water subsequently saturated Na₂CO₃solution and brine, after

that dried over anhydrous Na₂SO₄and evaporated in vacuum. It

mmol) to the ethynyl magnesium bromide solution (22.7 ml, 11.4

o

1

5

mmol) as brown solid (1.06g, yield 65%); mp 119ꢀ120 C; H

NMR (300 MHz, CDCl₃): δ 2.70 (d, 1H, J=2.3 Hz, ), 5.71

(m, 1H, HꢀCꢀOH), 6.61 (s, 1H, ArH), 7.23 (d, 2H, J=7.3 Hz, 65 was recrystallized from methanol to attain the pure compound

ArH), 7.38 (d, 2H, J=7.3 Hz, ArH) ppm; MS (ESI): m/z 217

and was used directly for next step.

1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀ

one (8a)

[M+H] ⁺.

10 1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol

(7d)

1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone

1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7d) ⁷⁰(8a) was obtained using above described method by adding 1ꢀ(3ꢀ

was obtained using above described method by adding 3ꢀ(3,4,5ꢀ

trimethoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6d) (1.87 g, 7.1

¹⁵mmol) to the ethynyl magnesium bromide solution (21.3 ml, 10.5

(3,4,5ꢀtrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol

(7a)

(1.15 g, 4 mmol) to the 2ꢀ iodoxy benzoic acid (1.68 g, 6 mmol)

in DMSO solution(10 mL) as pale yellow solid (856 mg, yield

o

1

o

mmol) as white solid (1.25 g, yield 61%); mp 128−129 C; H

NMR (500 MHz, CDCl₃): 2.70 (d, 1H, J=2.3 Hz,

3H, –OCH₃), 3.93 (s, 6H, –OCH₃), 5.68 (d, 1H, J=2.3 Hz, HꢀCꢀ

OH), 6.64 (s, 1H, ArH). 6.99 (s, 2H, ArH) ppm; MS (ESI): m/z

20 290 [M+H] ⁺.

75%); mp 125ꢀ126 C; ¹H NMR (400 MHz, CDCl₃): 3.58 (s,

), 3.90 (s, 75 1H,

), 3.91 (s, 3H, –OCH₃), 3.94 (s, 6H, –OCH₃), 6.89 (s,

1H, ArH), 7.02 (s, 2H, ArH) ppm; MS (ESI): m/z 287 [M+H] ⁺.

1ꢀ(3ꢀ(3, 4ꢀDimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone

(8b)

1ꢀ(3ꢀ(3,4ꢀDimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7e)

1ꢀ(3ꢀ(3,4ꢀDimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone

1ꢀ(3ꢀ(3,4ꢀDimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol

(7e) 80 (8b) was obtained using above described method by adding 1ꢀ(3ꢀ

(3,4ꢀdimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7b)

was obtained using above described method by adding 3ꢀ(3,4,ꢀ

dimethoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6e) (1.80 g, 7.7

²⁵mmol) to the ethynyl magnesium bromide solution (23.2 ml, 11.2

mmol) as white solid (1.30g, yield 65%); mp 122ꢀ123 ^oC; ¹H

(1.06 g, 4.1 mmol) to the 2ꢀ iodoxy benzoic acid (1.72 g, 6.2

mmol) in DMSO solution (10 mL) as pale yellow solid (904 mg,

yield 86%); mp 119ꢀ120 ^oC; ¹H NMR (500 MHz, CDCl₃): 3.36

), 3.91 (s, 85 (brs, 1H, ), 3.93 (s, 3H, –OCH₃), 3.96 (s, 6H, –OCH₃), 6.93ꢀ

NMR (300 MHz, CDCl₃): 2.66 (d, 1H, J=2.6,

3H, –OCH₃), 3.94 (s, 3H, –OCH₃), 5.63 (d, 1H, J=2.5 Hz, HꢀCꢀ

OH), 6.69 (s, 1H, ArH), 7.01 (d, 1H, J=8.0 Hz, ArH), 7.19 (s,

³⁰1H, ArH), 7.33 (dd, 1H, J=7.9 Hz, J=1.2 Hz, ArH) ppm; MS

(ESI): m/z 260 [M+H] ⁺.

1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7f)

1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7f) was

obtained using above described method by adding 3ꢀ(4ꢀ

³⁵methoxyphenyl)isoxazoleꢀ5ꢀcarbaldehyde (6f) (1.61 g, 7.9 mmol)

7.06 (m, 2H, ArH), 7.23ꢀ7.32 (m, 1H, ArH), 7.44 (s, 1H, ArH),

9.96 (brs, 1H, NH) ppm; MS (ESI): m/z 257 [M+H] ⁺.

1ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8c)

1ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8c) was

⁹⁰obtained using above described method by adding 1ꢀ(3ꢀ(4ꢀ

fluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7c) (920 mg, 4.3

mmol) to the 2ꢀ iodoxy benzoic acid (1.79g, 6.4 mmol) in DMSO

solution(10 mL) as pale yellow solid (720 mg, yield 79%); mp

o

to the ethynyl magnesium bromide solution (23.8 ml, 12 mmol)

114ꢀ115 C; ¹H NMR (300 MHz, CDCl₃): 3.59 (s, 1H,

),

o

as white solid (1.13 g, yield 62%); mp 120ꢀ121 C; ¹H NMR 95 6.91 (s, 1H, ArH), 7.29 (d, 2H, J=7.8 Hz, ArH), 7.53 (d, 2H,

(400 MHz, CDCl₃): 2.59 (d, 1H, J=2.1 Hz,

), 3.83 (s, 3H, –

J=7.8 Hz, ArH) ppm; MS (ESI): m/z 215 [M+H] ⁺.

1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone

(8d)

OCH₃), 5.80 (d, 1H, J=2.1 Hz, HꢀCꢀOH), 6.56 (s, 1H, ArH),

40 7.08 (d, 2H, J=8.1 Hz, ArH), 7.41 (d, 2H, J=8.1 Hz, ArH) ppm;

MS (ESI): m/z 230 [M+H] ⁺.

1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone

¹⁰⁰(8d) was obtained using above described method by adding 1ꢀ(3ꢀ

(3,4,5ꢀtrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7d) (1.12

g, 3.9 mmol) to the 2ꢀ iodoxy benzoic acid (1.64g, 5.8 mmol) in

DMSO (10 mL) solution as pale yellow solid (960 mg, yield

1ꢀ(3ꢀ(4ꢀFluorophenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7g)

1ꢀ(3ꢀ(4ꢀFluorophenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7g) was

obtained using above described method by adding 3ꢀ(4ꢀ

45 fluorophenyl)isoxazoleꢀ5ꢀcarbaldehyde (6g) (1.60 g, 8.4 mmol)

to the ethynyl magnesium bromide solution (25.1 ml, 12.6 mmol)

o

81%); mp 135ꢀ136 C; ¹H NMR (300 MHz, CDCl₃): 3.60 (s,

as white solid (1.09 g, yield 60%); mp 127ꢀ128 ^oC; ¹H NMR (400 105 1H,

MHz, CDCl₃): 2.59 (d, 1H, J=1.9 Hz, ), 5.41 (d, 1H, J=1.7

), 3.89 (s, 3H, –OCH₃), 3.94 (s, 6H, –OCH₃), 6.99 (s,

2H, ArH), 7.36 (s, 1H, ArH) ppm; MS (ESI): m/z 288 [M+H] ⁺.

