Scholarly article on N-(4-fluorophenyl)-3-methyl-4-nitrobenzothioamide 343975-38-2 from Bioorganic and Medicinal Chemistry p. 977

Accepted Manuscript

Synthesis and Biological Evaluation of cis-Restricted Triazole/Tetrazole Mim-

ics of Combretastatin-Benzothiazole Hybrids as Tubulin Polymerization Inhib-

itors and Apoptosis Inducers

A.V. Subba Rao, Konderu Swapna, Siddiq Pasha Shaik, V. Lakshma Nayak, T.

Srinivasa Reddy, Satish Sunkari, Thoukhir Basha shaik, Chandrakant Bagul,

Ahmed Kamal

PII:

S0968-0896(16)31395-5

DOI:

http://dx.doi.org/10.1016/j.bmc.2016.12.010

BMC 13437

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

1 August 2016

22 October 2016

8 December 2016

Please cite this article as: Subba Rao, A.V., Swapna, K., Pasha Shaik, S., Lakshma Nayak, V., Srinivasa Reddy, T.,

Sunkari, S., Basha shaik, T., Bagul, C., Kamal, A., Synthesis and Biological Evaluation of cis-Restricted Triazole/

Tetrazole Mimics of Combretastatin-Benzothiazole Hybrids as Tubulin Polymerization Inhibitors and Apoptosis

Inducers, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.12.010

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Graphical Abstract

Synthesis and Biological Evaluation of cis-

Restricted Triazole/Tetrazole Mimics of

Combretastatin-Benzothiazole Hybrids as

Tubulin Polymerization Inhibitors and

Apoptosis Inducers

A.V. Subba Rao,^a,bKonderu Swapna,^cSiddiq Pasha Shaik,^a,bV. Lakshma Nayak, ^aT. Srinivasa Reddy,^cSatish

Sunkari, ^a,bThoukhir Basha shaik,^aChandrakant Bagul,^cand Ahmed Kamal^*a,b,c

^aMedicinal Chemistry and Pharmacology, ^bAcademy of Scientific and Innovative Research (AcSIR), CSIR-

Indian Institute of Chemical Technology, Hyderabad, 500007, India;

^cDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education & Research (NIPER),

Hyderabad 500 037, India.

Bioorganic & Medicinal Chemistry

Synthesis and Biological Evaluation of cis-Restricted Triazole/Tetrazole Mimics

of Combretastatin-Benzothiazole Hybrids as Tubulin Polymerization Inhibitors

and Apoptosis Inducers

A.V. Subba Rao,^a,bKonderu Swapna,^cSiddiq Pasha Shaik,^a,bV. Lakshma Nayak, ^aT. Srinivasa Reddy,^c

Satish Sunkari, ^a,bThoukhir Basha shaik,^aChandrakant Bagul,^cand Ahmed Kamal^*a,b,c

^aMedicinal Chemistry and Pharmacology, ^bAcademy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology, Hyderabad,

500007, India;

^cDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education & Research (NIPER), Hyderabad 500 037, India.

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

A series of colchicine site binding tubulin inhibitors were synthesized by the modification of the

combretastatin pharmacophore. The ring B was replaced by the pharmocogically relevant

benzothiazole scaffolds, and the cis configuration of the olefinic bond was restricted by the

incorporation of a triazole and tetrazole rings which is envisaged by the structural resemblance

to a tubulin inhibitor like combretastatin (CA-4). These compounds were evaluated for their

antiproliferative activity on selected cancer cell lines and an insight in the structure activity

relationship was developed. The most potent compounds (9a and 9b) demonstrated an

antiproliferative effect comparable to that of CA-4. Mitotic cell cycle arrest in G2/M phase

revealed the disruption of microtubule dynamics that was confirmed by tubulin polymerization

assays and immunocytochemistry studies at the cellular level. Western blot analysis revealed

that these compounds accumulate more tubulin in the soluble fraction. The colchicine

competitive binding assay and the molecular docking studies suggested that the binding of these

mimics at the colchicine site of the tubulin is similar to that of CA-4. Moreover, the triggering of

apoptotic cell death after mitotic arrest was investigated by studying their effect by Hoechst

staining, Annexin-V-FITC assay, mitochondrial membrane potential, ROS generation and

caspase-3 activation.

Available online

Keywords:

Combretastatin A-4;

Benzothiazole;

Triazoles and Tetrazoles;

Antiproliferative activity;

Tubulin polymerization;

Colchicine competition assay;

Apoptosis;

1. Introduction

cycle, causing mitotic catastrophy and finally leading to

apoptotic cell death. Some of the well-known naturally occuring

Small molecules that disrupt microtubule/tubulin dynamics

are used widely in cancer treatment. Hence discovery and

development of newer small molecules with antitubulin activity

have attracted medicinal chemists for past few years.¹

Microtubules are one of the key structural components of the

cytoskeleton in eukaryotic cells comprising of α and β-tubulin

heterodimers. They play a crucial role in various cellular

processes and have emerged as an attractive and viable target in

the development of anticancer drugs mainly due to their

indispensability in mitotic cell division.²Generally, drugs that

target microtubules and bind to the tubulin binding sites thereby

stabilizing or destabilizing microtubule assembly. There are four

main binding sites of tubulin³which includes the taxane and

laulimalide/pelouside A sites for the microtubule stabilizing

agents,^4,5the vinca domain,⁶maytansine binding site⁷and the

colchicine domain^8,9for the destabilizing agents. Interfering with

the dynamic stability of microtubules, these agents act as spindle

poisons arresting the dividing cells in G2/M phase of the cell

tubulin binding ligands that affect the microtubule dynamics by

binding to distinct colchicine domain of tubulin are colchicine (1)

and combretastatin A-4 (2a) (Figure 1).^10,11Combretastatin A-4

(CA-4), natural vicinally diaryl cis- stilbenoid phenol isolated

from the bark of the South African tree Combretum caffrum is

one of the simplest and highly effective molecule among the

structurally diverse microtubule disturbing agents affecting

microtubule dynamics by binding to the colchicine site. CA-4

strongly inhibits tumor cell growth, including MDR cancer cell

lines,¹²and exhibits remarkable strong inhibition of tubulin

polymerization as well as potent cytotoxicity against murine

lymphocytic leukemia, human ovarian and colon cancer cell lines

resulting in vascular shutdown in solid tumor.^13-17The limitation

of poor aqueous solubility of CA-4^18,19circumvented with the

development of a prodrug, CA-4 phosphate (2b) and other

synthetic analogues like OXI4531 (2c), both of which are

currently undergoing clinical trials.²⁰However, evidences of

tumor re-growth, isomerization of cis double bond and

significant toxicity towards normal cells resulted in a surge of

research endeavors in the development of more effective and

safer CA-4 based new molecules acting on microtubules with

desired properties.^21-23In recent years CA-4 emerged as a

preferred lead compound in the development of new inhibitors of

tubulin polymerization because of its high potency and ease of

synthesis.^22,23Similar to other colchicine site binders, CA-4 has

three important pharmacophoric substructures, the two

hydrophobic rings (A and B) and the linking olefinic bridge with

appropriate dihedral angle resulting from the cis configuration. It

was previously postulated that the presence of trimethoxy

substitution on ring A is crucial for efficient binding at colchicine

site.¹⁰On the other hand, the ring B was found to be tolerant to

structural modification and a number of reports defined this

ranging from substituted phenyl ring to heterocyclic and

nonsubstituted aromatic rings.^13,24Importantly, the olefinic bond

with cis configuration plays a fundamental role in binding at the

colchicine site by positioning the rings at appropriate distance to

maximize interactions.²⁵Several attempts have been reported to

modify this cis-olefinic bond to prevent its isomerization under

amenable conditions. This mostly included either modification of

the olefinic bond by the introduction of saturation, substituents

and its replacement with a three to six membered ring system,

which resulted in cis restricted analogues of CA-4.^23,26-29

Figure 1: Colchicine binding microtubule inhibitors (1, 2a, 2b, 9a and 9b)

and antitumor benzothiazoles (3) and its prodrug (4).

Our recent research studies have been mainly focused on the

synthesis, evaluation and mechanistic aspects of newer molecules

based on different heterocyclic scaffolds as potential anticancer

agents.^30,31In particular, the targeting of tubulin polymerization

by new diversified ligands based on CA-4 has led to promising

and interesting results.³²Herein, we describe different

modifications on CA-4 scaffold, specifically the incorporation of

benzothiazole moieties of pharmacological relevance by

replacement of ring B. The rationale behind this is the

significance of these moieties as privileged structures and the

opportunity offered by them as anchors that could be harnessed

for further diversification, besides they are present in a number of

antitumor agents such as (5F-203) (3)³³and its prodrug, phortress

(4)³⁴which is water soluble and chemically stable, is found to

rapidly and quantitatively revert to its parent amine in mice, rats

and dogs (in vivo). Thus, clinical evaluation of phortress has

demonstrated potent and selective antitumor activity via a

different mechanism of action which provides substantial scope

for the development of benzothiazole-based derivatives as

anticancer agents. Moreover, the structural resemblance to CA-4,

a colchicine site binder was envisaged by the replacement of the

olefinic bond by a triazole and tetrazole rings to maintain the cis

configuration and provide optimal conformational geometry for

interaction with the colchicine binding site. Such replacements of

the olefinic bond with a ring system have been reported to

provide potential advantage of promoting tubulin binding activity

and tumor selectivity besides minimizing toxicity.¹³In addition,

the constitutive role of the trimethoxybenzene moiety (ring A) is

ambiguous in tubulin binding.³⁵Considering the biological

importance of these moieties, an attempt has been made in the

present study to synthesize cis restricted triazole/tetrazole

analogues of CA-4 and benzothiazole (CAB) hybrids. The

resulting cis-restricted benzothiazole mimics of CA-4 were

evaluated for antiproliferative activity and examined for their

structure activity relationship (SAR) followed by studies to

elucidate the mechanism of action which included cell cycle

progression, tubulin polymerization assay and molecular docking

studies. Further to confirm the induction of apoptotic cell death

by the potent mimics like 9a and 9b, studies such as Hoechst

staining performed.

2. Results and Discussion

2.1 Chemistry

Scheme 1

Synthesis of substituted 2-(4-amino-phenyl) benzothiazoles

(25-27a-d)

The synthesis of triazole and tetrazole mimics of CA-4 linked

benzothiazole hybrids (5-13a-d) was accomplished by the

synthetic route illustrated in Schemes 1, 2 and 3. The preparation

of

various

substituted

2-(4-aminophenyl)benzothiazole

precursors 25-27(a–d), was achieved by the Jacobson’s

thioanilide radical cyclization. Thus, reaction of substituted

anilines (14a–d) with substituted p-nitrobenzoylchlorides (15a-c)

in pyridine gave the benzanilides 16-18(a–d) which were further

converted to their corresponding thiobenzanilides 19-21(a–d)

using Lawesson’s reagent. These were cyclized by using

Jacobson’s method to afford nitrobenzothiazole derivatives 22-

24(a–d) using potassium ferricyanide and 10% NaOH solution

which upon reduction of these nitro compounds with stannous

chloride yielded the corresponding substituted 2-(4-amino-

phenyl) benzothiazoles 25-27(a–d) as illustrated in Scheme 1.

These key intermediates were utilized in the synthesis of the

triazole and tetrazole mimics of CA-4 benzothiazole hybrids.

Scheme 1: Reagents and conditions: (a) Pyridine, reflux, 3 h; (b) Lawesson’s

Scheme 3: Reagents and conditions: (a) Pyridine, reflux, 12 h; (b)

.

reagent, toluene, reflux, 8 h; (c) K₃Fe(CN)₆, aq. NaOH, EtOH, 90 °C, 2-3 h;

Lawesson’s reagent, toluene, reflux, 8 h; (c) NH₂NH₂8H₂O, EtOH, rt, 12 h;

.

(d) SnCl₂2H₂O, EtOH, 3 h.

(d) trimethylorthoformate, EtOH, reflux, 3 h; (e) NaNO₂, AcOH, rt, 4 h.

Scheme 2

The reaction of thioamides with hydrazine hydrate affords

amidrazones 39-41(a–d). These amidrazones were used for next

step without purification. Finally, the intramolecular cyclization

was performed by using trimethylorthoformate in refluxing

conditions with catalytic amount of H₂SO₄furnishing the desired

cis-restricted 1,2,4-triazoles²⁸8-10(a-d), whereas 1,2,3,4-

tetrazoles 11-13(a-d) were achieved by using sodium nitrate in

AcOH as illustrated in Scheme 3.

Synthesis of 1, 4-disubstituted 1,2,3-triazoles 5-7(a-d)

To access the 1,4-disubstituted 1,2,3-triazole mimics of CA-4

benzothiazole hybrids the same intermediates 25-27(a-d) were

employed as depicted in Scheme 2. These amines were further

converted to azides by using sodium nitrite in HCl followed by

sodium azide to afford corresponding azides 28-30(a–d). 1,4-

Disubstituted 1,2,3-triazoles 5-7(a–d) were synthesized by

exposing trimethoxy phenyl acetaldehyde (31) to azides in the

presence of 1,8-diazabicyclo[5.4.0]undec-7-ene DBU in DMSO³⁶

as illustrated in Scheme 2.

3. Biological activity

3.1 Antiproliferative activity

MTT assay³⁷was performed to evaluate the cytotoxic effects of

all newly synthesized analogues of 1,4-disubstituted 1,2,3-

triazoles and 3,4-disubstituted 1,2,4-triazoles and 1,5-

disubstituted 1,2,3,4-tetrazoles against selected human cancer cell

lines viz., prostate (DU-145, cervix (HeLa), lung adenocarcinoma

(A549), liver (HepG2) and breast (MCF-7), using CA-4 as

reference compound and the IC50 (μM) values are presented in

Table 1.

Most of the compounds from these series displayed potent

broad spectrum growth inhibitory effects against all the tested

cancer cell lines. In the SAR studies it was considered to

investigate whether the presence of 1,2,3-triazoles, 1,2,4-triazoles

and 1,2,3,4-tetrazoles influences the activity along with the other

substituents on the benzothiazole ring. It was observed that the

compounds (8-10a-d) containing 1,2,4-triazole ring on olefinic

bridge showed relatively strong inhibitory effect than the

bioisosters of 1,2,3-triazoles and 1,2,3,4-tetrazoles with IC₅₀

values in the range of 0.048-14.67 μM against some

representative cancer cell lines. Among them, compound 9a and

9b with a methyl substitution on C’-3 position of the 2-

phenylbenzothiazole moiety and methoxy and fluoro substitution

on C’-6 position of benzothiazole moiety exhibited promising

activity with a IC₅₀value of 0.054 and 0.048 μM against the lung

cancer cell line. The replacement of 1,2,4-triazole bridge with

1,2,3-triazole and 1,2,3,4- tetrazole resulted in moderate activity

and compound 7c (1,2,3-triazole) and 12b (1,2,3,4-tetrazole)

were the most potent drugs among them exhibiting IC₅₀values of

0.246 and 0.243 μM respectively against the lung cancer cell line.

The substitution effect on 2-phenylbenzothiazole ring by these

studies reaffirmed the importance of 1,2,4 –triazole ring on the

cytotoxic effect by these CA-4 linked benzothiazole hybrids. We

then proceeded to explore the influence of substituents on the

phenyl ring as well as benzothiazole ring with respect to

antiproliferative activity.

Scheme 2: Reagents and conditions: a) NaNO₂, aq. HCl, 0-5 °C, NaN₃, 1 h;

b) DBU, DMSO, 1 h.

Scheme 3

Synthesis of 3,4-disubstituted 1,2,4-triazoles and 1,5-

disubstituted 1,2,3,4-tetrazoles 8-10 (a-d) and 11-13(a-d)

The key intermediates of 2-(4-aminophenyl) benzothiazoles 25-

27(a–d) were coupled with trimethoxy benzoyl chloride (32) to

give corresponding trimethoxy substituted benzothiazole amides

33-35(a–d) which upon further conversion of their carbonyl

functionality to its analogous thiocarbonyl group employing

Lawesson’s reagent under refluxing toluene provides thioamides

36-38(a–d) in good yields.