Hz, HꢀCꢀOH), 6.81 (s, 1H, ArH), 7.29 (d, 2H, J=8.6 Hz, ArH),

50 7.36 (d, 2H, J=8.6 Hz, ArH) ppm; MS (ESI): m/z 218 [M+H] ⁺.

General method for synthesis of 1ꢀ(3ꢀArylꢀ1Hꢀpyrazolꢀ5ꢀ

1ꢀ(3ꢀ(3,

(8e)

4ꢀDimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone

1ꢀ(3ꢀ(3,4ꢀDimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8e)

yl)propꢀ2ꢀynꢀ1ꢀone (8aꢀc) and 1ꢀ(3ꢀAryl isoxazolꢀ5ꢀyl)propꢀ2ꢀ ¹¹⁰was obtained using above described method by adding 1ꢀ(3ꢀ(3,4ꢀ

ynꢀ1ꢀone (8dꢀg)

dimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7e) (1.17 g, 4.5

mmol) to the 2ꢀ iodoxy benzoic acid (1.90 g, 6.8 mmol) in

DMSO (10 mL) solution as pale yellow solid (847 mg, yield

A solution of 2ꢀiodoxy benzoic acid (IBX) (1.5 eq) and dimethyl

55 sulfoxide (DMSO) was stirred for 10 min at room temperature

until homogeneous solution. A solution of 1ꢀ(3ꢀarylꢀ1Hꢀpyrazolꢀ

o

73%); mp 127ꢀ128 C; ¹H NMR (300 MHz, CDCl₃): 3.57 (s,

5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7aꢀc) and 1ꢀ(3ꢀaryl isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ 115 1H,

), 3.95 (s, 3H, –OCH₃), 3.97 (s, 3H, –OCH₃), 6.84 (s,

1ꢀol (7dꢀg) (1 eq) in dimethyl sulfoxide was added slowly, and it

1H, ArH), 6.97 (d, 1H, J=8.5 Hz, ArH), 7.30 (dd, 1H, J=6.4 Hz,

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J=2.0 Hz, ArH), 7.42 (s, 1H, ArH) ppm; MS (ESI): m/z 258

[M+H] ⁺.

mg, 0.38 mmol) and 6ꢀamino indole (51 mg, 0.38 mmol) as

1

60 yellow solid, mp 227ꢀ229 ^oC; H NMR (300 MHz, CDCl₃):

1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8f)

1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8f) was

obtained using above described method by adding 1ꢀ(3ꢀ(4ꢀ

methoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7f) (1.01 g, 4.4

3.89 (s, 3H, ꢀOCH₃), 3.95 (s, 6H, ꢀOCH₃), 5.87 (d, 1H, J=7.5 Hz,

COCH=), 6.53 (brs, 1H, ArH), 6.94 (s, 1H, ArH), 7.03 (dd, 1H,

J=2.5 Hz, J=6.6 Hz, ArH), 7.08 (s, 2H, ArH), 7.24ꢀ7.28 (m, 1H,

ArH), 7.38 (d, 1H, J=8.5 Hz, ArH), 7.42 (s, 1H, ArH), 7.61 (dd,

5

mmol) to the 2ꢀ iodoxy benzoic acid (1.85 g, 6.6 mmol) in ⁶⁵1H, J=7.5 HZ, J=12.6 Hz, =CHꢀNH), 8.24 (brs, 1H, NH), 12.18

DMXO (10 mL) solution as pale yellow solid (751 g, yield 75%);

mp 119ꢀ120 ^oC; ¹H NMR (400 MHz, CDCl₃):

3.69 (s, 1H,

), 3.87 (s, 3H, –OCH₃), 6.76 (s, 1H, ArH), 7.00 (d, 2H, J=8.3

Hz, ArH), 7.74 (d, 2H, J=8.3 Hz, ArH) ppm; MS (ESI): m/z 228

[M+H] ⁺.

1ꢀ(3ꢀ(4ꢀFluorophenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8g)

1ꢀ(3ꢀ(4ꢀFluorophenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8g) was

¹⁵obtained using above described method by adding 1ꢀ(3ꢀ(4ꢀ

(d, 1H, J=12.5 Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz, CDCl₃):

181.6, 153.5, 151.4, 144.6, 142.5, 137.5, 127.6, 127.2, 116.0,

102.8, 95.7, 60.8, 56.1 ppm; HPLC purity: tR 6.99 min (98.3 %);

IR (KBr) (ν_max/cm^–): 3364, 2922, 2849, 1635, 1514, 1434, 1287,

⁷⁰1143, 1081, 1020, 981, 885; MS (ESI): m/z 419 [M+H]⁺; HRMS

(ESI) calcd for C₂₃H₂₃N₄O₄[M+H]⁺419.17138; found:

419.17139 and C₂₃H₂₂N₄O₄Na [M+Na]⁺441.15333; found:

441.15349.

10

fluorophenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀol (7g) (960 mg, 4.4

(Z)ꢀ3ꢀ(4ꢀ(Trifluoromethyl)phenylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

mmol) to the 2ꢀ iodoxy benzoic acid (1.86 g, 6.6 mmol) in ⁷⁵trimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9c)

DMSO (10 mL) solution as pale yellow solid (732mg, yield

77%); mp 116ꢀ117 C; H NMR (400 MHz, CDCl₃): 3.57 (s,

(Z)ꢀ3ꢀ(4ꢀ(Trifluoromethyl)phenylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

o

1

trimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9c) was

obtained according to above described method by the addition of

1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone

20 1H,

), 6.78 (s, 1H, ArH), 7.21 (d, 2H, J=7.6 Hz, ArH), 7.41

(d, 2H, J=7.6 Hz, ArH) ppm; MS (ESI): m/z 216 [M+H] ⁺.

General method for synthesis of (Z)ꢀ3ꢀArylaminoꢀ1ꢀ(5ꢀarylꢀ 80 (8a) (110 mg, 0.38 mmol) and 4ꢀ(trifluoromethyl)aniline (63 mg,

1Hꢀpyrazolꢀ3ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9aꢀg) and (Z)ꢀ3ꢀArylaminoꢀ

1ꢀ(5ꢀaryl isoxazolꢀ3ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9hꢀq)

²⁵The 1ꢀ(3ꢀArylꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8aꢀc) and 1ꢀ(3ꢀ

Aryl isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8dꢀg) and aryl amine were

0.38 mmol) as yellow solid, mp 238ꢀ240 ^oC; ¹H NMR (300 MHz,

CDCl₃): 3.92 (s, 3H, ꢀOCH₃), 3.96 (s, 6H, ꢀOCH₃), 6.02 (d,

1H, J=7.9 Hz, COCH=), 6.99 (s, 1H, ArH), 7.03 (s, 2H, ArH),

7.20 (d, 2H, J=8.5 Hz, ArH), 7.56 (dd, 1H, J=7.9 Hz, J=12.3,

dissolved in absolute ethanol and the mixture was stirred for 4ꢀ5 ⁸⁵=CHꢀNH), 7.63 (d, 2H, J=8.5 Hz, ArH), 11.97 (d, 1H, J=12.3

h at room temperature. After the completion of the reaction

(confirm by TLC), water was added to the reaction mixture and

³⁰the crude product filtered. It was recrystallized from methanol to

afford pure (Z)ꢀ3ꢀArylaminoꢀ1ꢀ(5ꢀarylꢀ1Hꢀpyrazolꢀ3ꢀyl)propꢀ2ꢀ

Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz, CDCl₃):

δ 191.7,

155.3, 154.1, 151.6, 144.7, 137.7, 136.1, 122.6, 115.0, 112.8,

94.1, 93.3, 60.9, 56.0 ppm; ppm; HPLC purity: tR 5.86 min (97.7

%)IR (KBr) (ν_max/cm^–): 3363, 2933, 2836, 1634, 1573, 1500,

enꢀ1ꢀones (9aꢀg) and (Z)ꢀ3ꢀArylaminoꢀ1ꢀ(5ꢀaryl isoxazolꢀ3ꢀ ⁹⁰1425, 1289, 1247, 1126, 1091, 980, 886; MS (ESI): m/z 448

yl)propꢀ2ꢀenꢀ1ꢀones (9hꢀq).

(Z)ꢀ3ꢀ(1HꢀIndolꢀ5ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ

³⁵1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9a)

[M+H]⁺; HRMS (ESI) calcd for C₂₂H₂₀F₃N₃O₄Na [M+Na]⁺

470.12981; found: 470.13022.

(Z)ꢀ3ꢀ(4ꢀMethoxyphenylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

(Z)ꢀ3ꢀ(1Hꢀindolꢀ5ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ1Hꢀ

trimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9d)

pyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone 9a was obtained according to above ⁹⁵(Z)ꢀ3ꢀ(4ꢀMethoxyphenylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ

described method by the addition of 1ꢀ(3ꢀ(3,4,5ꢀ

trimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8a) (110

⁴⁰mg, 0.38 mmol) and 5ꢀamino indole (51 mg, 0.38 mmol) as

1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9d) was obtained according to

above described method by the addition of 1ꢀ(3ꢀ(3,4,5ꢀ

trimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8a) (110

o

1

yellow solid, mp 162ꢀ164 C; H NMR (300 MHz, CDCl₃):

mg, 0.38 mmol) and 4ꢀmethoxyaniline (47 mg, 0.38 mmol) as

3.91 (s, 3H, ꢀOCH₃), 3.95 (s, 6H, ꢀOCH₃), 6.23 (d, 1H, J=6.8 Hz, ¹⁰⁰yellow solid, mp 260ꢀ262 C; ¹H NMR (300 MHz, CDCl₃):

o

COCH=), 6.55 (brs, 1H, ArH), 6.91 (s, 1H ArH), 7.00ꢀ7.08 (m,

3H, ArH), 7.24ꢀ7.33 (m, 1H, ArH), 7.37ꢀ7.48 (m, 2H, ArH),

45 7.64 (dd, 1H, J=6.8 Hz, 12.1 Hz, =CHꢀNH), 8.25 (brs, 1H, NH),

12.20 (d, 1H, J=12.8 Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz,

3.81 (s, 3H, ꢀOCH₃), 3.89 (s, 3H, ꢀOCH₃), 3.95 (s, 6H, ꢀOCH₃),

5.86 (d, 1H, J=7.6 Hz, COCH=), 6.68ꢀ6.97 (m, 3H, ArH), 7.04ꢀ

7.13 (m, 4H, ArH), 7.48 (dd, 1H, J=7.6 Hz, J=12.8 Hz, =CHꢀ

NH), 12.02 (d, 1H, J=12.8 Hz, NHꢀCH=) ppm; ¹³C NMR (400

CDCl₃):

δ 181.6, 169.9, 163.7, 154.3, 146.4, 135.9, 127.9, 117.8, 105 MHz,CDCl₃): δ 181.4, 170.7, 163.7, 156.9, 153.63, 147.2, 139.9,

116.3, 98.0, 94.6, 94.3, 61.1, 56.2 ppm; HPLC purity: tR 5.86

min (97.5 %); IR (KBr) (ν_max/cm^–): 3291, 2919, 1639, 1523,

50 1502, 1425, 1294, 1240, 1130, 1003, 816; MS (ESI): m/z 419

[M+H]⁺; HRMS (ESI) calcd for C₂₃H₂₃N₄O₄[M+H]⁺419;

C₂₃H₂₂N₄O₄Na [M+Na]⁺441.15333; found: 441.15341.

(Z)ꢀ3ꢀ(1HꢀIndolꢀ6ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ

1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9b)

55 (Z)ꢀ3ꢀ(1HꢀIndolꢀ6ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ1Hꢀ

pyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9b) was obtained according to

133.1, 122.5, 11.3, 115.1, 103.2, 97.9, 93.7, 61.0, 56.3, 55.6 ppm;

HPLC purity: tR 13.16 min (98.0 %); HPLC purity: tR 7.13 min

(95.2 %)IR (KBr) (ν_max/cm^–): 3390, 2921, 2850, 1628, 1504,

1436, 1285, 1256, 1179, 1078, 794; MS (ESI): m/z 410 [M+H]⁺;

¹¹⁰HRMS (ESI) calcd for C₂₂H₂₃N₃O₅Na [M+Na]⁺432.15324;

found: 432.15335

(Z)ꢀ1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9e)

(Z)ꢀ1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

above described method by the addition of 1ꢀ(3ꢀ(3,4,5ꢀ 115 trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9e) was obtained

trimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8a) (110

according to above described method by the addition of 1ꢀ(3ꢀ

10

|

Journal Name, [year], [vol], 00–00

This journal is © The Royal Society of Chemistry [year]

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DOI: 10.1039/C4OB02449D

o

1

(3,4,5ꢀtrimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8a)

yellow solid, mp 242ꢀ244 C; H NMR (300 MHz, CDCl₃):

(110 mg, 0.38 mmol) and 3,4,5ꢀtrimethoxyaniline (70 mg, 0.38 60 3.90 (s, 3H, ꢀOCH₃), 3.95 (s, 6H, ꢀOCH₃), 5.89 (d, 1H, J=7.6 Hz,

mmol) as yellow solid, mp 234ꢀ236 ^oC; ¹H NMR (300 MHz,

CDCl₃): 3.83 (s, 3H, ꢀOCH₃), 3.89 (s, 9H, ꢀOCH₃), 3.95 (s,

6H, ꢀOCH₃), 5.90 (d, 1H, J=7.4 Hz, COCH=), 6.34 (s, 2H,

ArH), 6.94 (s, 1H, ArH), 7.04 (s, 2H, ArH), 7.48 (dd, 1H, J=7.4

COCH=), 6.55 (brs, 1H, ArH), 6.91ꢀ7.00 (m, 2H, ArH), 7.07 (s,

2H, ArH), 7.14ꢀ7.23 (m, 2H, ArH), 7.57ꢀ7.69 (m, 2H, ArH),

8.17ꢀ8.26 (brs, 1H, NH), 12.19 (d, 1H, J=12.1 Hz, NHꢀCH=)

5

ppm; ¹³C NMR (300 MHz, CDCl₃+DMSOꢀd₆):