Further, compounds 5-13(a-d) allowed us to investigate the SAR

effects of electron-withdrawing (F) and electron-releasing

(OMe), as well as substituents like (H, Me and OMe) on the

phenyl ring at the C’3-position of the benzothiazole ring. It was

observed that among different substituents studied on phenyl ring

attached to benzothiazole ring, methyl group (R = Me) at C’3-

position exhibited significant increase in activity compared to

other substituents (H, OMe) with irrespective of triazole and

tetrazole rings. Based on this findings, we introduced some other

substituents on the benzothiazole ring and studied their effect on

the antiproliferative activity. Initially, we introduced a methoxy

group at the C-6 position and the resulting compound showed

pronounced activity on the A549 cell line compared to other cell

lines and this compound (9a) exhibited significant cytotoxic

activity with IC₅₀values of 0.054 μM which has 1,2,4-triazole

ring. The effect of different bridge rings revealed that the

bioisosteric replacement of the 1,2,4-triazole by a 1,2,3-

Table 1: ^aIC₅₀(μM) values for Combretastatin-Benzothiazole hybrids 5-13(a-d)

Bridge

Compound

DU-145^b

HeLa^c

A549^d

HepG2^e

MCF-7^f

1,2,3

5a

5b

5c

5d

6a

6b

6c

6d

7a

7b

7c

7d

12.30 ±0.8

8.12 ±0.5

11.48 ±0.6

3.46 ±0.4

6.30 ±0.5

1.41 ±0.1

7.61 ±0.9

8.54 ±0.5

15.85 ±2.12

3.38 ±0.4

7.76 ±0.6

9.77 ±0.9

10.69 ±1.05

6.02 ±0.7

0.24 ±0.01

11.00 ±1.1

12.38 ±0.97

7.76 ±0.8

22.95 ±1.73

10.71 ±0.9

19.95 ±1.1

9.12 ±0.03

22.00 ±0.9

10.81 ±0.8

18.78 ±0.09

20.77 ±0.06

15.48 ±1.32

16.14 ±1.14

4.36 ±0.09

27.78 ±2.12

Triazole

9.33 ±0.4

9.05 ±0.8

17.37 ±2

14.79 ±0.9

18.19 ±1.05

7.33 ±0.41

10.20 ±0.7

15.84 ±1.21

18.62 ±1.54

14.16 ±0.19

0.41 ±0.06

15.89 ±1.34

11.48 ±0.5

13.10 ±0.8

6.76 ±0.8

15.48 ±0.7

20.14 ±1.2

8.05 ±0.7

8.12 ±0.6

15.48 ±0.6

18.57 ±1.04

16.44 ±1.73

13.93 ±0.9

1.47 ±0.09

29.87 ±1.12

11.35 ±1.07

11.14 ±1.58

10.14 ±1.21

0.37 ±0.02

17.80 ±1.03

1,2,4

8a

11.22 ±0.9

8.21 ±0.5

10.02 ±0.98

7.00 ±0.4

4.13 ±0.5

0.21 ±0.01

0.16 ±0.01

0.15 ±0.06

0.93 ±0.04

0.21 ±0.01

8.76 ±0.3

2.81 ±0.5

6.45 ±0.4

5.51 ±0.2

7.24 ±0.9

6.58 ±0.7

5.04 ±0.2

0.091±0.04

0.054±0.01

0.048±0.009

0.26 ±0.06

0.23 ±0.04

9.06 ±0.4

1.22 ±0.06

7.24 ±0.4

3.46 ±0.3

11.64 ±0.75

10.41 ±0.62

9.65 ±0.34

1.27 ±0.03

0.67 ±0.06

0.30 ±0.02

2.54 ±0.4

9.50 ±0.9

12.52±0.64

8.21 ±0.7

3.15 ±0.1

1.13 ±0.05

1.41 ±0.06

5.35 ±0.3

2.99 ±0.3

14.67 ±0.87

2.49 ±0.3

8.57 ±0.5

10.54 ±0.89

Triazole

8b

8c

11.84 ±0.7

0.46 ±0.08

0.48 ±0.03

0.28 ±0.02

1.04 ±0.09

0.70 ±0.07

11.60 ±0.8

7.58 ±0.7

8d

9a

9b

9c

9d

1.25 ±0.06

9.05 ±0.63

2.75 ±0.6

10a

10b

10c

10d

10.21 ±0.6

11.53 ±0.8

9.20 ±0.8

7.41 ±0.9

1,2,3,4

11a

11b

11c

11d

12a

12b

12c

12d

13a

13b

13c

13d

CA-4

9.12 ±0.9

6.60 ±0.4

2.23 ±0.3

10.23 ±0.9

6.16 ±0.7

10.96 ±1.11

0.97 ±0.05

8.70 ±0.7

9.81 ±0.3

4.56 ±0.5

4.36 ±0.7

11.74 ±2.21

5.79 ±0.6

0.005±0.001

3.80 ±0.4

2.07 ±0.5

7.24 ±0.9

3.54 ±0.6

4.78 ±0.3

0.24±0.03

5.38 ±0.5

7.41 ±0.8

2.25 ±0.06

1.38 ±0.04

9.24 ±0.8

3.80 ±0.1

0.058±0.001

9.05 ±0.7

7.95 ±0.7

22.38 ±1.64

8.51 ±0.72

10.47 ±0.7

1.69 ±0.1

9.74 ±0.8

23.44 ±2

11.16 ±0.9

8.57 ±0.8

Tetrazole

4.57 ±0.5

19.95 ±0.9

7.24 ±0.8

31.62 ±2.79

8.68 ±0.8

15.88 ±1.53

1.14 ±0.05

11.74 ±0.7

14.79 ±1.76

5.67 ±0.3

12.64 ±1.09

3.23 ±0.2

17.15 ±1.32

17.54 ±1.27

3.45 ±0.3

1.67 ±0.08

1.24 ±0.01

17.00 ±1.43

15.96 ±1.61

0.009±0.001

b

4.77 ±0.8

1.52 ±0.1

13.45 ±0.6

10.82 ±1.52

0.069±0.006

24.73 ±1.54

16.64 ±0.5

0.043±0.001

^a50% Inhibitory concentration and the values are average of three individual experiments after 48 h of drug treatment. prostate cancer. ^ccervical cancer. ^dlung

cancer. ^eliver cancer. ^fbreast cancer.

triazole and 1,2,3,4-tetrazole ring on the olefinic bridge

resulted in total loss of antiproliferative activity in most of the

cell lines. It is important to point out that the introduction of an

additional methoxy group i.e., disubstitution at C5 and C7-

position, having 1,2,4-triazoles, produced 6-fold reduction in

antiproliferative activity against all the cancer cell lines.

Whereas in case of 1,2,3-triazoles and 1,2,3,4-tetrazoles a

slight reduction (1-2-fold) in antiproliferative activity observed

in all the cancer cell lines with some exceptions in certain cell

lines. Moreover, introduction of additional methoxy groups i.e

trisubstitution at C5, C6 and C7-position having 1,2,4-

triazoles, produced a 3-fold reduction in antiproliferative

activity against all the cancer cell lines. Surprisingly when

electron withdrawing substituents (F) are incorporated at C-6

position on the benzothiazole ring, a substantial increase in the

cytotoxic potency was observed in all cases (1,2,3-triazole,

1,2,4-triazole and 1,2,3,4- tetrazoles). Compound 9b with a

fluoro substituent on C-6 position of the benzothiazole ring

and a methyl substituent on the C’-3 position of the phenyl

ring is the most active hybrid exhibiting an IC₅₀ranging from

0.048 to 1.41 μM against different cancer cell lines. On other

hand, unsubstituted phenyl ring (R = H) of the benzothiazole

counterparts (5a-d, 8a-d and 11a-d), an identical pattern of

activity was observed wherein the 1,2,4-triazole ring plays a

relatively stronger influence on the activity compared to

compounds having moieties 1,2,3-triazole and 1,2,3,4-

tetrazoles. This compound 8d showed significant activity with

IC₅₀values in the range of 0.0991-3.155 μM compared to other

compounds like 5a-d and 11a-d. Moreover, we also introduced

electron–releasing group (R = OMe) on phenyl ring of

benzothiazole (7a-d, 10a-d and 13a-d), having 1,2,3-triazole

ring where in compound 7c showed significant activity with

IC₅₀values in the range of 0.246-4.365 μM, amongst the other

compounds like 10a-d and 13a-d. Interestingly, hybrids 9a and

9b exhibit promising antiproliferative activity against A549

cancer cell line.

but superior to CA-4 (Panel B and C). Compounds 9a and 9b

arrested 59.5 % and 65.5 % cells, respectively, in G2/M phase

compared to 58.7 % blocked by CA-4 as shown in Table 2.

Therefore, treatment with compounds 9a and 9b causes a clear

G2/M arrest pattern in a concentration-dependent manner, with

a concomitant decrease in cells found in other phases of the

cell cycle. These results demonstrate that the growth inhibitory

effect of these hybrids are induced because of the arrest in

G2/M phase during cell cycle progression in a concentration

dependent manner.

A

B

C

Overall, SAR studies of these cis-restricted triazole and

tetrazole mimics of CA-4 linked benzothiazole hybrids suggest

that the superior activity of 1,2,4-triazole ring in place of the

olefinic bridge compared to other heterocyclic moieties is in

agreement with some similar studies on CA-4 analogues

described in the earlier studies.⁸The considerations that play a

crucial role in the antiproliferative activity are summarized in

Fig. 2.

D

F

E

G

Figure 2: SAR of CA-4 benzothiazole hybrids.

3.2. Analysis of Cell cycle Arrest

Cell cycle analyses were performed using flow cytometry³⁸to

examine the effect of the cell cycle distribution profiles of

A549 cells by these hybrids (9a and 9b) as a manifestation of

their antiproliferative action. Initially, cell cycle analysis was

performed using the cell permeable DNA binding dye

propidium iodide to determine whether the antiproliferative

effect resulted from cell cycle arrest. For this purpose, A549

cells were treated with 9a and 9b at concentrations of 50 nM as

well as 100 nM for 48 h to study the distribution of cells at

different phases of cell cycle as shown in Fig. 3. The results

demonstrated that compared to untreated cells (panel A), the

cells treated with 9a (Panel D and E) and 9b (Panel F and G)

displayed significant arrest in G2/M phase with increasing

concentrations of 9a and 9b which were not only comparable

Figure 3: FACS analysis of cell cycle distribution of A549 cells after

treatment with CA-4, 9a and 9b at 50 nM and 100 nM for 48 h. Cell cycle

analysis was performed employing propidium iodide as indicated under

Materials and Methods.

Table 2: Distribution of cell cycle phases following

treatment with compounds (CA-4, 9a and 9b) in A549 cells

was quantified by flow cytometry.

% of cells in phase

Compound

IC₅₀^a± SD (in µM)

1.67±0.04

% of Tubulin inhibition

Panel: Compd

Con(nM)

G0/G1

S

G2/M

9a

9b

72.89

74.43

73.20

1.00±0.04

A: Control

B: CA-4

C: CA-4

D: 9a

78.5

56.1

26.1

63.7

21.5

60.6

19.8

9.4

12.1

31.3

58.7

25.9

59.5

25.1

65.5

CA-4

1.60±0.07

Note: ^aConcentration of drug to inhibit 50% of tubulin assembly. Values

indicated are the mean ± SD of two different experiments performed in

triplicates. Statistical analysis was performed using Graph Pad Prism

software version 5.01.

50

100

50

12.6

15.9

10.6

17.6

14.7

15.4

E: 9a

100

50

3.4. Immunocytochemistry

F: 9b

Spindle poisons exert their action through the disruption of

chromosome separation during mitosis.⁴⁰To validate the effect

of these compounds on the disruption of microtubule dynamics

in living cells, studies were carried out to examine the in situ

effects of 9a and 9b on cellular microtubules. A549 cells

seeded on sterile cover slips were treated with these

compounds and CA-4 was employed as a standard at a

concentration of 50 nM for 48 h. The confocal images depicted

in Fig. 5 showed that the untreated lung cancer cell line

displayed the normal distribution of microtubules. However,

cells treated with compounds 9a and 9b showed disrupted

microtubule organization (Fig. 5), thus demonstrating the

inhibition of tubulin polymerization. The density of

microtubules was pronounced at the cell periphery with

disorganized central networks and the standard CA-4 also

G: 9b

100

3.3. Inhibition of Tubulin Polymerization

The G2/M phase arrest of cells takes place due to the

perturbation in the mitosis cell division machinery and the

tubulin is one of the important structural proteins in the mitosis

process. Therefore, as 9a and 9b showed significant

antiproliferative activity as well as arrest in the G2/M phase in

the cell cycle analysis, it is likely that they would inhibit the

polymerization of tubulin.³⁹The progression in tubulin

polymerization was investigated by monitoring the increase in

fluorescence emission at 420 nM at 3 μM concentration

employing CA-4 as the positive control (Fig. 4). Compounds

9a and 9b inhibited tubulin polymerization by 72.89 %, 74.43

% respectively, which is comparable to CA-4. Furthermore, we

decided to elucidate the action of 9a and 9b towards tubulin by

determining their IC₅₀values at different concentrations. It is

observed that 9a and 9b significantly inhibit the tubulin

assembly with IC₅₀values of 1.67 and 1.00 μM as shown in

Table 3. These results suggest that antiproliferative activity as

well as cell cycle analysis correlates well with the effect on the

inhibition of tubulin polymerization and these results support

that compounds 9a and 9b are potent inhibitors of tubulin

assembly.

showed

disrupted

microtubule

organization.

This

immunofluorescence study showed that the level of tubulin

polymerization inhibition for both 9a and 9b was comparable

to that of CA-4.

FITC

DAPI

Merge

Untreated

9a

120

100

Control

9a

9b

80

60

40

20

0

CA-4

9b

1 4 7 1013161922252831343740434649525558

Time (min)

80

70

60

50

40

30

20

10

0

CA-4

Figure 5: A549 cells were treated with compounds 9a and 9b at 50 nM

concentration for 48 h followed by staining with α-tubulin antibody.

Microtubule organization was clearly observed by green colour tubulin

network like structures in untreated cells (UT) and was found to be

disrupted in cells treated with compounds 9a and 9b. CA-4 was used as

positive control.

Control

9a

9b

CA-4

Figure 4: Effect of compounds on tubulin polymerization: Tubulin

polymerization was monitored by the increase in fluorescence at 360 nm

(excitation) and 420 nm (emission) for 1 h at 37 °C. Synthesized

compounds 9a and 9b and CA-4 were included at a final concentration of

3 μM 9a and 9b and CA-4 used as positive control. Percentage of tubulin

polymerization inhibition compared to control.

3.5. Distribution of soluble versus polymerized tubulin in

cells

Microtubules exhibit dynamic equilibrium with free tubulin

monomers in the cells. Pharmacological agents exploit this

property of microtubules to exert their anticancer effects.⁴¹

Since these mimics inhibit the tubulin polymerization and

Table 3. Inhibition of tubulin polymerization (IC₅₀) of

mimics 9a, 9b and CA-4.

disturbs the microtubule dynamics, we elucidated the levels of

soluble versus polymerized forms of tubulin in A549 cells

following treatment with 1M of 9a and 9b for 48 h. In

addition, cells were treated with combretastatin A-4 (CA-4, 1

M) and paclitaxel (1 M) as positive controls and DMSO as

negative in parallel experiments. Subsequently, these soluble

and polymerized fractions were collected and subjected to

Western blot analysis. This analysis reveals that the amount of

tubulin protein in both soluble and polymerized fractions was

approximately the same in DMSO treated cells. The standard

combretastatin A-4 treated cells exhibited a shift of tubulin

from the polymerized fraction into the soluble fraction. In

comparison, paclitaxel a microtubule polymerization agent

showed more amount of tubulin in the polymerized fraction.

As expected the cells treated with 9a and 9b significantly

increased the tubulin content in the soluble fraction.

Specifically, 9b treated cells showed a more distinct shift in

tubulin balance, with almost all tubulin present in the soluble

fraction similar to that of the positive control. Therefore,

increased tubulin in soluble fraction of cells treated by these

hybrids corroborated with the inhibition of tubulin assembly

and arrest of cells in G2/M phase as shown in Fig. 6.

1.60

1.40

1.20

1.00

0.80

0.60

0.40

0.20

0.00

-0.20

9a

9b

CA-4

Vinblastin

0

5

10

15

20

Concentration (µM)

Figure 7: Fluorescence based colchicine competitive binding assay of

conjugates 9a and 9b were carried out at various concentrations containing

3 µM of tubulin and colchicine for 60 min at 37 °C. Combretastatin A4

was used as a positive control whereas vinblastine was used as negative

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

DMSO (control).

3.7. Hoechst staining

Apoptosis is one of the major pathways of programmed cell

death. Chromatin condensation, nuclear shrinking, and

fragmented nuclei are some of the characteristics of apoptotic

cells. Disruption of microtubule formation leads to cell cycle

arrest in the G2/M phase followed by apoptotic cell death.⁴³To

investigate the apoptotic inducing effect of these hybrids (9a &

9b) and CA-4, Hoechst staining assay was carried out on A549

cells. Hoechst 33258 is a cell membrane permeable nuclear

staining dye, which emits blue fluorescence and stains the live

cell nuclei as light blue, whereas the apoptotic cell nuclei

appear as bright blue due to chromatin condensation. A549

cells were treated with 9a, 9b and CA-4 at their IC₅₀

concentration (50 nM) for 48 h and stained with Hoechst. The

results from Fig. 8 indicated that in the control group, the

untreated cells did not show obvious morphological changes

(all the cells exhibited uniform rounded cell morphology),

however in compound treated cells exhibited typical apoptotic

morphology such as highly condensed nuclei (brightly stained)

as indicated by arrows. This observation demonstrates that

these hybrids 9a, 9b and CA-4 are able to induce apoptosis in

A549 cells.

Figure 6: A549 cells were treated with 1 µM of 9a and 9b for 48 h. CA-4

and paclitaxel were used as reference standards. Levels of tubulin was

detected by Western blot analysis.

3.6. Competitive Colchicine binding assay

These hybrids showed similar inhibitory effects on tubulin

polymerization to that of CA-4 and which prompted us to

investigate whether these compounds bind to the colchicine

site of the tubulin by employing fluorescence based assay.

Tubulin (3 µM) was incubated with various concentrations of

9a and 9b (0–20 µM) in the presence of colchicine (3 µM)

with CA-4 as positive control at 37 °C for 60 min and the

fluorescence of tubulin–colchicine complex was monitored at

435 nm by exciting at 350 nm. We observed an increase in

fluorescence of tubulin–colchicine complex in the presence of

these hybrids. This is in the agreement with a previous

investigation in which CA-4 is reported to give fluorescence

upon binding at the colchicine site.⁴²Therefore, the experiment

was carried out both in the presence and absence of colchicine

to obtain fluorescence values of the desired tubulin–colchicine

complex, the fluorescence values of tubulin–test compounds

complex was subtracted from the tubulin–test compounds–

colchicine complex. It was observed that at low concentration

these hybrids (9a and 9b) and CA-4 showed significant

affinity towards colchicine site, whereas at higher

concentrations (10 μM and 15 μM) 9b and CA-4 showed more

binding affinity while 9a showed lower binding affinity as

shown in Fig. 7. Compared to the positive control CA-4,

compound 9b exhibited more affinity towards colchicine site

whereas 9a has less affinity. Vinblastine was used as a

negative control, which is known to bind at a different site and

has no effect on tubulin–colchicine complex. Therefore this

study indicates that 9a and 9b bind at the colchicine site of the

tubulin.

A: Control

B: CA-4

C: 9a

D: 9b

Figure 8: Hoechst staining in A549 cells. A: Untreated control cells

(Control, A549), B: CA-4 (50 nM), C: 9a (50 nM) and D: 9b (50 nM).

3.8. Annexin V-FITC for apoptosis

a

To characterize the mode of cellular death induced by 9a and

9b, we performed a biparametric cytofluorimetric analysis

assay⁴⁴by using propidium iodide (PI), which stains DNA and

enters only dead cells, and fluorescent immunolabeling of the

protein annexin-V, which binds to phosphatidyl serine (PS) in

a highly selective manner. Dual staining for annexin-V and

with PI permits discrimination between live cells (annexin-

V−/PI−), early apoptotic cells (annexin-V+/PI−), late apoptotic

cells (annexin-V+/PI+) and necrotic cells (annexin-V−/PI+).

A549 cells were treated with these hybrids for 48 h at 50 and

100 nM concentrations and CA-4 was employed as a reference

compound to examine the apoptotic effect. After treatment, it

was observed that 9a and 9b significantly induced apoptosis in

A549 cells as shown in Fig. 9. As depicted in Fig. 9, both 9a

and 9b induced an accumulation of annexin-V positive cells in

comparison with the control in a concentration dependent

manner.