δ 171.3, 161.3,

Hz, J=12.6 Hz, =CHꢀNH), 11.96 (d, 1H, J=12.3 Hz, NHꢀCH=) 65 153.2, 147.3, 143.9, 132.2, 128.0, 125.7, 111.9, 107.4, 102.7,

ppm; ¹³C NMR (500 MHz, CDCl₃): 188.1, 152.8, 147.4, 140.5,

136.5, 132.9, 129.5, 125.7, 123.2, 121.9, 118.6, 112.7, 110.5,

102.2, 101.0, 92.3, 60.4, 55.4 ppm; HPLC purity: tR 5.58 min

(96.8 %) IR (KBr) (ν_max/cm^–):3240, 2920, 2849, 1638, 1589,

1554, 1489, 1281, 1235, 1128, 991, 875; MS (ESI): m/z 420ꢀ

[M+H]⁺; HRMS (ESI) calcd for C₂₃H₂₂N₃O₅[M+H]⁺420.15540;

¹⁰105.9, 96.4, 60.9, 55.9 ppm; HPLC purity: tR 5.24 min (97.1 %);

IR (KBr) (ν_max/cm^–): 3270, 3118, 1632, 1585, 1432, 1284, 1253,

1218, 1081, 985, 800; MS (ESI): m/z 470 [M+H]⁺; HRMS (ESI) ⁷⁰found: 420.15520 and C₂₃H₂₁N₃O₅Na [M+Na]⁺442.13734,

calcd for C₂₄H₂₈N₃O₇[M+H]⁺470.19243, found: 470.19251.

(Z)ꢀ3ꢀ(1HꢀIndolꢀ5ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4ꢀdimethoxyphenyl)ꢀ1Hꢀ

¹⁵pyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9f)

found: 442.13739.

(Z)ꢀ3ꢀ(1HꢀIndolꢀ6ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

trimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9i)

(Z)ꢀ3ꢀ(1HꢀIndolꢀ6ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

(Z)ꢀ3ꢀ(1HꢀIndolꢀ5ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4ꢀdimethoxyphenyl)ꢀ1Hꢀ

pyrazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9f) was obtained according to ⁷⁵trimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone

(9i)

was

above described method by the addition of 1ꢀ(3ꢀ(3,4ꢀ

dimethoxyphenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8b) (110

²⁰mg, 4.3 mmol) and 5ꢀ amino indole (57 mg, 4.3 mmol) as yellow

solid, mp 139ꢀ141 ^oC; ¹H NMR (500MHz, CDCl₃): 3.86 (s, 3H,

obtained according to above described method by the addition of

1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8d)

(110 mg, 0.38 mmol) and 6ꢀ amino indole (51 mg, 0.38 mmol) as

1

yellow solid, mp 134ꢀ136 ^oC; H NMR (300 MHz, CDCl₃):

ꢀOCH₃), 3.90 (s, 3H, ꢀOCH₃), 6.22 (d, 1H, J=7.4 Hz, COCH=), 80 3.90 (s, 3H, ꢀOCH₃), 3.93 (s, 6H, ꢀOCH₃), 6.25 (d, 1H, J=7.4 Hz,

6.40 (d, 1H, J=8.8 Hz, ArH), 6.85ꢀ7.02 (m, 1H, ArH), 7.07 (d,

1H, J=7.4 Hz, ArH), 7.17 (d, 1H, J=9.9 Hz, ArH), 7.20ꢀ7.31 (m,

²⁵1H, ArH), 7.36 (brs, 1H, ArH), 7.42ꢀ7.58 (m, 3H, ArH), 7.97

(dd, 1H, J=7.3 Hz, J=12.8 Hz, =CHꢀNH), 8.18 (s, 1H, NH),

COCH=), 6.55 (brs, 1H, ArH), 6.90 (s, 1H, ArH), 6.95ꢀ7.00 (dd,

1H, J=1.7 Hz, J=8.3 Hz, ArH), 7.05 (s, 2H, ArH), 7.16ꢀ7.24 (m,

2H, ArH), 7.59ꢀ7.69 (m, 2H, ArH, =CHꢀNH), 8.15ꢀ8.23 (brs,

1H, NH), 12.19 (d, 1H, J=13.7 Hz, NHꢀCH=) ppm; ¹³C NMR

11.03 (d, 1H, J=13.7 Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz, 85 (500 MHz, CDCl₃): 187.6, 153.1, 140.7, 137.6, 134.7, 133.2,

CDCl₃+ DMSOꢀd₆):

δ

182.3, 171.0, 161.9, 161.2, 145.9, 141.4,

132.2, 129.6, 126.4, 122.6, 121.8, 109.6, 106.3, 96.7, 60.9, 55.9

ppm; HPLC purity: tR 6.61 min (97.3 %); IR (KBr) (ν_max/cm^–):

3433, 2925, 2850, 1635, 1565, 1500, 1444, 1278, 1236, 1131,

1009, 873; MS (ESI): m/z 420 [M+H]⁺; HRMS (ESI) calcd for

127.5, 115.8, 114.3, 96.7, 96.3, 95.2, 94.7, 55.4 ppm; HPLC

³⁰purity: tR 5.39 min (97.4 %); IR (KBr) (ν_max/cm^–): 3389, 2922,

2850, 1631, 1513, 1465, 1259, 1136, 1025, 799; MS (ESI): m/z

389 [M+H]⁺, HRMS (ESI) calcd for C₂₂H₂₁N₄O₃[M+H]⁺⁹⁰C₂₃H₂₂N₃O₅[M+H]⁺420.15540; found: 420.15552 and

389.16121; found: 389.16135.

C₂₃H₂₁N₃O₅Na [M+Na]⁺442.13734, found: 442.13788.

(Z)ꢀ3ꢀ(4ꢀMethoxyphenylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

trimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9j)

(Z)ꢀ3ꢀ(4ꢀMethoxyphenylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

(Z)ꢀ1ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

³⁵trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9g)

(Z)ꢀ1ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9g) was obtained ⁹⁵trimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone

(9j)

was

according to above described method by using starting materials

1ꢀ(3ꢀ(4ꢀfluorophenyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8c) (110

⁴⁰mg, 5.3 mmol) and 3,4,5ꢀtrimethoxyaniline (94 mg, 5.3 mmol) as

obtained according to above described method by the addition of

1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8d)

(110 mg, 0.38 mmol) and 4ꢀmethoxyaniline (47 mg, 0.38 mmol)

1

o

yellow solid, mp 197ꢀ199 ^oC; H NMR (400 MHz, CDCl₃):

as yellow solid, mp 290ꢀ292 C; ¹H NMR (500 MHz, CDCl₃):

3.81 (s, 3H, ꢀOCH₃), 3.83 (s, 6H, ꢀOCH₃), 5.89 (d, 1H, J=7.5 Hz, 100 3.82 (s, 3H, ꢀOCH₃), 3.91 (s, 3H, ꢀOCH₃), 3.94 (s, 6H, ꢀOCH₃),

COCH=), 5.94 (s, 2H, ArH), 6.34 (s, 1H, ArH), 7.08ꢀ7.19 (m,

2H, ArH), 7.49 (dd, 1H, J=7.5 Hz, J=12.3 Hz, =CHꢀNH), 7.75ꢀ

45 7.84 (dd, 2H, J=3.2 Hz, J=6.6 Hz, ArH), 11.93 (d, 1H, J=12.4

6.22 (d, 1H, J=7.5 Hz, COCH=), 6.88 (s, 1H, ArH), 6.92 (d, 2H,

J=8.8 Hz, ArH), 7.04 (s, 2H, ArH), 7.09 (d, 2H, J=8.8 Hz, ArH),

7.48 (dd, 1H, J=7.6 Hz, J=12.8 Hz, =CHꢀNH), 12.01 (d, 1H,

J=12.7 Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz, CDCl₃):

Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz, CDCl₃):

δ 181.8,

170.0, 163.0, 154.3, 146.5, 135.8, 128.1, 116.4, 116.1, 97.9, 94.6, 105 182.2, 171.0, 163.5, 161.3, 154.2, 146.3, 135.9, 127.5, 119.9,

94.3, 61.0, 56.3 ppm; HPLC purity: tR 6.77 min (96.2 %); IR

(KBr) (ν_max/cm^–):3323, 2928, 2837, 1634, 1537, 1464, 1371,

50 1287, 1125, 992, 802; MS (ESI): m/z 398 [M+H⁺; HRMS (ESI)

calcd for C₂₁H₂₁N₃O₄F [M+H]⁺398.15139; found: 398.15146.

(Z)ꢀ3ꢀ(1HꢀIndolꢀ5ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

114.4, 96.8, 94.4, 61.0, 56.2, 55.4 ppm; HPLC purity: tR 7.03

min (95.7 %); IR (KBr) (ν_max/cm^–): 3428, 2938, 1642, 1567,

1500, 1462, 1270, 1123, 1078, 1006, 872; MS (ESI): m/z 411

[M+H]⁺; HRMS (ESI) calcd for C₂₂H₂₃N₂O₆[M+H]⁺411.15506;

¹¹⁰found: 411.15525 and C₂₂H₂₂N₂O₆Na [M+Na]⁺433.13701,

found: 433.13742.

trimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9h)

(Z)ꢀ3ꢀ(1HꢀIndolꢀ5ꢀylamino)ꢀ1ꢀ(3ꢀ(3,4,5ꢀ

(Z)ꢀ1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9k)

55 trimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀenꢀ1ꢀone

(9h)

was

obtained according to above described method by the addition of

(Z)ꢀ1ꢀ(3ꢀ(3,4,5ꢀTrimethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

1ꢀ(3ꢀ(3,4,5ꢀtrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8d) 115 trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9k) was obtained

(110 mg, 0.38 mmol) and 5ꢀ amino indole (51 mg, 0.38 mmol) as

according to above described method by the addition of 1ꢀ(3ꢀ

Journal Name, [year], [vol], 00–00 | 11

Organic & Biomolecular Chemistry

Page 12 of 15

View Article Online

DOI: 10.1039/C4OB02449D

(3,4,5ꢀtrimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8d) (110

according to above described method by the addition of 1ꢀ(3ꢀ(4ꢀ

mg, 0.38 mmol) and 3,4,5ꢀtrimethoxyaniline (70 mg, 0.38 mmol) 60 methoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8f) (110 mg, 0.48

o

as yellow solid, mp 291ꢀ293 C; ¹H NMR (300 MHz, CDCl₃):

3.90 (s, 12H, ꢀOCH₃), 3.91 (s, 6H, ꢀOCH₃), 6.27 (d, 1H, J=7.6

Hz, COCH=), 6.35 (s, 2H, ArH), 6.88 (s, 1H, ArH), 7.04 (s, 2H,

ArH), 7.51 (dd, 1H, J=7.5 Hz, J=12.5 Hz, =CHꢀNH), 11.95 (d,

1H, J=12.1 Hz, NHꢀCH=) ppm; ¹³C NMR (500 MHz, CDCl₃):

188.9, 152.9, 143.8, 137.6, 133.5, 132.4, 130.4, 129.1, 120.3,

107.9, 106.6, 105.8, 94.0, 60.8, 55.8 ppm; HPLC purity: tR 6.60

mmol) and 4ꢀ(trifluoromethyl)aniline (78 mg, 0.48 mmol) as

o

yellow solid, mp 129ꢀ130 C; ¹H NMR (400 MHz, DMSOꢀd₆):

5

3.85 (s, 3H, ꢀOCH₃), 6.21 (d, 1H, J=7.7 Hz, COCH=), 6.51 (d,

1H, J=8.4 Hz, ArH), 7.03ꢀ7.16 (m, 2H, ArH), 7.34 (d, 1H, J=8.4

⁶⁵Hz, ArH), 7.54 (d, 1H, J=8.9 Hz, ArH), 7.58ꢀ7.72 (m, 2H, ArH),

7.86 (d, 2H, J=8.6 Hz, ArH), 8.37 (t, 1H, J=12.8 Hz, =CHꢀNH),

10.63 (d, 1H, J=12.8 Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz,

¹⁰min (96.2 %); IR (KBr) (ν_max/cm^–): 3318, 2924, 2839, 1645,

1569, 1285, 1173, 1156, 1077, 864; MS (ESI): m/z 471 [M+H]⁺;

CDCl₃+ DMSOꢀd₆):

δ 178.2, 161.6, 144.4, 142.7, 142.1, 132.3,

125.6, 124.9, 117.3, 114.8, 113.9, 112.7, 95.7, 93.3, 53.4 ppm;

HRMS (ESI) calcd for C₂₄H₂₇N₂O₈[M+H]⁺471.17619; found: ⁷⁰HPLC purity: tR 6.40 min (96.7 %); MS (ESI): m/z 389 [M+H]⁺;

471.17667 and C₂₄H₂₆N₂O₈Na[M+Na]⁺493.15814, found:

493.15856.

HRMS (ESI) calcd for C₂₀H₁₆F₃N₂O₃[M+H]⁺389.11075; found:

389.11105 and C₂₀H₁₅F₃N₂O₃Na [M+Na]⁺411.09270, found:

411.09257.

¹⁵(Z)ꢀ1ꢀ(3ꢀ(3,4ꢀDimethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9l)

(Z)ꢀ1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(4ꢀ

(Z)ꢀ1ꢀ(3ꢀ(3,4ꢀDimethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9l) was obtained

according to above described method by the addition of 1ꢀ(3ꢀ(3,4ꢀ

²⁰dimethoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8e) (110 mg,

0.43 mmol) and 3,4,5ꢀtrimethoxyaniline (78 mg, 0.43 mmol) as

yellow solid, mp 152ꢀ154 C; H NMR (300 MHz, CDCl₃):

3.83 (s, 3H, ꢀOCH₃), 3.89 (s, 6H, ꢀOCH₃), 3.95 (s, 3H, ꢀOCH₃),

3.98 (s, 3H, ꢀOCH₃), 6.27 (d, 1H, J=7.7 Hz, COCH=), 6.35 (s,

25 2H, ArH), 6.84 (s, 1H, ArH), 6.97 (d, 1H, J=8.5 Hz, ArH), 7.33

(s, 1H, ArH), 7.42 (dd, 1H, J=1.9 Hz, J=6.4 Hz, ArH), 7.46ꢀ7.55

⁷⁵methoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9o)