Note: Determined by Annexin V–FITC assay; data are the quantification

of the FACS results shown in Figure 5.

3.9. Measurement of mitochondrial membrane potential

(∆Ψm)

Mitochondria play an essential role in the propagation of

apoptosis.⁴⁵It is well established that at an early stage,

apoptotic stimuli alter the mitochondrial transmembrane

potential (Δψmt). Δψmt was monitored by the fluorescence of

the

dye

5,5′,6,6′-tetrachloro-1,1′,3,3′-

tetraethylbenzimidazolcarbocyanine (JC-1). Mitochondrial

changes, including loss of mitochondrial membrane potential

(∆Ψm), are key events that take place during drug-induced

apoptosis. JC-1 has the unique property of forming orange

fluorescent aggregates locally and spontaneously under high

mitochondrial ΔΨM, whereas the monomeric form fluoresces

in green.⁴⁶When a collapse of ΔΨM occurs, JC-1 is only found

in monomeric form. In this study we have investigated the

involvement of mitochondria in the induction of apoptosis by

9a, 9b and 5F-203. Treated A549 cells in the presence of

compounds 9a and 9b after 48 h, it was observed that a

dramatic shift in fluorescence in a dose-dependent manner (50

and 100 nM) compared to the control cells, indicating

depolarization of mitochondrial membrane potential (Fig. 10).

A

C

B

E

D

Figure 9: Annexin V-FITC staining; A: Untreated control cells (A549), B:

CA-4 (100 nM), C: 9a (50 nM) and D: 9a (100 nM), E: 9b (50 nM) and F:

9b (100 nM).

Table 4: Apoptotic effects of compounds 9a, 9b and CA-4

in A549 lung cancer cells.^a

G

F

Sample

Control

CA-4

9a

Concentration

UL(%)

0.1

UR(%)

0.6

LL(%)

94.2

LR(%)

5.2

100 nM

50 nM

1.7

13.3

8.1

60.8

24.2

13.5

23.7

15.1

27.2

1.1

77.3

100 nM

50 nM

1.1

9.2

66.0

9a

0.8

5.8

78.3

9b

100 nM

0.7

8.2

63.9

9b

Figure 10: Drops in mitochondrial membrane potential (∆Ψm) was

assessed by JC-1 staining of A549 cells treated with compounds 9a and 9b

and samples were then subjected to flow cytometry analysis on a FACScan

(Becton Dickinson). A: Untreated control cells (A549), B: CA-4 (100 nM),

C: 5F-203 (100 nM) D: 9a (50 nM) and E: 9a (100 nM), F: 9b (50 nM)

and G: 9b (100 nM).

Figure 11: Production of ROS in human lung cancer cells (A549); A:

Untreated control cells (A549), B: CA-4 (100 nM), C: 5F-203 (100 nM)

D: 9a (50 nM) and E: 9a (100 nM), F: 9b (50 nM) and G: 9b (100 nM), the

cells were stained with DCFDA and analyzed by flow cytometry.

The ratio of DCF-positive cells for compounds 9a and 9b was

6.8 and 10.4 % and 12.3 and 20.9 % respectively at 50 and 100

nM concentrations, whereas CA-4 and 5F-203 18.0 and 10.0

respectively. The test results evidenced that these compounds

had enhanced the generation of ROS in A549 cells. Altogether,

these results indicate that these compounds induced apoptosis

through the mitochondrial pathway. In this context, it is

interesting to note that many other antimitotic compounds

induce apoptosis through the mitochondrial (intrinsic)

pathway.⁴⁸

3.10. Effect on intracellular ROS generation

Mitochondrial membrane depolarization is associated with

mitochondrial production of ROS.⁴⁷Therefore, we investigated

whether ROS production increased after treatment with the test

compounds. We analyzed the production of ROS by flow

cytometry utilizing fluorescence indicator: DCFDA (2,7-

dichlorodihydrofluorescein diacetate).⁴⁸Treatement of A549

cells in the presence of compounds 9a and 9b at 50 and 100

nM and 5F-203 100 nM concentrations for 48 h, the level of

ROS was significantly increased. The results presented in Fig.

11, where it can be observed that both 9a and 9b induced the

production of significant amounts of ROS in comparison with

control cells, which agrees with the previously described

dissipation of Δψm.

3.11. Effect on activation of caspase 3

Caspases, are a family of cysteine-aspartic proteases that are

crucial mediators of apoptosis. Among them, caspase-3 is well

understood among the mammalian caspases in terms of its

specificity and its role in apoptosis. Furthermore, there are

45,49,50

some reports

that indicate that the cell cycle arrest at

A

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

Hence, it was considered of interest to understand the

correlation of cytotoxicity with that to apoptosis by these

hybrids (9a and 9b).

Control

9a-50 nM

9b-50 nM

Caspase-3

B

C

β-actin

Figure 12: Effect of 9a and 9b on caspase-3 level. A549 cells were treated

with these compounds at 50 nM concentration for 48 h and western blot

analysis was performed. β-actin was used as internal loading control.

In this study, A549 cells were treated with them at 50 nM

concentration for 48 h and Western blot analysis was

performed. An increase in the levels of caspase-3 was observed

upon treatment of cells with these hybrids compare to control.

This results clearly indicates that 9a and 9b induced apoptosis

in A549 cells by activation of caspase-3 as shown in Fig. 12.

E

D

4. Molecular Modeling

To understand the binding mode and SAR for these hybrids,

some molecular modeling studies were performed on all

molecules. As these hybrids have a similarity to CA-4 (an

inhibitor of tubulin polymerization), we performed the docking

studies of on the tubulin protein and the tubulin crystal

structure was obtained from the Protein Data Bank (PDB ID

3E22).⁵¹Necessary corrections to the protein were carried out

using Protein Preparation Wizard from Schrodinger package.

Geometry of the molecule was optimized in Gaussian 09 using

PM3 semi-empirical method.⁵²Further docking studies were

performed using AutoDock 4.2 docking software.⁵³The

molecular docking studies have been performed on all the

molecules. The highest ranking pose of 9b shows that

molecule fits well in the colchicine binding domain (Fig. 13B).

The trimethoxyphenyl group was at the hydrophobic site deep

in the β chain and amino acid close in contact with this group

are Val238, Thr239, Cys241, Leu242, Ala250, Leu252,

F

G

Leu255, Ala316, Ala317, Val318, Thr353, Ala354, Ile378 of β

chain. The benzothiazole is extended towards the α,β interface

of the tubulin and forms close contact with Gln11, Ser178 and

Tyr224 of α chain and Glu247 and Gln248 residues of β chain.

However, 1-phenytriazole is in close contact with Asn101,

Ala180 and Val181 residues of α chain and Asn258, Met259

and Lys352 residues of β chain. Superimposed pose of 9b with

cocrystal ligand shows the trimethoxyphenyl ring is at the

same position whereas c ring of colchicine is at the same

position to that of triazole ring (Fig. 13C). Interestingly 9a and

9b have shown a similar binding pose (Fig. 13D). However the

3D geometry of 1,2,3-triazole and 1,2,4-triazole were totally

different, where 1,2,3-triazole is having slightly bent geometry

while 1,2,4-triazole is having L shaped. Due to this

geometrical difference, compounds from these series

accommodate different binding poses Fig. 13E. If we compare

the binding pose of 9b and 6b reveal that both compounds

having opposite binding pose. For 9b trimethoxy phenyl was in

hydrophobic site whereas for 6b trimethoxyphenyl group in at

interface of α- β chain. This may be the reason for the least

activity of compounds from this series. Besides geometry of

1,2,4-triazole and 1,2,3,4-tetrazole is similar, therefore their

binding poses are also similar (Fig. 13F). Thus the docking

studies suggest that these hybrids fit well in the colchicine

binding domain of the tubulin. Compounds with 1,2,3-triazole

moiety having different binding pose than the remaining

compounds and having lesser activity.

In the present investigation, a series of cis-restricted

triazole/tetrazole mimics of CA-4 linked benzothiazole hybrids

were designed, synthesized and evaluated for their cytotoxic

potential against selected human cancer cell lines. These

hybrids exhibited promising activity and the lead compounds

like 9a and 9b displayed potent IC₅₀values in the range of

0.054 and 0.048 μM respectively, against human lung cancer

cell line (A549). The importance of 1,2,4-triazole ring on CA-4

bridge in comparision to 1,2,3-triazole and 1,2,3,4-tetrazole

rings is evident from the SAR analysis. The methyl group on

C’-3 position of the benzothiazole moiety having either

electron withdrawing group (F) or electron releasing group

(OMe) is essential for imparting potent antiproliferative

activity. In the case of 9a having methoxy group on C-6

position as well as in 9b having a fluoro group on C-6 position

of benzothiazole moiety exhibited significant activity. The

SAR provided a useful insight that could be utilized in

developing improved leads based on these hybrid

scaffolds.FACS analysis demonstrated that these hybrids arrest

of cell cycle progression in G2/M phase in a concentration

dependent manner. Tubulin polymerization assay showed that

they are potent inhibitors of tubulin polymerization. Westren

blot analysis revealed that these hybrids remarkably inhibit

tubulin polymerization. To understand the mode of binding,

colchicine competitive binding assay and molecular docking

studies were undertaken which demonstrated that these hybrids

bind at the colchicine site of the tubulin. Interestingly,the

triggering of apoptotic cell death was investigated by studying

their effect by Hoechst staining, Annexin-V-FITC assay,

mitochondrial membrane potential, ROS generation and

caspase-3 activation. Moreover, 9b has a potential to be taken

up for preclinical studies for its development as a lead towards

the treatment of lung cancer.

6. Experimental Section

Chemistry

All reagents, starting materials and solvents were obtained

from commercial suppliers and used as such without further

purification. Reactions were monitored by thin layer

chromatography (TLC) with 0.25 mm pre-coated silica gel

plates (60 F254). Visualization was accomplished with either

UV light, iodine adsorbed on silica gel or by immersion in

ethanolic solution of phosphomolybdic acid (PMA), p-

anisaldehyde, 2, 4-DNP, KMnO4, ninhydrin solution followed

by heating with a heat gun for ~15 seconds. Column

chromatography was performed with Merck 60–120 mesh

silica gel. Deuterated solvents for NMR spectroscopic analyses

were used as received. All ¹H NMR and ¹³C NMR spectra were

obtained using a 300 MHz, 400 MHz, and 500 MHz

spectrometer and coupling constants were measured in Hertz.

All chemical shifts were quoted in ppm relative to TMS, using

the residual solvent peak as a reference standard. The

following abbreviations were used to explain the multiplicities:

s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,

br = broad. EI-MS spectra were recorded on Micro mass,

Quattro LC using ES I+ software with capillary voltage 3.98

kV and ESI mode positive ion trap detector. High -resolution

mass spectra (HRMS) were recorded on QSTAR XL Hybrid

MS/MS mass spectrometer. Chemical nomenclature was

generated using Chembiodrawultra 12.0.

Figure 13: A) Docking pose of 9b (yellow) in colchicine binding domain

of tubulin where α-chain is in green color and β-chain in pink color B)

Docking pose of 9b (Yellow) and protein in green color. C) Superimposed

pose of 9b with cocrystal ligand colchicine (pink) D) Superimposed pose

of 9b with 9a (cyan) E) Superimposed pose of 9b with 6b (blue) F)

Superimposed pose of 9b with 12b (green).

6.1.1 General Method A for the Synthesis of substituted

Benzanilides 16-18(a-d):

5. Conclusion

Substituted 4Nitrobenzoyl chlorides (15a-c) (0.029 mol) were

added slowly to a solution of the appropriately substituted

anilines (14ad) (0.04 mol) in pyridine (110 mL). The

resulting solution was stirred under reflux for 3 h and then

poured into ice water (400 mL).The precipitate formed was

collected and washed with 2N HCl (100 mL), followed by

water and methanol, to afford the respective benzanilides 16-

18(a-d) as a yellow solids.

6.1.9. N-(3,5-Dimethoxyphenyl)-3-methyl-4-nitrobenzamide

(17c):

The compound 17c was synthesized by following general

procedure A, as a yellow solid; Yield 72%; mp: 189-192 °C;

¹H NMR (500 MHz, CDCl₃): δ 8.03 (s, 1H), 7.98 (d, J = 8.4

Hz, 1H), 7.81 (s, 1H), 7.75 (dd, J = 8.4, 1.5 Hz, 1H), 6.87 (d, J

= 2.0 Hz, 2H), 6.29 (t, J = 2.2 Hz, 1H), 3.79 (s, 6H), 2.62 (s,

3H); MS (ESI): m/z 317 (M+H)⁺.

6.1.2. N-(4-Methoxyphenyl)-4-nitrobenzamide (16a):

The compound 16a was synthesized by following general

procedure A, as a yellow solid; Yield 85%; mp: 199-202 °C;

¹H NMR (300 MHz, DMSO): δ 10.47 (s, 1H), 8.36 (d, J = 8.8

Hz, 2H), 8.17 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 9.0 Hz, 2H),

6.95 (d, J = 9.0 Hz, 2H), 3.75 (s, 3H); MS (ESI): m/z 273

(M+H)⁺;

6.1.10.

N-(3,5-Dimethoxyphenyl)-2-methoxy-4-

nitrobenzamide (18c):

The compound 18c was synthesized by following general

procedure A, as a yellow solid; Yield 73%; mp: 176-179 °C;

¹H NMR (500 MHz, CDCl₃): δ 7.88 (d, J = 8.3 Hz, 2H), 7.66

(d, J = 1.4 Hz, 1H), 7.37 (dd, J = 8.3, 1.7 Hz, 1H), 6.88 (d, J =

2.1 Hz, 2H), 6.31 (t, J = 2.2 Hz, 1H), 4.02 (s, 3H), 3.81 (s, 6H);

MS (ESI): m/z 333 (M+H)⁺.

6.1.3.

N-(4-Methoxyphenyl)-3-methyl-4-nitrobenzamide

(17a):

The compound 17a was synthesized by following general

procedure A, as a yellow solid; Yield 71%; mp: 152-154 °C;

¹H NMR (300 MHz, CDCl₃): δ 8.00 (d, J = 8.4 Hz, 1H), 7.91

(s, 1H), 7.83 (s, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (d, J = 8.8

Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 3.82 (s, 3H), 2.64 (s, 3H);

MS (ESI): m/z 287 (M+H)⁺.

6.1.11.

4-Nitro-N-(3,4,5-trimethoxyphenyl)benzamide

(16d):

The compound 16d was synthesized by following general

procedure A, as a yellow solid; Yield 88%; mp: 220-224 °C;

¹H NMR (300 MHz, CDCl₃+DMSO): δ 10.12 (s, 1H), 8.32 (d,

J = 8.8 Hz, 2H), 8.20 (d, J = 8.8 Hz, 2H), 7.19 (s, 2H), 3.88 (s,

6H), 3.81 (s, 3H); MS (ESI): m/z 333 (M+H)⁺.

6.1.4. 2-Methoxy-N-(4-methoxyphenyl)-4-nitrobenzamide

(18a):

The compound 18a was synthesized by following general

procedure A, as a yellow solid; Yield 85%, mp: 156-158 ° C;

¹H NMR (300 MHz, CDCl₃): δ 7.91 (s, 1H), 7.86 (d, J = 8.3

Hz, 1H), 7.65 (s, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.3

Hz, 1H), 6.92 (d, J = 9.0 Hz, 2H), 4.00 (s, 3H), 3.82 (s, 3H);

MS (ESI): m/z 303 (M+H)⁺;

6.1.12.

3-Methyl-4-nitro-N-(3,4,5-

trimethoxyphenyl)benzamide (17d):

The compound 17d was synthesized by following general

procedure A, as a yellow solid; Yield 76%; mp: 210-214 °C;¹H

NMR (500 MHz, CDCl₃): δ 8.23 (s, 1H), 8.03 (s, 1H), 8.02 (s,

1H), 7.51 (s, 1H), 6.85 – 6.58 (m, 2H), 3.84 – 3.82 (m, 6H),

3.81 – 3.80 (m, 3H), 2.44 – 2.42 (m, 3H); MS (ESI): m/z 347

(M+H)⁺.

6.1.5. N-(4-Fluorophenyl)-4-nitrobenzamide (16b):

The compound 16b was synthesized by following general

1

procedure A, as a yellow solid; Yield 88%; mp: 164 °C; H

6.1.13.

2-Methoxy-4-nitro-N-(3,4,5-

NMR (300 MHz, CDCl₃): δ 8.35 (d, J = 8.7 Hz, 2H), 8.04 (d, J

= 8.6 Hz, 2H), 7.89 (s, 1H), 7.61 (dd, J = 8.6, 4.7 Hz, 2H), 7.10

(t, J = 8.6 Hz, 2H); (ESI): m/z 261 (M+H)⁺;

trimethoxyphenyl)benzamide (18d):

The compound 18d was synthesized by following general

procedure A, as a yellow solid; Yield 77%; mp: 178-180 °C;

¹H NMR (300 MHz, CDCl₃+DMSO): δ 9.42 (s, 1H), 7.86 (d, J

= 8.3 Hz, 1H), 7.73 (s, 1H), 7.58 (dd, J = 8.4, 1.4 Hz, 1H), 7.09

(s, 2H), 4.02 (s, 4H), 3.85 (s, 7H), 3.81 (s, 3H); MS (ESI): m/z

363 (M+H)⁺.

6.1.6.

N-(4-Fluorophenyl)-3-methyl-4-nitrobenzamide

(17b):

The compound 17b was synthesized by following general

procedure A, as a yellow solid; Yield 91%; mp: 160-162 °C;

¹H NMR (500 MHz, CDCl₃): δ 8.02 (d, J = 8.4 Hz, 1H), 7.96

(s, 1H), 7.84 (s, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.60 (dd, J =

8.8, 4.7 Hz, 2H), 7.11 – 7.05 (m, 2H), 2.65 (s, 3H); MS (ESI):

m/z 297 (M+Na)⁺.

6.2. General Method B for the Synthesis of Substituted

Thiobenzanilides 19-21(a-d):

To a solution of substituted benzanilides 16-18(a-d) (6g, 0.018

mol) in toluene (40 mL), Lawesson’s reagent (0.9 eq) was

added and reflux it for 8 h. After completion of the reaction,

cool to rt and solvent was removed in vaccuo and work up with

ethylacetate and water. Separation of the organic layer and

evaporation followed by column chromatography gave yellow

solids 19-21(a-d)

6.1.7.