(Z)ꢀ1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(4ꢀ

methoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9o) was obtained

according to above described method by the addition of 1ꢀ(3ꢀ(4ꢀ

methoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8f) (110 mg, 0.48

80 mmol) and 4ꢀmethoxyaniline (60 mg, 0.48 mmol) as yellow solid,

o

1

o

1

mp 117ꢀ119 C; H NMR (500 MHz, CDCl₃): 3.82 (s, 3H, ꢀ

OCH₃), 3.87 (s, 3H, ꢀOCH₃), 6.22 (d, 1H, J=7.6 Hz, COCH=),

6.82 (s, 1H, ArH), 6.92 (d, 2H, J=9.1 Hz, ArH), 7.00 (d, 2H,

J=8.3 Hz, ArH), 7.08 (d, 2H, J=8.3 Hz, ArH), 7.49 (dd, 1H,

(dd, 1H, J=7.6 Hz, J=12.6 Hz, =CHꢀNH), 11.96 (d, 1H, J= 12.6 ⁸⁵J=7.6 Hz, J=12.8 Hz, =CHꢀNH), 7.76 (d, 2H, J=9.1 Hz, ArH),

Hz, NHꢀCH=) ppm; ¹³C NMR (500 MHz, CDCl₃): 12.01 (d, 1H, J=12.8 Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz,

186.9,

DMSOꢀd₆): 179.9, 170.1, 163.8, 160.8, 148.2, 136.2, 133.6,

154.9, 152.9, 147.6, 143.8, 139.2, 133.1, 130.0, 129.4, 129.1,

³⁰124.9, 113.4, 110.7, 105.8, 93.3, 60.9, 56.7, 55.9 ppm; HPLC

purity: tR 5.8 min (98.0 %); IR (KBr) (ν_max/cm^–): 3376, 2930,

δ

127.6, 125.0, 121.1, 119.1, 114.1, 109.8, 109.0, 99.3, 97.6, 93.2,

55.2 ppm; HPLC purity: tR 7.30 min (97.2 %)IR (KBr) (ν_max/cm^–

1631, 1589, 1468, 1289, 1236, 1125, 991, 889; MS (ESI): m/z 90 ): 3412, 2933, 1638, 1498, 1434, 1232, 1125, 1075, 1007, 871,

441 [M+H]⁺; HRMS (ESI) calcd for C₂₃H₂₅N₂O₇[M+H]⁺

441.16563; found: 441.16368 and C₂₃H₂₄N₂O₇Na [M+Na]⁺

³⁵463.14757, found: 463.14629.

(Z)ꢀ3ꢀ(1HꢀIndolꢀ6ꢀylamino)ꢀ1ꢀ(3ꢀ(4ꢀmethoxyphenyl)isoxazolꢀ

5ꢀyl)propꢀ2ꢀenꢀ1ꢀone (9m)

805; MS (ESI): m/z 351 [M+H]⁺; HRMS (ESI) calcd for

C₂₀H₁₉N₂O₄[M+H]⁺351.13393; found: 351.13399 and

C₂₀H₁₈N₂O₄Na [M+Na]⁺373.11588, found: 373.11604.

(Z)ꢀ1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

95 trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9p)

(Z)ꢀ3ꢀ(1HꢀIndolꢀ6ꢀylamino)ꢀ1ꢀ(3ꢀ(4ꢀmethoxyphenyl)isoxazolꢀ5ꢀ

yl)propꢀ2ꢀenꢀ1ꢀone (9m) was obtained according to above

(Z)ꢀ1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(3,4,5ꢀ

trimethoxyphenylamino)propꢀ2ꢀenꢀ1ꢀone (9p) was obtained

according to above described method by the addition of 1ꢀ(3ꢀ(4ꢀ

methoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8f) (110 mg, 0.48

⁴⁰described

method

by

the

addition

of

1ꢀ(3ꢀ(4ꢀ

methoxyphenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8f) (110 mg, 0.48

mmol) and 6ꢀaminoindole (64 mg, 0.48 mmol) as yellow solid, ¹⁰⁰mmol) and 3,4,5ꢀtrimethoxyaniline (90 mg, 0.48 mmol) as yellow

o

1

mp 136ꢀ138 ^oC; ¹H NMR (300 MHz, DMSOꢀd₆): 3.86 (s, 3H, ꢀ

OCH₃), 6.13 (d, 1H, J=7.4 Hz, COCH=), 6.40 (d, 1H, J=7.9 Hz,

45 ArH), 6.85ꢀ7.11 (m, 4H, ArH), 7.18ꢀ7.26 (m, 1H, ArH), 7.33 (s,

1H, ArH), 7.52 (dd, 1H, J=6.2 Hz, J=8.4 Hz, ArH), 7.78ꢀ7.96

solid, mp 149ꢀ151 C; H NMR (300 MHz, CDCl₃): 3.83 (s,

3H, ꢀOCH₃), 3.88 (s, 3H, ꢀOCH₃), 3.89 (s, 6H, ꢀOCH₃), 6.27 (d,

1H, J=7.7 Hz, COCH=), 6.34 (s, 2H, ArH), 6.82 (s, 1H, ArH),

7.00 (d, 2H, J=8.9 Hz, ArH), 7.50 (dd, 1H, J=7.7 Hz, J=12.6 Hz,

(m, 3H, ArH, =CHꢀNH), 8.08 (s, 1H, ꢀNH), 10.97 (d, 1H, J=12.6 105 =CHꢀNH), 7.77 (d, 2H, J=8.9 Hz, ArH), 11.97 (d, 1H, J=12.1

Hz, NHꢀCH=) ppm; ¹³C NMR (500 MHz, CDCl₃+ DMSOꢀd₆): Hz, NHꢀCH=) ppm; ¹³C NMR (300 MHz,CDCl₃):

179.0,

δ

180.2, 170.1, 163.2, 160.6, 147.5, 133.4, 131.8, 127.9, 126.9,

50 125.9, 119.3, 113.9, 111.9, 107.5, 101.0, 96.3, 92.6, 54.8 ppm;

HPLC purity: tR 9.34 min (96.3 %); MS (ESI): m/z 360 [M+H]⁺;

159.7, 152.65, 150.9, 144.1, 135.9, 133.6, 128.1, 121.4, 120.2,

114.7, 110.9, 60.9, 56.2, 55.5 ppm; HPLC purity: tR 9.34 min

(95.7 %); IR (KBr) (ν_max/cm^–): 3244, 2921, 2847, 1649, 1615,

HRMS (ESI) calcd for C₂₁H₁₈N₃O₃[M+H]⁺360.13427; found: ¹¹⁰1563, 1437, 1285, 1111, 1066, 838, 771; MS (ESI): m/z 411

360.13449 and C₂₁H₁₇N₃O₃Na [M+Na]⁺382.11621, found:

382.11648.

55 (Z)ꢀ1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(4ꢀ

(trifluoromethyl)phenylamino)propꢀ2ꢀenꢀ1ꢀone (9n)

(Z)ꢀ1ꢀ(3ꢀ(4ꢀMethoxyphenyl)isoxazolꢀ5ꢀyl)ꢀ3ꢀ(4ꢀ

(trifluoromethyl)phenylamino)propꢀ2ꢀenꢀ1ꢀone (9n) was obtained

[M+H]⁺; HRMS (ESI) calcd for C₂₂H₂₃N₂O₆[M+H]⁺411.15506;

found: 411.15496 and C₂₂H₂₂N₂O₆Na [M+Na]⁺433.13701,

found: 433.13668.

(Z)ꢀ3ꢀ(1HꢀIndolꢀ5ꢀylamino)ꢀ1ꢀ(3ꢀ(4ꢀfluorophenyl)isoxazolꢀ5ꢀ

115 yl)propꢀ2ꢀenꢀ1ꢀone (9q)

(Z)ꢀ3ꢀ(1HꢀIndolꢀ5ꢀylamino)ꢀ1ꢀ(3ꢀ(4ꢀfluorophenyl)isoxazolꢀ5ꢀ

yl)propꢀ2ꢀenꢀ1ꢀone (9q) was obtained according to above

12

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DOI: 10.1039/C4OB02449D

with iceꢀcold 70% ethanol at 4 ^oC for 30 min, ethanol was

removed by centrifugation and cells were stained with 1 mL of

DNA staining solution [0.2 mg of propidium Iodide (PI), and 2

mg RNase A] for 30 min as described earlier . The DNA contents

⁶⁵of 20,000 events were measured by flow cytometer (BD FACS

Canto II). Histograms were analyzed using FCS express 4 plus.

Tubulin Polymerization Assay

described

method

by

the

addition

of

1ꢀ(3ꢀ(4ꢀ

fluorophenyl)isoxazolꢀ5ꢀyl)propꢀ2ꢀynꢀ1ꢀone (8g) (110 mg, 0.5

mmol) and 5ꢀaminoindole (68 mg, 0.5 mmol) as yellow solid, mp

o

1

130ꢀ131 C; H NMR (300 MHz, CDCl₃+ DMSOꢀd₆): 6.18

5

(dd, 1H, J=7.3 Hz, J=1.7 Hz COCH=), 6.49 (s, 1H, ArH), 6.93

(s, 1H, ArH), 6.99 (td, 1H, J=8.5 Hz, ArH), 7.16ꢀ7.23 (m, 2H,

ArH), 7.39ꢀ7.45 (m, 3H, ArH), 7.67 (dd, 1H, J=7.3 Hz, J=12.8

Hz, =CHꢀNH), 7.80ꢀ7.86 (dd, 2H, J=2.3 Hz, J=7.6 Hz, ArH),

10.14 (brs, 1H, NH), 12.22 (d, 1H, J=12.7 Hz, NHꢀCH=); ¹³C

An in vitro assay for monitoring the timeꢀdependent

polymerization of tubulin to microtubules was performed

⁷⁰employing a fluorescenceꢀbased tubulin polymerization assay kit

(BK011, Cytoskeleton, Inc.) according to the manufacturer’s

protocol. The reaction mixture in a final volume of 10 µl in PEM

buffer (80 mM PIPES, 0.5 mM EGTA, 2 mM MgCl₂, pH 6.9) in

384 well plates contained 2 mg/mL bovine brain tubulin, 10 µM

⁷⁵fluorescent reporter, 1 mM GTP in the presence or absence of

10 NMR (500 MHz, DMSOꢀd₆):

δ

170.0, 148.3, 147.5, 147.2,

141.9, 138.8,128.5, 126.3, 123.2, 121.7, 119.4, 109.4, 108.8,

105.7, 101.4 ppm; HPLC purity: tR 9.04 min (98.8 %); IR (KBr)

(ν_max/cm^–): 3247, 2934, 2833, 1632, 1588, 1487, 1368, 1290,

1247, 1126, 991, 829, 790; MS (ESI): m/z 348 [M+H]⁺; HRMS

15 (ESI) calcd for C₂₀H₁₅FN₃O₂[M+H]⁺348.11428; found:

348.11454.

o

Biology

test compounds at 37 C. Tubulin polymerization was followed

Cell Cultures, Maintenance and Antiproliferative Evaluation

All cell lines used in this study were purchased from the

²⁰American Type Culture Collection (ATCC, United States). Aꢀ

549, HeLa, MCFꢀ7 and HCTꢀ116 were grown in Dulbecco's

modified Eagle's medium (containing 10% FBS in a humidified

atmosphere of 5% CO₂at 37 °C). Aꢀ549 cells were cultured in

Eagle's minimal essential medium (MEM) containing nonꢀ

25 essential amino acids, 1 mM sodium pyruvate, 10 mg/mL bovine

insulin, and 10% FBS. Cells were trypsinized when subꢀconfluent

from T25 flasks/60 mm dishes and seeded in 96ꢀwel plates. The

synthesized test compounds were evaluated for their in vitro

antiproliferative in four different human cancer cell lines. A

³⁰protocol of 48 h continuous drug exposure was used, and a MTT

cell proliferation assay was used to estimate cell viability or

growth. The cell lines were grown in their respective media

containing 10% fetal bovine serum and were seeded into 96ꢀwell

microtiter plates in 200 ꢁL aliquots at plating densities depending

³⁵on the doubling time of individual cell lines. The microtiter plates

were incubated at 37 °C, 5% CO₂, 95% air, and 100% relative

humidity for 24 h prior to addition of experimental drugs.

Aliquots of 2 ꢁL of the test compounds were added to the wells

already containing 198 ꢁL of cells, resulting in the required final

⁴⁰drug concentrations. For each compound, four concentrations

(0.1, 1, 2, 10 and 50 ꢁM) were evaluated, and each was done in

triplicate wells. Plates were incubated further for 48 h, and the

assay was terminated by the addition of 10 ꢁL of 5% MTT and

incubated for 60 min at 37 °C. Later, the plates were airꢀdried.

45 Bound stain was subsequently eluted with 100 ꢁL of DMSO, and

the absorbance was read on multimode plate reader (Tecan

M200) at a wavelength of 560 nm. Percent growth was calculated

on a plate by plate basis for test wells relative to control wells.

The above determinations were repeated thrice. The growth

50 inhibitory effects of the compounds were analyzed by generating

dose response curves as a plot of the percentage surviving cells

versus compound concentration. The sensitivity of the cancer

cells to the test compound was expressed in terms of IC₅₀, a value

defined as the concentration of compound that produced 50%

55 reduction as compared to the control absorbance. IC₅₀values are

indicated as means ± SD of three independent experiments.

Cell Cycle Analysis

by monitoring the fluorescence enhancement due to the

incorporation of a fluorescence reporter into microtubules as

polymerization proceeds. Fluorescence emission at 420 nm

⁸⁰(excitation wavelength is 360 nm) was measured for 1 h at 1ꢀmin

intervals in a multimode plate reader (Tecan M200). To

determine the IC₅₀values of the compounds against tubulin

polymerization, the compounds were preꢀincubated with tubulin

at varying concentrations (0.1, 1, 2, 5 and 10 µM). Assays were

⁸⁵performed under similar conditions as employed for

polymerization assays as described above.

Immunohistochemistry of Tubulin and Analysis of Nuclear

Morphology

A549 cells were seeded on glass cover slip, incubated for 24 h in

⁹⁰the presence or absence of test compounds 9a, 9b and 9f at

concentrations of 3 ꢁM. Cells grown on cover slips were fixed in

3.5% formaldehyde in phosphateꢀbuffered saline (PBS) pH 7.4

for 10 minutes at room temperature. Cells were permeablized for

6 minutes in PBS containing 0.5% Triton Xꢀ100 (Sigma) and

⁹⁵0.05% Tweenꢀ20 (Sigma). The permeablized cells were blocked

with 2% BSA (Sigma) in PBS for 1h. Later, the cells were

incubated with primary antibody for tubulin from (sigma) at

(1:200) diluted in blocking solution for 4h at room temperature.