N-(4-Fluorophenyl)-2-methoxy-4-nitrobenzamide

(18b):

The compound 18b was synthesized by following general

procedure A, as a yellow solid; Yield 82%; mp: 175-178 °C;

¹H NMR (300 MHz, CDCl₃+DMSO): δ 10.18 (s, 1H), 7.88 (d,

J = 8.4 Hz, 1H), 7.81 – 7.71 (m, 3H), 7.68 (dd, J = 8.4, 1.2 Hz,

1H), 7.06 (t, J = 8.7 Hz, 2H), 4.05 (s, 3H); MS (ESI): m/z

291(M+H)⁺.

6.2.1. N-(4-methoxyphenyl)-4-nitrobenzothioamide (19a):

The compound 19a was synthesized by following general

procedure B, as a yellow solid; Yield 84%; mp: 173-175 °C;

MS (ESI): m/z 289 (M+H)⁺.

6.1.8. N-(3,5-Dimethoxyphenyl)-4-nitrobenzamide (16c):

The compound 16c was synthesized by following general

procedure A, as a yellow solid; Yield 88%; mp: 211-213 °C;

¹H NMR (300 MHz, CDCl₃+DMSO): δ 10.22 (s, 1H), 8.31 (d,

J = 8.9 Hz, 2H), 8.20 (d, J = 8.9 Hz, 2H), 7.07 (d, J = 2.2 Hz,

2H), 6.25 (t, J = 2.2 Hz, 1H), 3.80 (s, 6H); MS (ESI): m/z 303

(M+H)⁺.

6.2.2.

N-(4-methoxyphenyl)-3-methyl-4-

nitrobenzothioamide (20a):

The compound 20a was synthesized by following general

procedure B, as a yellow solid; Yield 72%; mp: 160-162 °C;

MS (ESI): m/z 303 (M+H)⁺.

6.2.3.

2-Methoxy-N-(3-methoxyphenyl)-4-

(30 mL) taken in a 250 mL RB flask at 90 °C over a period of

1 h. The resulting solution was stirred at 90 °C for a further 2 h

and then cooled to room temperature. The precipitate formed

was filtered and washed with water. Products were purified by

column chromatography (ethylacetate/hexane) and to furnish

the 4-nitrophenyl-benzothiazoles 22-24(a-d) as yellow solids.

nitrobenzothioamide (21a):

The compound 21a was synthesized by following general

procedure B, as a yellow solid; Yield 69%; mp: 170-171 °C;

MS (ESI): m/z 319 (M+H)⁺.

6.2.4. N-(4-fluorophenyl)-4-nitrobenzothioamide (19b):

6.3.1. 6-Methoxy-2-(4-nitrophenyl)benzo[d]thiazole (22a):

The compound 19b was synthesized by following general

procedure B, as a yellow solid; Yield 80%; mp: 143-145 °C;

MS (ESI): m/z 277 (M+H)⁺.

The compound 22a was synthesized by following general

procedure C, as a yellow solid; Yield 62%; mp: 216-217 °C;

¹H NMR (300 MHz, CDCl₃): δ 8.34 (d, J = 8.9 Hz, 2H), 8.21

(d, J = 8.9 Hz, 2H), 8.00 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 2.5

Hz, 1H), 7.15 (dd, J = 9.0, 2.5 Hz, 1H), 3.92 (s, 3H); MS

(ESI): m/z 287 (M+H)⁺.

6.2.5. N-(4-fluorophenyl)-3-methyl-4-nitrobenzothioamide

(20b):

The compound 20b was synthesized by following general

procedure B, as a yellow solid; Yield 77%; mp: 164-165 °C;

MS (ESI): m/z 291 (M+H)⁺.

6.3.2.

6-Methoxy-2-(3-methyl-4-

nitrophenyl)benzo[d]thiazole (23a):

The compound 23a was synthesized by following general

procedure C, as a yellow solid; Yield 64%; mp: 195-197 °C;

¹H NMR (300 MHz, CDCl3): δ 8.10 (d, J = 8.5 Hz, 1H), 8.04

(s, 1H), 7.99 (t, J = 8.4 Hz, 2H), 7.39 (d, J = 2.4 Hz, 1H), 7.15

(dd, J = 9.0, 2.5 Hz, 1H), 3.93 (s, 3H), 2.72 (s, 3H); MS (ESI):

m/z 301 (M+H)⁺.

6.2.6. N-(3-fluorophenyl)-2-methoxy-4-nirobenzothioamide

(21b):

The compound 21b was synthesized by following general

procedure B, as a yellow solid; Yield 73%; mp: 166-168 °C;

MS (ESI): m/z 307 (M+H)⁺.

6.2.7.

(19c):

N-(3,5-dimethoxyphenyl)-4-nitrobenzothioamide

6.3.3.

6-Methoxy-2-(3-methoxy-4-

nitrophenyl)benzo[d]thiazole (24a):

The compound 19c was synthesized by following general

procedure B, as a yellow solid; Yield 90%; mp: 138-140 °C;

MS (ESI): m/z 319 (M+H)⁺.

The compound 24a was synthesized by following general

procedure C, as a yellow solid; Yield 66%; mp: 201-203 °C;

¹H NMR (300 MHz, CDCl₃):δ 7.99 (dd, J = 11.3, 8.7 Hz, 2H),

7.90 (d, J = 1.2 Hz, 1H), 7.60 (dd, J = 8.4, 1.4 Hz, 1H), 7.40

(d, J = 2.4 Hz, 1H), 7.16 (dd, J = 9.0, 2.5 Hz, 1H), 4.12 (s, 3H),

3.93 (s, 3H); MS (ESI): m/z 317 (M+H)⁺.

6.2.8.

N-(3,5-dimethoxyphenyl)-3-methyl-4-

nitrobenzothioamide (20c):

The compound 20c was synthesized by following general

procedure B, as a yellow solid; Yield 74%; mp: 178-180 °C;

MS (ESI): m/z 333 (M+H)⁺.

6.3.4. 6-Fluoro-2-(4-nitrophenyl)benzo[d]thiazole (22b):

The compound 24b was synthesized by following general

procedure C, as a yellow solid; Yield 65%; mp: 194-195 °C;

¹H NMR (300 MHz, CDCl₃): δ 8.39 – 8.32 (m, 2H), 8.27 –

8.20 (m, 2H), 8.08 (dd, J = 8.9, 4.8 Hz, 1H), 7.64 (dd, J = 7.9,

2.5 Hz, 1H), 7.38 – 7.27 (m, 1H); MS (ESI): m/z 275 (M+H)⁺.

6.2.9.

N-(3,5-dimethoxyphenyl)-2-methoxy-4-

nitrobenzothioamide (21c):

The compound 21c was synthesized by following general

procedure B, as a yellow solid; Yield 84%; mp: 198-200 °C;

MS (ESI): m/z 349 (M+H)⁺.

6.3.5. 6-Fluoro-2-(3-methyl-4-nitrophenyl)benzo[d]thiazole

(23b):

6.2.10. 4-Nitro-N-(3,4,5-trimethoxyphenyl)benzothioamide

The compound 23b was synthesized by following general

procedure C, as a yellow solid; Yield 67%; mp: 220-224 °C;

¹H NMR (500 MHz, CDCl₃): δ 8.10 (d, J = 8.5 Hz, 1H), 8.06

(dd, J = 8.7, 5.0 Hz, 2H), 7.99 (dd, J = 8.5, 1.6 Hz, 1H), 7.62

(dd, J = 8.0, 2.5 Hz, 1H), 7.28 (dt, J = 8.8, 2.3 Hz, 1H), 2.71 (s,

3H); MS (ESI): m/z 289 (M+H)⁺.

(19d):

The compound 19d was synthesized by following general

procedure B, as a yellow solid; Yield 84%; mp: 198-200 °C;

MS (ESI): m/z 349 (M+H)⁺.

6.2.11.

3-Methyl-4-nitro-N-(3,4,5-

trimethoxyphenyl)benzothioamide (20d):

6.3.6.

6-Fluoro-2-(3-methoxy-4-

nitrophenyl)benzo[d]thiazole (24b):

The compound 20d was synthesized by following general

procedure B, as a yellow solid; Yield 72%; mp: 136-138 °C;

MS (ESI): m/z 363 (M+H)⁺.

The compound 24b was synthesized by following general

procedure C, as a yellow solid; Yield 63%; mp: 189-201 °C;

¹H NMR (500 MHz, CDCl₃): δ 8.06 (dd, J = 9.0, 4.8 Hz, 1H),

7.96 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 1.3 Hz, 1H), 7.61 (ddd, J

= 11.9, 8.2, 2.0 Hz, 2H), 7.32 – 7.25 (m, 1H), 4.11 (s, 3H); MS

(ESI): m/z 305 (M+H)⁺.

6.2.12.

2-Methoxy-4-nitro-N-(3,4,5-

trimethoxyphenyl)benzothioamide (21d):

The compound 21d was synthesized by following general

procedure B, as a yellow solid; Yield 76%; mp 136-138 °C;

MS (ESI): m/z 379 (M+H)⁺.

6.3.7.

5,7-Dimethoxy-2-(4-nitrophenyl)benzo[d]thiazole

(22c):

6.3. General Method C for the Jacobson Synthesis of

The compound 22c was synthesized by following general

procedure C, as a yellow solid; Yield 60%; mp: 238-239 °C;

¹H NMR (300 MHz, CDCl₃): δ 8.37 – 8.32 (m, 2H), 8.24 (d, J

= 8.9 Hz, 2H), 7.22 (d, J = 1.9 Hz, 1H), 6.56 (d, J = 1.9 Hz,

1H), 3.99 (s, 3H), 3.92 (s, 3H); MS (ESI): m/z 317 (M+H)⁺.

Substituted 2-(4-Nitrophenyl) benzothiazoles 22-24(a-d):

A solution of the substituted thiobenzanilides 19-21(a-d)

(0.017 mol) in aqueous sodium hydroxide (8 eq in 50 mL of

water) containing ethanol (3 mL) was added dropwise to a

preheating solution of potassium ferricyanide (4 eq) in water

6.3.8.

5,7-Dimethoxy-2-(3-methyl-4-

The compound 26a was synthesized by following general

procedure D, as a yellow solid; Yield 95%; mp 151-153 °C; ¹H

NMR (300 MHz, CDCl₃): δ 7.87 (d, J = 8.9 Hz, 1H), 7.78 (d, J

= 1.2 Hz, 1H), 7.69 (dd, J = 8.2, 1.9 Hz, 1H), 7.31 (d, J = 2.5

Hz, 1H), 7.04 (dd, J = 8.9, 2.6 Hz, 1H), 6.71 (d, J = 8.3 Hz,

1H), 3.91 (s, 1H), 3.87 (s, 3H), 2.23 (s, 3H); MS (ESI): m/z

271 (M+H)⁺.

nitrophenyl)benzo[d]thiazole (23c):

The compound 23c was synthesized by following general

procedure C, as a yellow solid; Yield 65%; mp: 223-225 °C;

¹H NMR (500 MHz, CDCl₃): δ 8.00 (dd, J = 1.9, 1.2 Hz, 1H),

7.82 (d, J = 8.4 Hz, 1H), 7.06 – 7.02 (m, 2H), 6.83 (d, J = 2.4

Hz, 1H), 3.93 (s, 3H), 3.80 (s, 3H), 2.65 (s, 3H); MS (ESI): m/z

330 (M+H)⁺.

6.4.3. 2-Methoxy-4-(6-methoxybenzo[d]thiazol-2-yl)aniline

(27a):

6.3.9.

5,7-Dimethoxy-2-(3-methoxy-4-

nitrophenyl)benzo[d]thiazole (24c):

The compound 27a was synthesized by following general

procedure D, as a yellow solid; Yield 85%; mp 181-183 °C; ¹H

NMR (300 MHz, CDCl₃+DMSO): δ 7.78 (d, J = 8.9 Hz, 1H),

7.47 (s, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.03 (dd, J = 8.9, 2.4 Hz,

1H), 6.74 (d, J = 8.1 Hz, 1H), 5.32 (s, 1H), 3.92 (s, 3H), 3.85

(s, 3H); MS (ESI): m/z 287 (M+H)⁺.

The compound 24c was synthesized by following general

procedure C, as a yellow solid; Yield 64%; mp: 219-221 °C;

¹H NMR (500 MHz, CDCl₃):δ 7.95 (d, J = 8.4 Hz, 1H), 7.89

(d, J = 1.4 Hz, 1H), 7.63 (dd, J = 8.4, 1.6 Hz, 1H), 7.20 (d, J =

1.9 Hz, 1H), 6.55 (d, J = 1.9 Hz, 1H), 4.10 (s, 3H), 3.98 (s,

3H), 3.92 (s, 3H);MS (ESI): m/z 347 (M+H)⁺.

6.4.4. 4-(6-Fluorobenzo[d]thiazol-2-yl)aniline (28b):

6.3.10. 5,6,7-Trimethoxy-2-(4-nitrophenyl)benzo[d]thiazole

(22d):

The compound 28b was synthesized by following general

procedure D, as a yellow solid; Yield 88%; mp 152-153 °C; ¹H

NMR (300 MHz, CDCl₃): δ 7.91 (dd, J = 8.9, 4.8 Hz, 1H),

7.85 (d, J = 8.6 Hz, 2H), 7.52 (dd, J = 8.2, 2.5 Hz, 1H), 7.17

(td, J = 8.9, 2.6 Hz, 1H), 6.73 (d, J = 8.6 Hz, 2H), 4.02 (s, 2H);

MS (ESI): m/z 245 (M+H)⁺.

The compound 22d was synthesized by following general

procedure C, as a yellow solid; Yield 66%; mp 160-168 °C; ¹H

NMR (300 MHz, CDCl₃): δ 8.36 (d, J = 8.8 Hz, 2H), 8.23 (d, J

= 8.8 Hz, 2H), 7.40 (s, 1H), 4.14 (s, 3H), 3.99 (d, J = 6.4 Hz,

6H); MS (ESI): m/z 347 (M+H)⁺.

6.4.5.

4-(6-Fluorobenzo[d]thiazol-2-yl)-2-methylaniline

(29b):

6.3.11.

5,6,7-Trimethoxy-2-(3-methyl-4-

nitrophenyl)benzo[d]thiazole (23d):

The compound 29b was synthesized by following general

procedure D, as a yellow solid; Yield 92%; mp 203-205 °C; ¹H

NMR (300 MHz, CDCl₃): δ 7.91 (dd, J = 8.9, 4.8 Hz, 1H),

7.85 (d, J = 8.6 Hz, 2H), 7.52 (dd, J = 8.2, 2.5 Hz, 1H), 7.17

(td, J = 8.9, 2.6 Hz, 1H), 6.73 (d, J = 8.6 Hz, 2H), 4.02 (s, 2H);

MS (ESI): m/z 259 (M+H)⁺.

The compound 23d was synthesized by following general

procedure C, as a yellow solid; Yield 69%; mp 176-179 °C; ¹H

NMR (300 MHz, CDCl₃): δ 7.96 (d, J = 8.4 Hz, 1H), 7.86 (s,

1H), 7.61 (d, J = 8.3 Hz, 1H), 7.63 – 7.38 (m, 1H), 7.37 (s,

1H), 4.11 (d, J = 5.7 Hz, 6H), 3.97 (d, J = 5.6 Hz, 6H); MS

(ESI): m/z 361 (M+H)⁺.

6.4.6.

4-(6-Fluorobenzo[d]thiazol-2-yl)-2-methoxyaniline

(30b):

6.3.12.

5,6,7-Trimethoxy-2-(3-methoxy-4-

nitrophenyl)benzo[d]thiazole (24d):

The compound 30b was synthesized by following general

procedure D, as a yellow solid; Yield 83%; mp 191-193 °C; ¹H

NMR (500 MHz, CDCl₃): δ 7.91 (dd, J = 8.9, 4.8 Hz, 1H),

7.56 (d, J = 1.8 Hz, 1H), 7.51 (dd, J = 8.1, 2.6 Hz, 1H), 7.41

(dd, J = 8.1, 1.9 Hz, 1H), 7.16 (td, J = 8.9, 2.6 Hz, 1H), 6.72

(dd, J = 8.1, 4.1 Hz, 1H), 4.16 (s, 2H), 3.97 (s, 3H). MS (ESI):

m/z 276 (M+H)⁺.

The compound 24d was synthesized by following general

procedure C, as a yellow solid; Yield 66%; mp 185-187 °C; ¹H

NMR (300 MHz, CDCl₃): δ 7.96 (d, J = 8.4 Hz, 1H), 7.86 (s,

1H), 7.61 (d, J = 8.3 Hz, 1H), 7.63 – 7.38 (m, 1H), 7.37 (s,

1H), 4.11 (d, J = 5.7 Hz, 6H), 3.97 (d, J = 5.6 Hz, 6H); MS

(ESI): m/z 377 (M+H)⁺.

6.4.7. 4-(5,7-Dimethoxybenzo[d]thiazol-2-yl)aniline (28c):

6.4. General Method D for the Reduction of Substituted

2(4Nitrophenyl)benzothiazoles 25-27(a-d):

The compound 28c was synthesized by following general

procedure D, as a yellow solid; Yield 90%; mp 150-152 °C; ¹H

NMR (500 MHz, CDCl₃): δ 7.90 – 7.85 (m, 2H), 7.14 (d, J =

2.0 Hz, 1H), 6.75 – 6.69 (m, 2H), 6.45 (d, J = 2.0 Hz, 1H),

3.98 (s, 1H), 3.95 (s, 3H), 3.88 (s, 3H); MS (ESI): m/z 286

(M)⁺.

To a solution of substituted 2(4nitrophenyl)benzothiazoles

(3 g, 8.66 mmol) in ethanol, tin(II) chloride dihydrate (3 eq,

25.98 mmol) was added and refluxed it for 3 h. The solvent

was removed under vacuum and the resulting oil taken up in

choloform (75 mL) was quenched with aq. NaHCO₃solution.

The resulting organic layer was separated and evaporated to

leave a residue of the amine which was purified by column

chromatography (eluent: ethylacetate/hexane).

6.4.8.

4-(5,7-Dimethoxybenzo[d]thiazol-2-yl)-2-

methylaniline (29c):

The compound 29c was synthesized by following general

procedure D, as a yellow solid; Yield 91%; mp: 142-145 °C;

¹H NMR (300 MHz, CDCl₃): δ 7.84 (s, 1H), 7.75 (dd, J = 8.3,

2.0 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 6.73 (d, J = 8.3 Hz, 1H),

6.47 (d, J = 2.0 Hz, 1H), 3.97 (s, 3H), 3.96 – 3.93 (m, 1H),

3.91 (s, 3H), 2.26 (s, 3H); MS (ESI): m/z 301 (M+H)⁺.

6.4.1. 4-(6-Methoxybenzo[d]thiazol-2-yl)aniline (25a):

The compound 25a was synthesized by following general

procedure D, as a yellow solid; Yield 92%; mp 191-193 °C; ¹H

NMR (300 MHz, CDCl₃): δ 7.88 (t, J = 8.6 Hz, 3H), 7.34 (d, J

= 2.4 Hz, 1H), 7.07 (dd, J 8.9, 2.5 Hz, 1H), 6.75 (d, J = 8.6 Hz,

2H), 3.99 (s, 1H), 3.90 (s, 3H); MS (ESI): m/z 257 (M+H)⁺.

6.4.9.

4-(5,7-Dimethoxybenzo[d]thiazol-2-yl)-2-

methoxyaniline (30c):

6.4.2.

4-(6-Methoxybenzo[d]thiazol-2-yl)-2-methylaniline

(26a):

The compound 30c was synthesized by following general

procedure D, as a yellow solid; Yield 82%; mp: 173-175 °C;

¹H NMR (300 MHz, CDCl₃): δ 7.58 (d, J = 1.6 Hz, 1H), 7.47

(dd, J = 8.1, 1.6 Hz, 1H), 7.13 (d, J = 1.9 Hz, 1H), 6.73 (d, J =

8.1 Hz, 1H), 6.45 (d, J = 1.9 Hz, 1H), 4.13 (s, 2H), 3.96 (d, J =

6.8 Hz, 6H), 3.89 (s, 3H); MS (ESI): m/z 317 (M+H)⁺.

The compound 34a was synthesized by following general

procedure F, as a pale yellow solid; Yield 72%; mp 203-204

°C; H NMR (300 MHz, CDCl₃): δ 8.19 (d, J = 8.4 Hz, 1H),

7.98 (s, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H),

7.73 (s, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.12 (s, 2H), 7.11 – 7.07

(m, 1H), 3.95 (s, 6H), 3.93 (s, 3H), 3.90 (s, 3H), 2.43 (s, 3H);

MS (ESI): m/z 465 (M+H)⁺.

1

6.4.10.

4-(5,6,7-Trimethoxybenzo[d]thiazol-2-yl)aniline

(28d):

The compound 28d was synthesized by following general

procedure D, as a yellow solid; Yield 89%; mp: 145-147 °C;

¹H NMR (300 MHz, CDCl₃): δ 7.85 (d, J = 8.6 Hz, 2H), 7.30

(s, 1H), 6.73 (d, J = 8.6 Hz, 2H), 4.09 (s, 3H), 3.99 (s, 2H),

3.94 (d, J = 4.4 Hz, 6H); MS (ESI): m/z 317 (M+H)⁺.

6.6.3.

3,4,5-Trimethoxy-N-(2-methoxy-4-(6-

methoxybenzo[d]thiazol-2-yl)phenyl)benzamide (35a):

The compound 35a was synthesized by following general

procedure F, as a pale yellow solid; Yield 76%; mp 198-201

1

°C; H NMR (500 MHz, CDCl₃): δ 8.61 (d, J = 8.4 Hz, 1H),

6.4.11.

2-Methyl-4-(5,6,7-trimethoxybenzo[d]thiazol-2-

8.57 (s, 1H), 7.93 (d, J = 8.9 Hz, 1H), 7.75 (d, J = 1.6 Hz, 1H),

7.60 (dd, J = 8.4, 1.6 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.13 (s,

2H), 7.08 (dt, J = 15.9, 7.9 Hz, 1H), 4.08 (s, 3H), 3.96 (s, 6H),

3.93 (s, 3H), 3.90 (s, 3H). MS (ESI): m/z 481 (M+H)⁺.

yl)aniline (29d):

The compound 29d was synthesized by following general

procedure D, as a yellow solid; Yield 90%; mp: 150-152 °C;

¹H NMR (500 MHz, CDCl₃): δ 7.79 (s, 1H), 7.71 (dd, J = 8.2,

2.0 Hz, 1H), 7.30 (s, 1H), 6.71 (d, J = 8.2 Hz, 1H), 4.09 (s,

3H), 3.94 (d, J = 7.0 Hz, 8H). MS (ESI): m/z 331 (M+H)⁺.

6.6.4.

N-(4-(6-Fluorobenzo[d]thiazol-2-yl)phenyl)-3,4,5-

trimethoxybenzamide (33b):

The compound 33b was synthesized by following general

procedure F, as a pale yellow solid; Yield 75%; mp 156-178

°C; H NMR (300 MHz, CDCl₃): δ 8.07 (d, J = 8.6 Hz, 2H),

6.4.12.

yl)aniline (30d):

2-Methoxy-4-(5,6,7-trimethoxybenzo[d]thiazol-2-

1

7.99 (dd, J = 8.6, 5.1 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H), 7.58

(dd, J = 8.1, 2.5 Hz, 1H), 7.26 – 7.18 (m, 1H), 7.10 (s, 2H),

3.93 (d, J = 4.2 Hz, 9H); MS (ESI): m/z 439 (M+H)⁺.

The compound 30d was synthesized by following general

procedure D, as a yellow solid; Yield 87%; mp 141-143 °C; ¹H

NMR (300 MHz, CDCl₃): δ 7.56 (s, 1H), 7.47 (dd, J = 8.1, 1.7

Hz, 1H), 7.31 (s, 1H), 6.75 (d, J = 8.1 Hz, 1H), 4.14 (s, 2H),

4.11 (s, 3H), 4.02–3.90 (m, 9H); MS (ESI): m/z 347 (M+H)⁺.

6.6.5. N-(4-(6-Fluorobenzo[d]thiazol-2-yl)-2-methylphenyl)-

3,4,5-trimethoxybenzamide (34b):

6.5. General Method E for the Conversion amines to azides

25-27(a-d):

The compound 34b was synthesized by following general

procedure F, as a pale yellow solid; Yield 72%; mp 201-203

°C; H NMR (300 MHz, CDCl₃): δ 8.22 (d, J = 8.5 Hz, 1H),

8.01 (dd, J = 8.9, 4.8 Hz, 2H), 7.92 (dd, J = 8.4, 1.9 Hz, 1H),

7.78 (s, 1H), 7.60 (dd, J = 8.1, 2.5 Hz, 1H), 7.22 (dd, J = 8.9,

2.6 Hz, 1H), 7.14 (s, 2H), 3.96 (s, 6H), 3.94 (s, 3H), 2.45 (s,

3H); MS (ESI): m/z 453 (M+H)⁺.

1

Substituted anilines 25-27(a-d) (150 mg, 1 mmol) was

dissolved in 2N HCl (7 mL) in a 25 mL round-bottomed flask

and cooled to 0°C in an ice bath for 5min. To this stirred

solution aq.NaNO₂(3 mmol) was added portion wise and

mixture was stirred for 30 min followed by NaN₃(3 mmol)

was added to the reaction mixture. The resulting solution was

stirred at room temperature for 1 h. After completion of the

reaction, neutralized with aq.NaHCO₃solution (25 ml)

followed by extract with etyhyl acetate. The crude product was

purified by column chromatography with hexane/ethyl acetate

(1:9) to afford the pure products as yellow solids 28-30(a-d).

6.6.6.

N-(4-(6-Fluorobenzo[d]thiazol-2-yl)-2-

methoxyphenyl)-3,4,5-trimethoxybenzamide (35b):

The compound 35b was synthesized by following general

procedure F, as a pale yellow solid; Yield 69%; mp 205-208

°C; ¹H NMR (300 MHz, CDCl₃): δ 8.68–8.54 (m, 2H), 7.98 (s,

1H), 7.76 (s, 1H), 7.62 (d, J = 9.8 Hz, 2H), 7.25 (d, J = 10.6

Hz, 2H), 7.13 (s, 2H), 4.08 (s, 3H), 3.95 (d, J = 9.6 Hz, 9H).

MS (ESI): m/z 469 (M+H)⁺.

6.6. General Method F for the Synthesis of Substituted N-

(4-(benzo[d]thiazol-2-yl)phenyl)-3,4,5-

trimethoxybenzamide 33-35(a-d):

6.6.7.

N-(4-(5,7-Dimethoxybenzo[d]thiazol-2-yl)phenyl)-

Trimethoxy benzoyl chloride (32) (0.027 mol) were added

slowly to a solution of the appropriately substituted anilines

25-27(a-d) (0.027 mol) in pyridine (15 mL). The resulting

solution was stirred under reflux for 8 h and then poured into

ice water (100mL).The precipitate formed was collected and

washed with 2N HCl (100 mL), followed by water and

methanol, to afford the respective benzanilides 33-35(a-d) as a

white solids.

3,4,5-trimethoxybenzamide (33c):

The compound 33a was synthesized by following general

procedure F, as a pale yellow solid; Yield 68%; mp 180-185

1

°C; H NMR (300 MHz, CDCl₃): δ 8.09 (d, J = 8.6 Hz, 2H),

7.98 (s, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.17 (d, J = 2.0 Hz, 1H),

7.10 (s, 2H), 7.04 (s, 1H), 6.50 (d, J = 1.9 Hz, 1H), 3.97 (s,

3H), 3.94 – 3.89 (m, 9H), 3.76 (s, 3H); MS (ESI): m/z 481

(M+H)⁺.

6.6.1. 3,4,5-Trimethoxy-N-(4-(6-methoxybenzo[d]thiazol-

2yl)phenyl)benzamide (33a):

6.6.8.

N-(4-(5,7-Dimethoxybenzo[d]thiazol-2-yl)-2-

methylphenyl)-3,4,5-trimethoxybenzamide (34c):

The compound 33a was synthesized by following general

procedure F, as a pale yellow solid; Yield 72%; mp180-183

°C; H NMR (300 MHz, CDCl₃+DMSO): δ) 7.99 (q, J = 8.9

Hz, 4H), 7.89 (d, J = 8.9 Hz, 1H), 7.74 (s, 1H), 7.43 (d, J = 2.4

Hz, 1H), 7.33 (s, 2H), 7.08 (dd, J = 8.9, 2.5 Hz, 1H), 3.96 (s,

6H), 3.90 (s, 3H), 3.87 (s, 3H); MS (ESI): m/z 451 (M+H)⁺.

The compound 34c was synthesized by following general

procedure F, as a pale yellow solid; Yield 70%; mp 210-213

1

°C; H NMR (300 MHz, CDCl₃): δ 8.19 (d, J = 8.4 Hz, 1H),

8.00 (s, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.74 (s, 1H), 7.18 (d, J =

1.8 Hz, 1H), 7.12 (s, 2H), 6.50 (d, J = 1.8 Hz, 1H), 3.97 (s,

3H), 3.95 (s, 6H), 3.93 (s, 3H), 3.90 (s, 3H), 2.43 (s, 3H); MS

(ESI): m/z 495 (M+H)⁺.

6.6.2. 3,4,5-Trimethoxy-N-(4-(6-methoxybenzo[d]thiazol-2-

yl)-2-methylphenyl)benzamide (34a):

6.6.9.

N-(4-(5,7-dimethoxybenzo[d]thiazol-2-yl)-2-

The compound 38a was synthesized by following general

procedure G, as a pale yellow solid; Yield 69%; mp 203-204

°C; MS (ESI): m/z 497 (M+H)⁺.

methoxyphenyl)-3,4,5-trimethoxybenzamide (35c):

The compound 35c was synthesized by following general

procedure F, as a pale yellow solid; Yield 72%; mp 201-203

°C; ¹H NMR (500 MHz, CDCl₃): δ 8.61 (d, J = 8.4 Hz, 1H),

8.57 (s, 1H), 7.74 (d, J = 1.7 Hz, 1H), 7.67 (dd, J = 8.4, 1.8 Hz,

1H), 7.17 (d, J = 2.0 Hz, 1H), 7.13 (s, 2H), 6.50 (d, J = 2.0 Hz,

1H), 4.06 (s, 3H), 3.96 (d, J = 3.6 Hz, 9H), 3.93 (s, 3H), 3.90

(s, 3H). MS (ESI): m/z 511 (M+H)⁺.

6.7.4.

N-(4-(6-Fluorobenzo[d]thiazol-2-yl)phenyl)-3,4,5-

trimethoxybenzothioamide (36b):

The compound 36b was synthesized by following general

procedure G, as a pale yellow solid; Yield 68%; mp 188-201

°C; MS (ESI): m/z 439 (M+H)⁺.

6.7.5. N-(4-(6-Fluorobenzo[d]thiazol-2-yl)-2-methylphenyl)-

3,4,5-trimethoxybenzothioamide (37b):

6.6.10.

3,4,5-Trimethoxy-N-(4-(5,6,7-

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzamide (33d):

The compound 37b was synthesized by following general

procedure G, as a pale yellow solid; Yield 64%; mp 210-

213°C; MS (ESI): m/z 469 (M+H)⁺.

The compound 33d was synthesized by following general

procedure F, as a pale yellow solid; Yield 71%; mp 145-150

°C; H NMR (300 MHz, CDCl₃): δ 8.12 (d, J = 8.6 Hz, 2H),

1

7.78 (d, J = 8.6 Hz, 1H), 7.48 (s, 1H), 7.17 (d, J = 2.0 Hz, 1H),

7.10 (s, 2H), 7.04 (d, J = 1.9 Hz, 1H), 4.09 (s, 3H), 3.93 (s,

3H), 3.89 (s, 3H), 3.87 (s, 3H), 3.78 (s, 6H); MS (ESI): m/z

511 (M+H)⁺.

6.7.6.

4-Fluoro-N-(2-methoxy-4-(5,6,7-

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzothioamide

(38b):

The compound 38b was synthesized by following general

procedure G, as a pale yellow solid; Yield 67%; mp 223-

225°C; MS (ESI): m/z 485 (M+H)⁺.

6.6.11.

3,4,5-Trimethoxy-N-(2-methyl-4-(5,6,7-

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzamide (34d):

The compound 34d was synthesized by following general

procedure F, as a pale yellow solid; Yield 71%; mp 160-165

°C; H NMR (300 MHz, CDCl₃): δ 8.17 (d, J= 8.4 Hz, 1H),

8.06 – 7.95 (m, 1H), 7.94 – 7.80 (m, 1H), 7.77 (s, 1H), 7.34 (s,

1H), 7.03 (s, 2H), 4.11 (s, 3H), 3.96 (s, 3H), 3.93 (s, 3H), 3.87

(s, 3H), 3.78 (s, 6H), 2.43 (s, 3H); MS (ESI): m/z 525 (M+H)⁺.

6.7.7.

N-(4-(5,7-Dimethoxybenzo[d]thiazol-2-yl)phenyl)-

3,4,5-trimethoxybenzothioamide (36c):

1

The compound 36c was synthesized by following general

procedure G, as a pale yellow solid; Yield 69%; mp 199-200

°C; MS (ESI): m/z 497 (M+H)⁺.

6.7.8.

N-(4-(5,7-Dimethoxybenzo[d]thiazol-2-yl)-2-

6.6.12.

3,4,5-Trimethoxy-N-(2-methoxy-4-(5,6,7-

methylphenyl)-3,4,5-trimethoxybenzothioamide (37c):

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzamide (35d):

The compound 37c was synthesized by following general

procedure G, as a pale yellow solid; Yield 67%; mp 175-176

°C; MS (ESI): m/z 511 (M+H)⁺.

The compound 35d was synthesized by following general

procedure F, as a pale yellow solid; Yield 73%; mp 176-178

°C; ¹H NMR (300 MHz, CDCl₃+DMSO): δ 10.16 (s, 1H), 7.95

– 7.65 (m, 4H), 7.19 (s, 2H), 4.07 (s, 3H), 3.87 (s, 6H), 3.78 (s,

3H), 3.23 (m, 9H); MS (ESI): m/z 541 (M+H)⁺.

6.7.9.

3,5-Dimethoxy-N-(2-methoxy-4-(5,6,7-

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzothioamide

(38c):

6.7. General Method G for the Synthesis of Substituted N-

(4-(benzo[d]thiazol-2-yl)phenyl)-3,4,5-

trimethoxybenzothioamide 36-38(a-d):

The compound 38c was synthesized by following general

procedure G, as a pale yellow solid; Yield 69%; mp 187-189

°C; MS (ESI): m/z 527 (M+H)⁺.

To a solution of substituted of N-(4-(benzo[d]thiazol-2-yl)

phenyl)-3,4,5-trimethoxybenzamide 33-35(a-d) (1g, 0.018

mol) in toluene, Lawesson’s reagent (0.9eq) was added and

reflux it for 8h. After cooling solvent was removed in

rotavapor and workedup with ethylacetate and water.

Separation of the organic layer and evaporation followed by

column chromatography gave yellow solids 36-38(a-d).

6.7.10.

3,4,5-Trimethoxy-N-(4-(5,6,7-

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzothioamide

(36d):

The compound 36d was synthesized by following general

procedure G, as a pale yellow solid; Yield 67%; mp 175-

176^°C;MS (ESI): m/z 527 (M+H)⁺.

6.7.1. 3,4,5-Trimethoxy-N-(4-(6-methoxybenzo[d]thiazol-2-

yl)phenyl)benzothioamide (36a):

6.7.11.

3,4,5-Trimethoxy-N-(2-methyl-4-(5,6,7-

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzothioamide

(37d):

The compound 36a was synthesized by following general

procedure G, as a pale yellow solid; Yield 68%; mp 206-210

°C; MS (ESI): m/z 467 (M+H)⁺.

The compound 37d was synthesized by following general

procedure G, as a pale yellow solid; Yield 72%; mp 199-200

°C; MS (ESI): m/z 541 (M+H)⁺.

6.7.2. 3,4,5-Trimethoxy-N-(4-(6-methoxybenzo[d]thiazol-2-

yl)-2-methylphenyl)benzothioamide (37a):

6.7.12.

3,4,5-Trimethoxy-N-(2-methoxy-4-(5,6,7-

The compound 37a was synthesized by following general

procedure G, as a pale yellow solid; Yield 66%; mp 212-214

°C; MS (ESI): m/z 481 (M+H)⁺.