Subsequently the antibodies were removed and the cells were

100 washed thrice with PBS. Cells were then incubated with FITC

labeled antiꢀmouse secondary antibody (1:500) for 1h at room

temperature. Cells were washed thrice with PBS and mounted in

medium containing DAPI. Images were captured using the

Olympus confocal microscope and analyzed with Provision

105 software.

Western blot analysis of soluble versus polymerized tubulin

A549 cells were seeded in 12ꢀwell plates at 1×105 cells per well

in complete growth medium. Following treatment of cells with

respective compounds 9a, 9b and nocodazole for duration of 24

110 h, cells were washed with PBS and subsequently soluble and

insoluble tubulin fractions were collected. To collect the soluble

tubulin fractions, cells were permeablized with 200 ꢀL of preꢀ

warmed lysis buffer [80 mM PipesꢀKOH (pH 6.8), 1 mM MgCl2,

1 mM EGTA, 0.2% Triton Xꢀ100, 10% glycerol, 0.1% protease

115 inhibitor cocktail (SigmaꢀAldrich)] and incubated for 3 min at 30

oC. Lysis buffer was gently removed, and mixed with 100 µL of

3×Laemmli’s sample buffer (180 mM TrisꢀCl pH 6.8, 6% SDS,

15% glycerol, 7.5% βꢀmercaptoethanol and 0.01% bromophenol

Aꢀ549 cells in 60 mm dishes were incubated for 24 h in the

presence or absence of test compounds 9a, 9b and 9f at 3 µM

60 concentrations. Cells were harvested with trypsinꢀEDTA, fixed

Journal Name, [year], [vol], 00–00 | 13

Organic & Biomolecular Chemistry

Page 14 of 15

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DOI: 10.1039/C4OB02449D

blue). Samples were immediately heated to 95 oC for 3 min. To

nocodazole at 2 µM. A549 cells were seeded in six well plates

collect the insoluble tubulin fraction, 300 ꢀL of 1×Laemmli’s 60 with the confluence of per (2.5 × 10^ꢀ6) well and are treated with

sample buffer was added to the remaining cells in each well, and

the samples were heated to 95oC for 3 min. Equal volumes of

samples were run on an SDSꢀ10 % polyacrylamide gel and were

transferred to a nitrocellulose membrane employing semidry

the compounds at 2 µM concentration. The cells were washed

with PBS after incubation for 48 h and then scraped in to the PBS

and centrifuged at 2000 rpm for 10 min at 4 °C. Pellet was

resuspended in 80 ml of lysis buffer. Then the pellet was passed

5

transfer at 50 mA for 1h. Blots were probed with mouse antiꢀ ⁶⁵through insulin syringe followed by incubation of suspension on

human αꢀtubulin diluted 1:2,000 ml (Sigma) and stained with

rabbit antiꢀmouse secondary antibody coupled with horseradish

¹⁰peroxidase, diluted 1:5000 ml (Sigma). Bands were visualized

using an enhanced Chemiluminescence protocol (Pierce) and

radiographic film (Kodak).

ice for 20–30 min centrifuged the lysate at 13,200 rpm for 20 min

at 4 °C and transferred the supernatant to fresh tubes. In a 96 well

black polystyrene plate, 50 ml cell lysate, 50 ml of 2X assay

buffer and 2 ml of caspaseꢀ3substrate were taken. The reaction

⁷⁰was allowed to take place for 1 h. The fluorescence generated by

the release of the fluorogenic group AFC on cleavage by caspaseꢀ

3 was measured by excitation at 400 nm and emission at 505 nm

for every 5 min over 1 h. Protein was estimated by Bradford’s

method and normalized consequently.

Dotꢀblot assay

Cells were trypsinized when subꢀconfluent from T25 flasks/60

¹⁵mm dishes and seeded in 6ꢀwell plates. The pyrazoleꢀoxadiazole

conjugates were evaluated for their activity against Cyclin B1.

A549 cells were treated with 2 µM concentrations of (9a, 9b and ⁷⁵Assessment of apoptosis by PI

9f) for 24 h. Susbequently, cells were harvested and proteins were

quantified using Amido Black followed by densitomerty analysis.

²⁰Equal amount of protein were blotted on nitrocellulose membrane

using BioꢀDot SF microfiltration apparatus (BioꢀRad). Briefly,

The A549 cells exposed to compound 9a, 9b and 9f at 2 µM

concentration after 24 h and cells were washed twice with PBS

and fixed with 4% formaldehyde for10 min. The fixed cells were

then washed again with PBS and stained with and 10 ꢁg/mL of PI

nitrocellulose membrane and 3 filters papers (Whatmann 3) were 80 for 10 min. After washed once with PBS the cells were examined

soaked in IX TBS solution for 10 min. Later, the filter papers,

membrane were arranged in the apparatus and connected to

²⁵vacuum pump (Millipore). The membranes were rehydrated using

100 µl of 1XTBS by vacuum filtration. Subsequently, 50 µl

under a fluorescence microscope.

Acknowledgement

V.S.R, A.B.S and G.B.K acknowledge CSIRꢀUGC, New Delhi and for

the award of senior research fellowship. We also acknowledge CSIR for

volumes of equal protien samples were blotted on the membrane ⁸⁵financial support under the 12^thFive Year plan project “Affordable

and washed with 200 µl of 1X TBS through application of

vacuum. The blot was blocked with 5% blotto for 1 h at room

³⁰temperature. Immunoblot analysis was performed as described

previously using UVP, biospectrum 810 imaging system.

Competitive tubulin binding assay

The various concentrations (0 ꢁM, 5 ꢁM, 10ꢁM and 15 ꢁM ) of

conjugates 9a, 9b and 9f were coincubated with 3 ꢁM colchicines

³⁵in 30 mM Tris buffer containing 3 ꢁM tubulin at 37oC for 60

min. The standard nocodazole (III) was employed as a positive

Cancer Therapeutics (ACT)” (CSC0301).

⁹⁰Notes and references

^aMedicinal Chemistry and Pharmacology, CSIR ‒ Indian Institute of

Chemical Technology, Hyderabadꢀ 500 007, India; Phone: (+) 91ꢀ40ꢀ

27193157; Fax: (+) 91ꢀ40ꢀ27193189; Eꢀmail: ahmedkamal@iict.res.in

^bCentre for Chemical Biology

CSIRꢀIndian Institute of Chemical

control whereas peclitaxel was used as the negative control. After 95 Technology, Hyderabadꢀ 500 007, India

incubation, the fluorescence of tubulinꢀcolchicine complex was

measured by using Tecan multimode reader at excitation

⁴⁰wavelength of 380 nm and emission wavelength of 435 nm;

whereas 30 mM Tris buffer was used as a blank. The raw

fluorescence values were normalized first by subtracting the 100

fluorescence of the buffer and then setting the fluorescence of 3

ꢁM tubulin with 3 ꢁM colchicine to 100%. Values represented

45 were ± SD of at least three independent experiments.

†

Electronic Supplementary Information (ESI) available: [¹H NMR , ¹³C

NMR and HRMS spectra and of final compounds are provided]. See

DOI: 10.1039/b000000x/

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Synthesis of (Z)-(arylamino)-pyrazolyl/isoxazolyl-2-propenones as tubulin targeting anticancer agents and apoptotic inducers

DOI: 10.1039/c4ob02449d

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