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzothioamide

(38d):

The compound 38d was synthesized by following general

procedure G, as a pale yellow solid; Yield 69%; mp 211-213

°C; MS (ESI): m/z 557 (M+H)⁺.

6.7.3.

4-Methoxy-N-(2-methoxy-4-(5,6,7-

trimethoxybenzo[d]thiazol-2-yl)phenyl)benzothioamide

(38a):

6.8. General Method H for the Synthesis of Substituted

amidrazones 39-41(a-d):

To a stirred solution of substituted thioamides 36-38(a-d) (1

eq) diisolved in 20 mL of EtOH and NH₂NH₂·H₂O (5 eq) was

added slowly with vigorously stirring at room temperature for

16 h. The progress of the reaction was monitored by TLC for

completion. After completion of the reaction, the solvent and

excess hydrazine was evaporated under vacuum, and the syrup

was dissolved in CHCl₃(30 mL). The organic layer was

washed with water (1 x 10 mL) and brine (1 x 10 mL), and was

dried over Na₂SO₄. The organic layer was concentrated under

reduced pressure to afford 39-41(a-d) as yellow solids which

were directly used in the next step without purification.

(s, 6H); MS (ESI): m/z 505 (M+H)⁺; HRMS (ESI m/z) for

C₂₆H₂₅N₄O₅S calcd 505.15402, found 505.15324 (M+H)⁺.

6.9.4. 5-Fluoro-2-(4-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-

triazol-1-yl)phenyl)benzo[d]thiazole (5b):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

1

pale yellow solid. Mp: 182-184 °C; (138 mg), Yield: 81%; H

NMR (500 MHz, CDCl₃): δ 8.25 (s, 2H), 8.23 (d, J = 1.7 Hz,

1H), 8.04 (dd, J = 8.9, 4.8 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H),

7.62 (dd, J = 8.0, 2.5 Hz, 1H), 7.29 – 7.24 (m, 1H), 7.16 (s,

2H), 3.97 (s, 6H), 3.91 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ

165.82, 161.72, 159.76, 153.79, 150.75, 148.68, 138.54,

136.21, 136.12, 133.64, 128.88, 125.53, 124.47, 124.40,

120.66, 117.06, 115.52, 115.33, 108.12, 107.90, 103.14, 61.04,

56.34; MS (ESI): m/z 463 (M+H)⁺; HRMS (ESI m/z) for

C₂₄H₂₀N₄O₃FS calcd 463.12347, found 463.12304 (M+H)⁺.

6.9. General procedure I for Synthesis of 1, 4-di substituted

1,2,3-triazoles 5-7(a-d):

To stirred solution of 0.05 mmol of catalyst DBU in DMSO

(1.0 mL), was added 0.75 mmol of 2-phenyl benzothiazole

azides 28-30(a-d) and 0.5 mmol of 3,4,5-tri methoxyphenyl

acetaldehyde (31) and the reaction mixture was stirred at 25 °C

for 1 h. The crude reaction mixture was worked up with

aqueous NH₄Cl solution and the aqueous layer was extracted

with dichloromethane (2 x 20 mL). The combined organic

layers were dried (Na₂SO₄), filtered and concentrated. Pure

organo-click products 5-7(a-d) were obtained by column

chromatography (silica gel, mixture of hexane/ethyl acetate).

6.9.5. 6-Fluoro-2-(3-methyl-4-(4-(3,4,5-trimethoxyphenyl)-

1H-1,2,3-triazol-1-yl)phenyl)benzo[d]thiazole (6b):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

yellow solid. Mp: 153-155 °C; (140 mg), Yield: 84%; ¹H NMR

(500 MHz, CDCl₃): δ 8.13 (s, 1H), 8.05 (dd, J = 8.8, 4.7 Hz,

1H), 8.02 (d, J = 1.8 Hz, 1H), 8.00 (s, 1H), 7.63 (dd, J = 8.0,

2.5 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.30 – 7.25 (m, 1H),

7.17 (s, 2H), 3.96 (s, 6H), 3.91 (s, 3H), 2.43 (s, 3H); ¹³C NMR

(125 MHz, CDCl₃): δ 165.94, 161.72, 159.76, 153.78, 150.71,

147.80, 138.40, 138.24, 136.27, 136.18, 134.62, 134.58,

130.35, 126.51, 125.90, 124.50, 124.43, 120.80, 115.51,

115.32, 108.11, 107.89, 103.06, 61.02, 56.31, 18.33; MS

(ESI): m/z 477 (M+H)⁺; HRMS (ESI m/z) for C₂₅H₂₂N₄O₃FS

calcd 477.13912, found 477.13924 (M+H)⁺.

6.9.1.

5-Methoxy-2-(4-(4-(3,4,5-trimethoxyphenyl)-1H-

1,2,3-triazol-1-yl)phenyl)benzo[d]thiazole (5a):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

1

pale yellow solid. Mp: 152-155 °C; (134 mg), Yield: 80%; H

NMR (300 MHz, CDCl₃): δ 8.23 (d, J = 2.9 Hz, 2H), 8.19 (s,

1H), 7.95 (dd, J = 13.7, 8.8 Hz, 3H), 7.37 (d, J = 2.4 Hz, 1H),

7.16 (s, 2H), 7.12 (dd, J = 9.0, 2.5 Hz, 1H), 3.97 (s, 6H), 3.91

(s, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 163.49, 158.11,

153.75, 148.63, 148.57, 138.47, 138.07, 136.59, 134.01,

128.53, 125.59, 123.93, 120.50, 117.08, 116.08, 104.13,

103.11, 61.01, 56.31, 55.83; MS (ESI): m/z 475 (M+H)⁺;

HRMS (ESI m/z) for C₂₅H₂₃N₄O₄S calcd 475.14345, found

475.14310 (M+H)⁺.

6.9.6.

6-Fluoro-2-(3-methoxy-4-(4-(3,4,5-

trimethoxyphenyl)-1H-1,2,3-triazol-1-

yl)phenyl)benzo[d]thiazole (7b):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

white solid. Mp: 139-1141 °C; (124 mg), Yield: 76%; ¹H NMR

(300 MHz, CDCl₃): δ 8.40 (s, 1H), 8.09 – 7.99 (m, 2H), 7.94

(s, 1H), 7.72 (dd, J = 8.3, 1.6 Hz, 1H), 7.63 (dd, J = 8.0, 2.4

Hz, 1H), 7.33 – 7.23 (m, 2H), 7.16 (s, 2H), 4.11 (s, 3H), 3.97

(s, 6H), 3.91 (s, 3H); ¹³C NMR (75 MHz, CDCl₃+DMSO): δ

168.77, 162.95, 159.35, 158.81, 158.27, 153.72, 151.86,

145.86, 145.60, 138.92, 133.23, 127.55, 126.13, 123.82,

121.70, 121.29, 120.44, 116.65, 112.86, 111.53, 109.07,

103.78, 60.79, 56.57, 55.96; MS (ESI): m/z 493 (M+H)⁺;

HRMS (ESI m/z) for C₂₅H₂₂N₄O₄FS calcd 493.13403, found

463.13327 (M+H)⁺.

6.9.2.

6-Methoxy-2-(3-methyl-4-(4-(3,4,5-

trimethoxyphenyl)-1H-1,2,3-triazol-1-

yl)phenyl)benzo[d]thiazole (6a):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

1

pale yellow solid. Mp: 145-147 °C; (135 mg), Yield: 82%; H

NMR (300 MHz, CDCl₃): δ 8.25 – 8.19 (m, 1H), 8.11 (s, 1H),

8.03 – 7.90 (m, 3H), 7.52 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 2.1

Hz, 1H), 7.16 (d, J = 6.3 Hz, 2H), 7.15 – 7.10 (m, 1H), 3.96 (s,

6H), 3.91 (s, 6H), 2.41 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ

163.64, 158.15, 153.79, 148.66, 147.77, 136.69, 135.09,

134.53, 130.10, 128.61, 126.47, 125.70, 124.03, 120.80,

120.61, 117.08, 116.15, 104.16, 103.07, 61.04, 56.33, 55.88,

18.29; MS (ESI): m/z 489 (M+H)⁺; HRMS (ESI m/z) for

C₂₆H₂₅N₄O₄S calcd 489.15910, found 489.15912 (M+H)⁺.

6.9.7. 5,7-Dimethoxy-2-(4-(4-(3,4,5-trimethoxyphenyl)-1H-

1,2,3-triazol-1-yl)phenyl)benzo[d]thiazole (5c):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

1

white solid. Mp: 138-141 °C; (127 mg), Yield: 79%; H NMR

6.9.3.

6-Methoxy-2-(3-methoxy-4-(4-(3,4,5-

(500 MHz, CDCl₃): δ 8.25 (s, 1H), 8.23 (s, 2H), 7.93 (d, J =

8.6 Hz, 2H), 7.20 (d, J = 1.9 Hz, 1H), 7.15 (s, 2H), 6.53 (d, J =

1.9 Hz, 1H), 3.97 (d, J = 4.3 Hz, 9H), 3.91 (d, J = 3.3 Hz, 6H);

¹³C NMR (125 MHz, CDCl₃): δ 167.23, 160.63, 155.76,

154.36, 153.74, 148.56, 138.46, 138.28, 133.94, 128.65,

125.57, 120.48, 117.05, 116.43, 103.09, 97.59, 97.31, 61.01,

56.30, 56.01, 55.82; MS (ESI): m/z 505 (M+H)⁺; HRMS (ESI

m/z) for C₂₆H₂₅N₄O₅S calcd 505.15402, found 505.15364

(M+H)⁺.

trimethoxyphenyl)-1H-1,2,3-triazol-1-

yl)phenyl)benzo[d]thiazole (7a):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

pale yellow solid. Mp: 191-194 °C; (125 mg), Yield: 78%; H

NMR (300 MHz, CDCl₃): δ 8.22 (s, 1H), 7.78 (d, J = 9.0 Hz,

1H), 7.53 (s, 1H), 7.41 (dd, J = 8.1, 1.4 Hz, 1H), 7.29 (d, J =

2.3 Hz, 1H), 7.13 (d, J = 8.1 Hz, 1H), 7.02 (dd, J = 9.0, 2.4 Hz,

1H), 6.75 (s, 2H), 3.93 (s, 3H), 3.86 (d, J = 2.6 Hz, 6H), 3.68

1

6.9.8.

5,7-Dimethoxy-2-(3-methyl-4-(4-(3,4,5-

white off solid. Mp: 166-169 °C; (119 mg), Yield: 79%; H

trimethoxyphenyl)-1H-1,2,3-triazol-1-

yl)phenyl)benzo[d]thiazole (6c):

NMR (300 MHz, CDCl₃): δ 8.39 (s, 1H), 8.01 (d, J = 8.3 Hz,

1H), 7.90 (d, J = 1.5 Hz, 1H), 7.73 (dd, J = 8.3, 1.7 Hz, 1H),

7.38 (s, 1H), 7.16 (s, 2H), 4.13 (s, 3H), 4.09 (s, 3H), 3.97 (t, J

= 2.9 Hz, 12H), 3.91 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ

165.88, 154.26, 153.70, 150.41, 148.54, 146.70, 140.03,

138.35, 138.17, 133.80, 128.49, 125.56, 120.49, 117.12,

102.98, 100.69, 61.50, 60.98, 60.62, 56.28, 56.24; MS (ESI):

m/z 565 (M+H)⁺; HRMS (ESI m/z) for C₂₈H₂₉N₄O₇S calcd

565.17515, found 565.17533 (M+H)⁺.

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

white off solid. Mp: 163-167 °C; (120 mg), Yield: 76%; H

1

NMR (500 MHz, CDCl₃): δ 8.21 (s, 1H), 8.12 (dd, J = 8.2, 1.8

Hz, 1H), 8.01 (d, J = 7.0 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H),

7.27 (s, 1H), 7.17 (s, 2H), 6.62 (s, 1H), 4.10 (s, 3H), 3.97 (s,

6H), 3.91 (s, 2H), 2.44 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ

178.08, 172.78, 163.03, 160.53, 155.66, 154.26, 153.77,

152.62, 147.75, 146.46, 139.60, 133.24, 130.12, 128.05,

126.32, 125.74, 121.11, 120.90, 109.82, 104.47, 103.08, 97.60,

97.37, 61.01, 57.02, 56.30, 56.03, 55.84, 18.30; MS (ESI): m/z

519 (M+H)⁺; HRMS (ESI m/z) for C₂₇H₂₇N₄O₅S calcd

519.16967, found 519.16994 (M+H)⁺.

6.10. General Method J for the Synthesis of Substituted

1,2,4-Triazole 8-10(a-d):

The amidrazones 39-41(a-d) (1 mmol) was dissolved in

Ethanol (10 mL) and trimethylorthoformate (5 mmol) was

added at room temperature. Several drops of sulfuric acid were

added as catalyst, and the solution was stirred vigorously at

refluxing conditions for 3 h. After neutralization, the alcohol

was evaporated and the triazole was purified by column

chromatography (CHCl₃/MeOH) solvent system to obtain

respective triazoles 8-10(a-d) as a yellow solids.

6.9.9.

5,7-Dimethoxy-2-(3-methoxy-4-(4-(3,4,5-

trimethoxyphenyl)-1H-1,2,3-triazol-1-

yl)phenyl)benzo[d]thiazole (7c):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

white solid. Mp: 120-122 °C; (112 mg), Yield: 72%; H NMR

6.10.1.

6-Methoxy-2-(4-(3-(3,4,5-trimethoxyphenyl)-4H-

1

1,2,4-triazol-4-yl)phenyl)benzo[d]thiazole (8a):

(300 MHz, CDCl₃): δ 8.15 (s, 1H), 8.04 (d, J = 8.1 Hz, 1H),

8.00 (s, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H),

7.17 (s, 2H), 6.55 (d, J = 2.0 Hz, 1H), 3.98 (d, J = 7.0 Hz,

10H), 3.92 (d, J = 3.9 Hz, 7H), 2.41 (s, 3H); MS (ESI): m/z

535 (M+H)⁺; HRMS (ESI m/z) for C₂₇H₂₇N₄O₆S calcd

535.16458, found 535.16487 (M+H)⁺.

This compound was prepared according to the method J.

(154mg), Yield: 75%; White yellow solid, mp: 88-92 °C; H

1

NMR (300 MHz, CDCl₃): δ 8.40 (s, 1H), 8.19 (d, J = 8.4 Hz,

2H), 7.99 (d, J = 9.0 Hz, 1H), 7.41 (d, J = 8.6 Hz, 3H), 7.15

(dd, J = 9.0, 2.3 Hz, 1H), 6.73 (s, 2H), 3.93 (s, 3H), 3.87 (s,

3H), 3.67 (s, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 162.78,

158.28, 153.22, 148.55, 144.38, 139.50, 136.65, 136.04,

134.87, 128.48, 126.45, 124.08, 121.05, 116.28, 105.93,

104.10, 60.92, 56.00, 55.84; MS (ESI): m/z 475 (M+H)⁺;

HRMS (ESI m/z) for C₂₅H₂₃N₄O₄S calcd 475.14345, found

475.14122 (M+H)⁺.

6.9.10. 5,6,7-Trimethoxy-2-(4-(4-(3,4,5-trimethoxyphenyl)-

1H-1,2,3-triazol-1-yl)phenyl)benzo[d]thiazole (5d):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

1

white off solid. Mp: 151-154 °C; (120 mg), Yield: 77%; H

NMR (500 MHz, CDCl₃): δ 8.25 (s, 1H), 8.22 (d, J = 8.4 Hz,

2H), 7.94 (d, J = 8.4 Hz, 2H), 7.37 (s, 1H), 7.16 (s, 2H), 4.12

(s, 3H), 3.97 (t, J = 4.4 Hz, 12H), 3.91 (s, 3H); ¹³C NMR (75

MHz, CDCl₃): δ 165.88, 154.26, 153.70, 150.41, 148.54,

146.70, 140.03, 138.35, 138.17, 133.80, 128.49, 125.56,

120.49, 117.12, 102.99, 100.69, 61.50, 60.98, 60.62, 56.28,

56.24; MS (ESI): m/z 535 (M+H)⁺; HRMS (ESI m/z) for

C₂₇H₂₇N₄O₆S calcd 535.16458, found 535.16416 (M+H)⁺.

6.10.2.

6-Methoxy-2-(3-methyl-4-(3-(3,4,5-

trimethoxyphenyl)-4H-1,2,4-triazol-4-

yl)phenyl)benzo[d]thiazole (9a):

This compound was prepared according to the method J.

(152mg), Yield: 74%; White yellow solid, mp: 96-100 °C; H

1

NMR (500 MHz, CDCl₃): δ 8.28 (s, 1H), 8.08 (s, 1H), 8.04 (d,

J = 8.1 Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.44 (d, J = 8.1 Hz,

1H), 7.39 (d, J = 2.3 Hz, 1H), 7.15 (dd, J = 9.0, 2.4 Hz, 1H),

6.75 (s, 1H), 3.92 (s, 3H), 3.83 (s, 3H), 3.62 (s, 6H), 2.07 (s,

3H); ¹³C NMR (125 MHz, CDCl₃): δ 162.96, 158.32, 153.24,

148.58, 144.52, 139.49, 136.69, 136.36, 135.66, 135.55,

129.98, 128.15, 126.13, 124.09, 121.49, 116.32, 104.77,

104.15, 60.91, 55.93, 55.89, 17.67; MS (ESI): m/z 489

(M+H)⁺; HRMS (ESI m/z) for C₂₆H₂₅N₄O₄S calcd 489.15910,

found 489.15642 (M+H)⁺.

6.9.11.

5,6,7-Trimethoxy-2-(3-methyl-4-(4-(3,4,5-

trimethoxyphenyl)-1H-1,2,3-triazol-1-

yl)phenyl)benzo[d]thiazole (6d):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

1

white off solid. Mp: 174-177 °C; (124 mg), Yield: 81%; H

NMR (500 MHz, CDCl₃): δ 8.12 (s, 1H), 8.02 (d, J = 8.2 Hz,

1H), 8.00 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.39 (s, 1H), 7.17

(s, 2H), 4.13 (s, 3H), 3.98 (s, 3H), 3.96 (s, 9H), 3.91 (s, 3H),

2.41 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 166.01, 154.28,

153.72, 150.38, 147.71, 146.72, 140.09, 138.29, 137.91,

134.83, 134.49, 130.04, 126.43, 125.72, 125.60, 120.82,

120.60, 102.97, 100.73, 61.51, 60.98, 60.63, 56.29, 56.25,

18.26; MS (ESI): m/z 535 (M+H)⁺; HRMS (ESI m/z) for

C₂₇H₂₇N₄O₆S calcd 535.16458, found 535.16416 (M+H)⁺.

6.10.3.

5-Methoxy-2-(3-methoxy-4-(3-(3,4,5-

trimethoxyphenyl)-4H-1,2,4-triazol-4-

yl)phenyl)benzo[d]thiazole (10a):

This compound was prepared according to the method J.

(152mg), Yield: 74%; White yellow solid, mp: 96-100 °C; H

1

NMR (300 MHz, CDCl₃): δ 8.29 (s, 1H), 7.98 (d, J = 9.0 Hz,

1H), 7.85 (s, 1H), 7.63 (dd, J = 8.1, 1.4 Hz, 1H), 7.38 (d, J =

2.3 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.14 (dd, J = 9.0, 2.4 Hz,

1H), 6.75 (s, 2H), 3.92 (s, 3H), 3.84 (d, J = 2.6 Hz, 6H), 3.66

(s, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 163.07, 158.33,

154.59, 153.10, 148.47, 139.31, 136.72, 136.35, 135.64,

135.55, 129.78, 128.45, 126.38, 125.25, 121.89, 116.32,

110.34, 104.77, 104.13, 60.91, 56.20, 55.97, 55.88; MS (ESI):

6.9.12.

5,6,7-Trimethoxy-2-(3-methoxy-4-(4-(3,4,5-

trimethoxyphenyl)-1H-1,2,3-triazol-1-

yl)phenyl)benzo[d]thiazole (7d):

Prepared following the procedure I and purified by column

chromatography using EtOAc/hexane and was isolated as a

m/z 505 (M+H)⁺; HRMS (ESI m/z) for C₂₆H₂₅N₄O₅S calcd

This compound was prepared according to the method J. (132

;

505.15402, found 505.15154 (M+H)⁺.

mg), Yield: 64%; White yellow solid, mp: 188-190 °C ¹H

NMR (500 MHz, CDCl₃): δ 8.28 (s, 1H), 8.12 (s, 1H), 8.06

(dd, J = 8.2, 1.5 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.20 (d, J =

1.9 Hz, 1H), 6.75 (s, 2H), 6.55 (d, J = 1.9 Hz, 1H), 3.98 (s,

3H), 3.92 (s, 3H), 3.83 (s, 3H), 3.62 (s, 6H), 2.07 (s, 3H); ¹³C

NMR (125 MHz, CDCl₃): δ 166.74, 160.78, 155.73, 154.40,

153.24, 152.93, 144.48, 139.46, 136.40, 135.92, 135.51,

130.09, 128.20, 126.23, 121.45, 116.57, 104.73, 97.57, 60.92,

56.07, 55.91, 17.69; MS (ESI): m/z 519 (M+H)⁺; HRMS (ESI

6.10.4. 6-Fluoro-2-(4-(3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-

triazol-4-yl)phenyl)benzo[d]thiazole (8b):

This compound was prepared according to the method J. (145

1

mg), Yield: 70%; White Yellow solid, mp: 120-122 °C; H

NMR (300 MHz, CDCl₃): δ 8.41 (s, 1H), 8.22 (d, J = 8.5 Hz,

2H), 8.06 (dd, J = 9.0, 4.8 Hz, 1H), 7.64 (dd, J = 8.0, 2.4 Hz,

1H), 7.44 (d, J = 8.5 Hz, 2H), 7.34 – 7.24 (m, 1H), 6.73 (s,

2H), 3.87 (s, 3H), 3.67 (s, 6H); ¹³C NMR (75 MHz, CDCl₃): δ

165.16, 165.12, 162.46, 159.18, 153.26, 152.93, 150.69,

150.67, 144.34, 139.58, 136.57, 136.28, 136.13, 134.42,

128.76, 126.53, 124.66, 124.54, 121.04, 115.73, 115.40,

108.20, 107.84, 105.99, 60.93, 56.01; MS (ESI): m/z 463

(M+H)⁺; HRMS (ESI m/z) for C₂₄H₂₀N₄O₃FS calcd 463.12347,

found 463.12012 (M+H)⁺.

m/z)

for

C₂₇H₂₇N₄O₄S

calcd

519.16967,

found

519.16720(M+H)⁺.

6.10.9.

5,7-Dimethoxy-2-(3-methoxy-4-(3-(3,4,5-

trimethoxyphenyl)-4H-1,2,4-triazol-4-

yl)phenyl)benzo[d]thiazole (10c):

This compound was prepared according to the method J. (132

;

mg), Yield: 64%; White yellow solid, mp: 188-190 °C ¹H

6.10.5. 6-Fluoro-2-(3-methyl-4-(3-(3,4,5-trimethoxyphenyl)-

4H-1,2,4-triazol-4-yl)phenyl)benzo[d]thiazole (9b):

NMR (500 MHz, CDCl₃) δ 8.30 (s, 1H), 7.86 (d, J = 1.5 Hz,

1H), 7.68 (dd, J = 8.1, 1.7 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H),

7.20 (d, J = 1.9 Hz, 1H), 6.75 (s, 2H), 6.55 (d, J = 1.9 Hz, 1H),

3.98 (s, 3H), 3.92 (s, 3H), 3.83 (s, 6H), 3.65 (s, 6H); ¹³C NMR

(75 MHz, CDCl₃): δ 166.82, 160.73, 155.59, 154.53, 154.35,

153.07, 139.21, 136.56, 128.64, 125.48, 121.86, 120.03,

116.55, 110.43, 105.01, 97.52, 60.91, 56.16, 56.04, 55.92,

55.83; MS (ESI): m/z 535 (M+H)⁺; HRMS (ESI m/z) for

C₂₇H₂₇N₄O₆S calcd 535.16458, found 535.16216(M+H)⁺.

This compound was prepared according to the method J. (148

1

mg), Yield: 72%; White yellow solid, mp: 199-200 °C; H

NMR (500 MHz, CDCl₃): δ 8.28 (s, 1H), 8.10 (s, 1H), 8.07 –

8.03 (m, 2H), 7.63 (dd, J = 8.0, 2.5 Hz, 1H), 7.47 (d, J = 8.1

Hz, 1H), 7.31 – 7.26 (m, 1H), 6.75 (s, 2H), 3.83 (s, 3H), 3.63

(s, 6H), 2.09 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 165.31,

161.83, 159.85, 153.27, 152.96, 150.68, 144.41, 139.54,

136.54, 136.15, 135.12, 130.27, 128.28, 126.38, 124.63,

124.56, 121.41, 115.70, 115.51, 108.17, 107.95, 104.81, 60.93,

55.94, 17.72; MS (ESI): m/z 477 (M+H)⁺; HRMS (ESI m/z) for

C₂₅H₂₂N₄O₃FS calcd 477.13912, found 477.13675 (M+H)⁺.

6.10.10. 5,6,7-Trimethoxy-2-(4-(3-(3,4,5-trimethoxyphenyl)-

4H-1,2,4-triazol-4-yl)phenyl)benzo[d]thiazole (8d):

This compound was prepared according to the method J.

(142mg), Yield: 69%; White yellow solid, mp: 162-164 °C; ¹H

NMR (500 MHz, CDCl₃): δ 8.39 (s, 1H), 8.19 (d, J = 8.4 Hz,

2H), 7.41 (d, J = 8.4 Hz, 2H), 7.37 (s, 1H), 6.72 (s, 2H), 4.12

(s, 3H), 3.97 (s, 3H), 3.86 (s, 6H), 3.66 (s, 6H); ¹³C NMR (75

MHz, CDCl₃): δ 165.21, 154.41, 153.20, 152.90, 150.33,

146.71, 144.41, 144.37, 139.45, 136.21, 134.73, 128.47,

126.51, 121.04, 120.68, 105.86, 100.75, 61.52, 60.93, 60.68,

56.32, 55.98; MS (ESI): m/z 535 (M+H)⁺; HRMS (ESI m/z) for

C₂₇H₂₇N₄O₆S calcd 535.16458, found 535.16226 (M+H)⁺.

6.10.6.

5-Fluoro-2-(3-methoxy-4-(3-(3,4,5-

trimethoxyphenyl)-4H-1,2,4-triazol-4-

yl)phenyl)benzo[d]thiazole (10b):

This compound was prepared according to the method J. (148

1

mg), Yield: 72%; White yellow solid, mp: 199-200 °C; H

NMR (500 MHz, CDCl₃): δ 8.31 (s, 1H), 8.05 (dd, J = 9.0, 4.8

Hz, 1H), 7.86 (d, J = 1.7 Hz, 1H), 7.65 (dd, J = 8.1, 1.8 Hz,

1H), 7.62 (dd, J = 8.0, 2.5 Hz, 1H), 7.36 – 7.33 (m, 1H), 7.31 –

7.28 (m, 1H), 6.75 (s, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.66 (s,

6H); ¹³C NMR (75 MHz, CDCl₃): δ 165.40, 162.48, 159.20,

154.64, 153.13, 150.56, 136.33, 136.19, 128.72, 125.70,

124.63, 124.51, 121.73, 120.26, 115.75, 115.42, 110.59,

108.20, 107.85, 105.22, 60.90, 56.23, 55.98; MS (ESI): m/z

493 (M+H)⁺; HRMS (ESI m/z) for C₂₅H₂₂N₄O₄FS calcd

493.13171, found 493.13403 (M+H)⁺.

6.10.11.

5,6,7-Trimethoxy-2-(3-methyl-4-(3-(3,4,5-

trimethoxyphenyl)-4H-1,2,4-triazol-4-

yl)phenyl)benzo[d]thiazole (9d):

This compound was prepared according to the method J. (139

1

mg), Yield: 68%; White yellow solid, mp: 104-106 °C; H

NMR (400 MHz, CDCl₃): δ 8.28 (s, 1H), 8.09 (s, 1H), 8.04

(dd, J = 8.2, 1.7 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.37 (s, 1H),

6.75 (s, 2H), 4.12 (s, 3H), 3.98 (s, 3H), 3.96 (s, 3H), 3.83 (s,

3H), 3.62 (s, 6H), 2.07 (s, 3H); ¹³C NMR (75 MHz, DMSO): δ

163.47, 153.37, 152.76, 152.45, 148.65, 137.49, 135.38,

135.19, 133.57, 123.92, 117.28, 115.44, 98.08, 96.99, 60.10,

55.67, 29.00; MS (ESI): m/z 549 (M+H)⁺; HRMS (ESI m/z) for

C₂₈H₂₉N₄O₆S calcd 549.18023, found 549.17997 (M+H)⁺.

6.10.7. 5,7-Dimethoxy-2-(4-(3-(3,4,5-trimethoxyphenyl)-4H-

1,2,4-triazol-4-yl)phenyl)benzo[d]thiazole (8c):

This compound was prepared according to the method J. (137

1

mg), Yield: 67%; White yellow solid, mp: 197-198 °C; H

NMR (300 MHz, CDCl₃): δ 8.42 (s, 1H), 8.21 (d, J = 8.4 Hz,

2H), 7.42 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 1.6 Hz, 1H), 6.72 (s,

2H), 6.55 (d, J = 1.7 Hz, 1H), 3.98 (s, 3H), 3.92 (s, 3H), 3.86

(s, 3H), 3.65 (s, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 166.51,

160.74, 155.73, 154.37, 153.23, 152.88, 144.37, 139.62,

136.26, 134.87, 128.59, 126.50, 120.91, 116.55, 106.01, 97.65,

97.55, 60.89, 56.00, 55.81; MS (ESI): m/z 505 (M+H)⁺; HRMS

(ESI m/z) for C₂₆H₂₅N₄O₅S calcd 505.15402, found 505.15196

(M+H)⁺.

6.10.12.

5,6,7-Trimethoxy-2-(3-methoxy-4-(3-(3,4,5-

trimethoxyphenyl)-4H-1,2,4-triazol-4-

yl)phenyl)benzo[d]thiazole (10d):

This compound was prepared according to the method J. (139

1

mg), Yield: 68%; White yellow solid, mp: 104-106 °C; H

NMR (300 MHz, CDCl₃): δ 8.30 (s, 1H), 7.83 (d, J = 1.4 Hz,

1H), 7.66 (dd, J = 8.1, 1.6 Hz, 1H), 7.41 – 7.30 (m, 2H), 6.76

(s, 2H), 4.13 (s, 3H), 3.98 (d, J = 6.5 Hz, 6H), 3.83 (d, J = 9.2

Hz, 6H), 3.66 (s, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 165.48,

154.56, 154.43, 153.05, 150.23, 146.69, 144.92, 140.25,

139.21, 136.46, 128.65, 125.37, 121.84, 120.71, 119.93,

6.10.8.

5,7-Dimethoxy-2-(3-methyl-4-(3-(3,4,5-

trimethoxyphenyl)-4H-1,2,4-triazol-4-

yl)phenyl)benzo[d]thiazole (9c):

110.29, 105.00, 100.72, 61.51, 60.89, 60.66, 56.32, 56.14,

55.91; MS (ESI): m/z 565 (M+H)⁺; HRMS (ESI m/z) for

C₂₈H₂₉O₇N₄S calcd 565.17515, found 565.16529 (M+H)⁺.

9.0, 4.8 Hz, 1H), 7.63 (dd, J = 8.0, 2.5 Hz, 1H), 7.62 – 7.59 (m,

2H), 7.31 – 7.28 (m, 1H), 6.81 (s, 2H), 3.90 (s, 3H), 3.70 (s,

6H); ¹³C NMR (125 MHz, CDCl₃): δ 165.07, 161.87, 159.90,

153.60, 150.70, 140.72, 136.33, 136.23, 135.24, 128.65,

126.07, 124.73, 124.66, 118.12, 115.75, 115.55, 108.17,

107.96, 106.37, 61.05, 56.20; MS (ESI): m/z 464 (M+H)⁺;

HRMS (ESI m/z) for C₂₃H₁₉N₅O₃FS calcd 464.11871, found

464.11628 (M+H)⁺.

6.11. General Method K for the Synthesis of Substituted

1,2,3,4-Triazole 11-13(a-d):

The amidrazones 39-41(a-d) was dissolved in 5 ml of AcOH

and NaNO₂(2 eq) was added portion wise slowly at room

temperature for 10 min., and the reaction mixture was stirred

for 4 h. The progress of the reaction was monitored by TLC for

completion. After completion of the reaction, neutralize with

saturated NaHCO₃solution, and extracted with ethyl acetate (3

x 15 mL). The combined extract was washed with water (1 x

10 mL) as well as brine (1x10 mL) and was dried over

anhydrous Na₂SO₄. The organic layer was concentrated under

reduced pressure and the crude product was purified by

column chromatography (silica gel; 60-120 mesh) using ethyl

acetate-hexane (4:6) as eluent to afford compound 11-13(a-d)

as a pale yellow solids.

6.11.5. 6-Fluoro-2-(3-methyl-4-(5-(3,4,5-trimethoxyphenyl)-

1H-tetrazol-1-yl)phenyl)benzo[d]thiazole (12b):

This compound was prepared according to the method K. (136

mg), Yield: 66%; Brown color solid, mp: 162-164 °C;¹H NMR

(300 MHz, CDCl₃): δ 8.17 (s, 1H), 8.12 – 8.03 (m, 2H), 7.64

(dd, J = 8.0, 2.5 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.30 (dd, J

= 8.9, 2.6 Hz, 1H), 6.84 (s, 2H), 3.86 (s, 3H), 3.65 (s, 6H), 2.11

(s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 153.82, 153.48, 150.63,

136.49, 136.02, 135.85, 130.21, 128.19, 126.31, 124.67,

124.55, 118.02, 115.75, 115.42, 108.19, 107.83, 105.39, 60.93,

55.98, 17.56; MS (ESI): m/z 478 (M+H)⁺; HRMS (ESI m/z) for

C₂₄H₂₁N₅O₃FS calcd 478.13436, found 478.13234 (M+H)⁺.

6.11.1.

6-Methoxy-2-(4-(5-(3,4,5-trimethoxyphenyl)-1H-

tetrazol-1-yl)phenyl)benzo[d]thiazole (11a):

6.11.6.

6-Fluoro-2-(3-methoxy-4-(5-(3,4,5-

This compound was prepared according to the method K. (145

mg), Yield: 71%; Brown color solid, mp: 170-175 °C; H

trimethoxyphenyl)-1H-tetrazol-1-

yl)phenyl)benzo[d]thiazole (13b):

1

NMR (300 MHz, CDCl₃): δ 8.23 (d, J = 8.5 Hz, 2H), 7.98 (d, J

= 9.0 Hz, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 2.3 Hz,

1H), 7.14 (dd, J = 9.0, 2.5 Hz, 1H), 6.81 (s, 2H), 3.92 (s, 3H),

3.89 (s, 3H), 3.70 (s, 6H); ¹³C NMR (75 MHz, CDCl₃): δ

162.65, 158.34, 153.54, 153.50, 148.59, 140.72, 136.72,

135.84, 135.69, 128.33, 125.97, 124.14, 120.19, 118.13,

116.31, 106.39, 104.11, 60.96, 56.15, 55.83; MS (ESI): m/z

476 (M+H)⁺; HRMS (ESI m/z) for C₂₄H₂₂N₅O₄S calcd

476.13870, found 476.13701 (M+H)⁺.

This compound was prepared according to the method K. (136

mg), Yield: 66%; Brown color solid, mp: 162-164 °C; H

1

NMR (500 MHz, CDCl₃): δ 8.06 (dd, J = 9.0, 4.8 Hz, 1H),

7.88 (d, J = 1.7 Hz, 1H), 7.77 – 7.75 (m, 1H), 7.63 (dd, J = 8.0,

2.5 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 8.9, 2.6 Hz,

1H), 6.86 (s, 2H), 3.86 (s, 3H), 3.78 (s, 3H), 3.68 (s, 6H); ¹³C

NMR (125 MHz, CDCl₃): δ 165.26, 161.89, 159.92, 154.56,

153.37, 150.59, 140.46, 137.31, 136.39, 136.30, 128.88,

125.78, 124.72, 124.64, 120.38, 118.93, 115.76, 115.57,

110.71, 108.17, 107.96, 105.26, 60.98, 56.31, 56.08; MS

(ESI): m/z 494 (M+H)⁺; HRMS (ESI m/z) for C₂₄H₂₁N₅O₄FS

calcd 494.12928, found 494.12721 (M+H)⁺.

6.11.2.

6-Methoxy-2-(3-methyl-4-(5-(3,4,5-

trimethoxyphenyl)-1H-tetrazol-1-

yl)phenyl)benzo[d]thiazole (12a):

This compound was prepared according to the method K. (136

mg), Yield: 66%; Brown color solid, mp: 160-164 °C; H

6.11.7. 5,7-Dimethoxy-2-(4-(5-(3,4,5-trimethoxyphenyl)-1H-

tetrazol-1-yl)phenyl)benzo[d]thiazole (11c):

1

NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H), 8.07 (dd, J = 8.2, 1.7

Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H),

7.39 (d, J = 2.5 Hz, 1H), 7.15 (dt, J = 4.9, 2.5 Hz, 1H), 6.84 (s,

2H), 3.92 (s, 3H), 3.86 (s, 3H), 3.65 (s, 6H), 2.10 (s, 3H); ¹³C

NMR (100 MHz, CDCl₃): δ 158.80, 154.34, 149.83, 149.48,

144.54, 136.62, 132.73, 132.33, 131.54, 125.95, 124.08,

122.10, 120.13, 114.10, 112.38, 101.35, 100.08, 56.97, 52.00,

51.87, 13.57; MS (ESI): m/z 490 (M+H)⁺; HRMS (ESI m/z) for

C₂₅H₂₄N₅O₄S calcd 490.15435, found 490.15241 (M+H)⁺.

This compound was prepared according to the method K. (135

mg), Yield: 66%; Brown color solid, mp: 191- 195 °C; H

1

NMR (300 MHz, CDCl₃): δ 8.26 (d, J = 8.6 Hz, 2H), 7.59 (d, J

= 8.6 Hz, 2H), 7.20 (d, J = 1.9 Hz, 1H), 6.81 (s, 2H), 6.55 (d, J

= 1.9 Hz, 1H), 3.99 (s, 3H), 3.92 (s, 3H), 3.89 (s, 3H), 3.70 (s,

6H); ¹³C NMR (75 MHz, CDCl₃): δ 166.41, 160.74, 155.74,

154.37, 153.54, 153.49, 140.70, 136.09, 135.64, 128.44,

126.00, 118.10, 116.63, 106.36, 97.66, 97.59, 60.97, 56.14,

56.01, 55.80; MS (ESI): m/z 506 (M+H)⁺; HRMS (ESI m/z) for

C₂₅H₂₄N₅O₅S calcd 506.14927, found 506.14715 (M+H)⁺.

6.11.3.

6-Methoxy-2-(3-methoxy-4-(5-(3,4,5-

trimethoxyphenyl)-1H-tetrazol-1-

yl)phenyl)benzo[d]thiazole (13a):

6.11.8.

5,7-Dimethoxy-2-(3-methyl-4-(5-(3,4,5-

trimethoxyphenyl)-1H-tetrazol-1-

yl)phenyl)benzo[d]thiazole (12c):

Prepared following the procedure K and purified by column

chromatography using EtOAc/hexane and was isolated as a

Brown colour solid. (104 mg), Yield: 68%; Mp: 172-175 °C;

¹H NMR (300 MHz, CDCl₃): δ 8.26 (d, J = 8.6 Hz, 2H), 7.59

(d, J = 8.6 Hz, 2H), 7.20 (d, J = 1.9 Hz, 1H), 6.81 (s, 2H), 6.55

(d, J = 1.9 Hz, 1H), 3.99 (s, 3H), 3.92 (s, 3H), 3.89 (s, 3H),

3.70 (s, 6H). MS (ESI): m/z 506 (M+H)⁺; HRMS (ESI m/z) for

C₂₅H₂₄N₅O₅S calcd 506.14856, found 506.14678 (M+H)⁺.

This compound was prepared according to the method K. (137

mg), Yield: 67%; Brown color solid, mp: 210-213 °C; H

1

NMR (500 MHz, CDCl₃): δ 8.19 (d, J = 8.4 Hz, 1H), 8.00 (s,

1H), 7.94 (d, J = 8.4 Hz, 1H), 7.74 (s, 1H),7.12 (s, 1H), 6.50 (s,

2H), 3.97 (d, J = 0.6 Hz, 3H), 3.95 (d, J = 1.1 Hz, 6H), 3.93 (d,

J = 0.7 Hz, 3H), 3.90 (d, J = 0.7 Hz, 3H), 2.43 (s, 3H); ¹³C

NMR (75 MHz, CDCl₃): δ 168.75, 165.42, 160.38, 155.75,

154.22, 153.40, 141.40, 138.33, 130.58, 130.15, 129.16,

129.01, 126.21, 122.59, 106.37, 104.52, 97.35, 96.79, 60.99,

56.36, 55.94, 55.76, 17.83. MS (ESI): m/z 520 (M+H)⁺; HRMS

(ESI m/z) for C₂₆H₂₆N₅O₅S calcd 520.15867, found 520.15778

(M+H)⁺.

6.11.4.

6-Fluoro-2-(4-(5-(3,4,5-trimethoxyphenyl)-1H-

tetrazol-1-yl)phenyl)benzo[d]thiazole (11b):

This compound was prepared according to the method K. (134

1

mg), Yield: 65%; Brown color solid, mp: 195-197 °C; H

NMR (500 MHz, CDCl₃): δ 8.27 – 8.24 (m, 2H), 8.05 (dd, J =

6.11.9.

5,7-Dimethoxy-2-(3-methoxy-4-(5-(3,4,5-

incubator. After incubation cells were treated with test

compounds for 48 h. After 48 h of incubation, 10 µl MTT (3-

(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide)

(5 mg/mL) was added to each well and the plates were further

incubated for 4 h. Then the supernatant from each well was

carefully removed, formazon crystals were dissolved in 100 µl

of DMSO and absorbance at 570 nm wavelength was recorded.

trimethoxyphenyl)-1H-tetrazol-1-

yl)phenyl)benzo[d]thiazole (13c):

This compound was prepared according to the method K. (137

mg), Yield: 67%; Brown color solid, mp: 210-213 °C; H

1

NMR (500 MHz, CDCl₃): δ 8.61 (d, J = 8.4 Hz, 1H), 8.57 (s,

1H), 7.74 (d, J = 1.7 Hz, 1H), 7.67 (dd, J = 8.4, 1.8 Hz, 1H),

7.17 (d, J = 2.0 Hz, 1H), 7.13 (s, 2H), 6.50 (d, J = 2.0 Hz, 1H),

4.06 (s, 3H), 3.96 (d, J = 3.6 Hz, 9H), 3.93 (s, 3H), 3.90 (s,

3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.88, 165.05, 160.35,

155.68, 154.26, 153.33, 148.19, 141.36, 130.40, 130.18,

129.23, 121.12, 119.49, 108.05, 104.57, 97.33, 96.74, 60.97,

56.40, 56.23, 55.92, 55.76. MS (ESI): m/z 536 (M+H)⁺; HRMS

(ESI m/z) for C₂₆H₂₆N₅O₅S calcd 536.15455, found 536.15326

(M+H)⁺.

7.2. Cell cycle analysis

Flow cytometric analysis (FACS) was performed to evaluate

the distribution of the cells through the cell-cycle phases. A549

cells were incubated for 48 h with compounds 9a and 9b at

concentrations of 50 and 100 nM. Untreated and treated cells

were harvested, washed with phosphate-buffered saline (PBS),

fixed in ice-cold 70% ethanol, and stained with propidium

iodide (Sigma–Aldrich). Cell-cycle analysis was performed by

flow cytometry (Becton Dickinson FACS Caliber instrument)

6.11.10. 5,6,7-Trimethoxy-2-(4-(5-(3,4,5-trimethoxyphenyl)-

1H-tetrazol-1-yl)phenyl)benzo[d]thiazole (11d):

56

.

This compound was prepared according to the method K. (138

mg), Yield: 67%; Brown color solid, mp: 150-152 °C; H

7.3. Tubulin polymerization assay

1

A fluorescence based in vitro tubulin polymerization assay was

performed according to the manufacturer’s protocol (BK011,

Cytoskeleton, Inc.). Briefly, the reaction mixture in a total

volume of 10 µl contained PEM buffer, GTP (1 µM) in the

presence or absence of test compounds. Tubulin

polymerization was followed by a time dependent increase in

fluorescence due to the incorporation of a fluorescence reporter

into microtubules as polymerization proceeds. Fluorescence

emission at 420 nm (excitation wavelength is 360 nm) was

measured by using a Varioscan multimode plate reader

(Thermo scientific Inc.). CA-4 was used as reference

compound and to determine the IC₅₀values of the compounds

against tubulin polymerization, the compounds were pre-

incubated with tubulin at varying concentrations (0.5, 1, 2, 3, 5

µM). Assays were performed under similar conditions as

employed for polymerization assays as described above. ⁵⁷

NMR (500 MHz, CDCl₃): δ 8.24 (d, J = 8.5 Hz, 2H), 7.58 (d, J

= 8.5 Hz, 2H), 7.37 (s, 1H), 6.81 (s, 2H), 4.12 (s, 3H), 3.98 (s,

3H), 3.96 (s, 3H), 3.89 (s, 3H), 3.70 (s, 6H); ¹³C NMR (75

MHz, CDCl₃): δ 165.10, 154.45, 153.52, 150.35, 146.71,

140.65, 140.31, 135.98, 135.54, 128.33, 126.05, 120.77,

120.21, 118.09, 106.32, 100.83, 61.50, 60.98, 60.66, 56.32,

56.13; MS (ESI): m/z 536 (M+H)⁺; HRMS (ESI m/z) for

C₂₆H₂₆N₅O₆S calcd 536.15983, found 536.15782 (M+H)⁺.

6.11.11.

5,6,7-Trimethoxy-2-(3-methyl-4-(5-(3,4,5-

trimethoxyphenyl)-1H-tetrazol-1-

yl)phenyl)benzo[d]thiazole (12d):

This compound was prepared according to the method K. (141

1

mg), Yield: 69%; Brown color solid, mp: 200-202 °C; H

NMR (500 MHz, CDCl₃): δ 8.20 (d, J = 8.6 Hz, 1H), 7.98 (s,

1H), 7.92 (dd, J = 8.4, 2.0 Hz, 1H), 7.35 (s, 1H), 7.12 (s, 2H),

3.96 (s, 3H), 3.95 (s, 9H), 3.95 (s, 3H), 3.93 (s, 3H), 2.44 (s,

3H); ¹³C NMR (100 MHz, CDCl₃): δ 161.39, 150.44, 149.22,

148.89, 146.32, 142.73, 140.43, 136.25, 135.44, 132.40,

131.76, 131.39, 125.96, 124.19, 122.09, 117.36, 116.69,

100.69, 96.75, 57.53, 56.90, 56.68, 52.33, 51.88, 13.68; MS

(ESI): m/z 550 (M+H)⁺; HRMS (ESI m/z) for C₂₇H₂₈N₅O₆S

calcd 550.16923, found 550.16784 (M+H)⁺.

7.4. Immunocytochemistry

A549 cells were seeded on a glass cover slip, incubated for 48

h in the presence or absence of test compounds 9a, 9b and CA-

4 at a concentration of 50 nM. Cells grown on coverslips were

fixed in 3.5% formaldehyde in phosphate-buffered saline

(PBS) pH 7.4 for 10 minutes at room temperature. Cells were

permeabilized for 6 minutes in PBS containing 0.5% Triton X-

100 (Sigma) and 0.05% Tween-20 (Sigma). The permeabilized

cells were blocked with 2% BSA (Sigma) in PBS for 1 h.

Later, the cells were incubated with the primary antibody for

tubulin from Sigma at 1:200 diluted in blocking solution for 4

h at room temperature. Subsequently the antibodies were

removed and the cells were washed thrice with PBS. Cells

were then incubated with the FITC labeled anti-mouse

secondary antibody (1: 500) for 1 h at room temperature. Cells

were washed thrice with PBS and mounted in medium

containing DAPI. Images were captured using an Olympus

confocal microscope and analyzed with Provision software.

6.11.12.

5,6,7-Trimethoxy-2-(3-methoxy-4-(5-(3,4,5-

trimethoxyphenyl)-1H-tetrazol-1-

yl)phenyl)benzo[d]thiazole (13d):

This compound was prepared according to the method K. (141

1

mg), Yield: 69%; Brown color solid, mp: 200-202 °C; H

NMR (300 MHz, CDCl₃): δ 7.86 (d, J = 1.4 Hz, 1H), 7.77 (dd,

J = 8.1, 1.6 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.38 (s, 1H),

6.87 (s, 2H), 4.13 (d, J = 4.8 Hz, 3H), 3.98 (d, J = 5.9 Hz, 6H),

3.87 (s, 3H), 3.78 (s, 3H), 3.68 (s, 6H); ¹³C NMR (75 MHz,

CDCl₃): δ 165.29, 154.47, 153.30, 150.24, 146.70, 140.34,

137.60, 128.80, 125.38, 120.84, 120.06, 118.91, 110.43,

105.13, 100.77, 61.52, 60.95, 60.68, 56.33, 56.23, 56.00; MS

(ESI): m/z 566 (M+H)⁺; HRMS (ESI m/z) for C₂₇H₂₈N₅O₆S

calcd 566.16423, found 550.16554 (M+H)⁺.

7.5. Western blot analysis of soluble versus polymerized

tubulin.

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

complete growth medium. Following treatment of cells with

respective compounds (9a, 9b and CA-4) for a duration of 48

h, cells were washed with PBS and subsequently soluble and

insoluble tubulin fractions were collected. To collect the

soluble tubulin fractions, cells were permeabilized with 200 μL

of pre-warmed lysis buffer [80 mM Pipes-KOH (pH 6.8), 1

mM MgCl₂, 1 mM EGTA, 0.2% Triton X-100, 10% glycerol,

7. Biology

7.1. Antiproliferative activity

The cytotoxic activity of the compounds was determined by

using MTT assay.^54,55Cells were seeded in 100 µl DMEM,

supplemented with 10% FBS in each well of 96-well

microculture plates and incubated for 24 h at 37 °C in a CO₂

0.1% protease inhibitor cocktail (Sigma-Aldrich)] and

incubated for 3 min at 30 °C. Lysis buffer was gently removed,

and mixed with 100 μL of 3 × Laemmli’s sample buffer (180

mM Tris-Cl pH 6.8, 6% SDS, 15% glycerol, 7.5% β-

mercaptoethanol and 0.01% bromophenol blue). Samples were

immediately heated to 95 °C for 3 min. To collect the insoluble

tubulin fraction, 300 μL of 1 × Laemmli’s sample buffer was

added to the remaining cells in each well, and the samples

were heated to 95 °C for 3 min. Equal volumes of samples

were run on an SDS-10% polyacrylamide gel and were

transferred to a nitrocellulose membrane employing semidry

transfer at 50 mA for 1 h. Blots were probed with mouse anti-

human α-tubulin diluted 1:2000 ml (Sigma) and stained with a

rabbit anti-mouse secondary antibody coupled with horseradish

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

using an enhanced chemiluminescence protocol (Pierce) and

radiographic film (Kodak).

and suspended in pre warmed medium. The cells were then

subjected to flow cytometric analysis on a flow cytometer

(Becton Dickinson) in the FL1, FL2 channel to detect

mitochondrial potential ^60.

7.10. ROS generation

The production of ROS (reactive oxygen species) was

measured

by

flow

cytometry

using

DCFDA

61.

(2',7'dichlorofluorescindiacetate) as previously described

In

this study A549 cells were treated with compounds 9a and 9b

at 50 and 100 nM concentrations and 5F-203 100 nM

concentration for 48 h. After treatment, cells were incubated

with DCFDA (2 µM) at 37 °C for 30 min and then measured

with the flow cytometer (FACS).

7.11. Western blot analysis of caspase 3

After treatment with test compounds 9a and 9b at 50 and 100

nM concentrations for 48 h. Protein was isolated with RIPA

(radioimmunoprecipitation assay) buffer. Protein (50 µg per

lane) was applied in 10 % SDS-PAGE (Sodium dodecyl

7.6. Colchicine competition assay

The test compounds (9a and 9b) of various concentrations 5

µM, 10 µM, 15 µM and 20 µM were incubated with 3 µM

tubulin in the presence and absence of 3 µM colchicine in 30

mM Tris buffer for 60 min at 37 °C. CA-4 was used as a

positive control whereas vinblastine was used as negative

control which binds at the taxane site. After incubation the

fluorescence of tubulin–colchicine complex was determined by

using Tecan multimode reader with excitation wavelength of

350 nm and emission wavelength of 435 nm. 30 mM Tris

buffer was used as blank which was subtracted from all the

samples and the fluorescence values are normalized to DMSO

(control).⁵⁸

sulfate

Polyacrylamide

gel

electrophoresis).

After

electrophoresis, the protein was transferred to a polyvinylidine

difluoride (PVDF) membrane (Thermo Scientific Inc.) and

blocked with BSA (bovine serum albumin). The membrane

was washed with TBST for 5 min, then primary antibody was

added. After 24 h, the membrane was incubated with the

corresponding horseradish peroxidase labeled secondary

antibody and incubated for another 1 h. Membranes were

washed with TBST three times for 15 min and the protein blots

were visualized with chemiluminescence reagent (Thermo

Scientific Inc.). Images were captured by using the

chemiluminescence (vilber lourmat) ^62.

7.7. Hoechst staining

Acknowledgents

Human lung cancer A549 cells (5 X10⁴cells/well) at their

growth phase were seeded in 12 well plates and allowed to

adhere for 48 h. The culture medium containing the test

compounds 9a and 9b at their IC₅₀concentration were added to

the cells and incubated for 48 h at 37 ºC. After 48 h, culture

medium was removed; cells were washed with PBS and fixed

with 4 % para formaldehyde solution at 4 ºC for 10 min. The

cells were washed twice with PBS and stained with Hoechst

33258 (5 µg/mL) for 30 min at room temp. The excess dye was

removed by washing twice with PBS and cells were examined

for morphological changes under fluorescence microscope

using 350 nm excitation and 460 nm emission (Nikon,

magnification 10X).

The authors A.V.S.R and S. P. S. would like to thank to CSIR,

New Delhi, India for the award of research fellowships. The

authors are also thankful to CSIR for financial support under

the 12^thFive Year plan project “Affordable Cancer

Therapeutics (ACT)” (CSC0301).

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