Synthesis of new S, N, O-alkylated benzo [d]

imidazo [2,1-b]thiazole and the study of their

biological applications
Cite as: AIP Conference Proceedings 2213, 020108 (2020); https://doi.org/10.1063/5.0000174
Published Online: 25 March 2020

Mazin H. Al-Jamal, Naeemah Al-Lami, and Ruqia M. Al-Azy

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AIP Conference Proceedings 2213, 020108 (2020); https://doi.org/10.1063/5.0000174 2213, 020108

© 2020 Author(s).
Synthesis of new S, N, O-alkylated Benzo [d] imidazo [2,1-
b]thiazole and the Study of Their Biological Applications
Mazin H. Al-Jamal 1, a), Naeemah Al-Lami1, b) and Ruqia M. Al-Azy2, c)
1
Chemistry Department, College of sciences, Baghdad University, Jadriyah, Baghdad, Iraq.
2
Biotechnology Department, College of Sciences, University of Al-Nahrain, Jadriyah, Baghdad, Iraq.
a)
mg2msimawah3@gmail.com
b)
naeema_chem_2008@yahoo.com
c)
ruqaia.alezzy83@yahoo.com

Abstract. A series of benzo[d]imidazo[2,1-b]thiazole[BIT] derivatives were designed, synthesized and rectified for
their cytotoxicity against cell line of HepG2 human liver carcinoma. Preliminary results showed that two of three
examined [BIT] compounds a6 and b1b showed an importantant antiproliferation toward human cancer cell line
(HepG2), where the IC50 values were 104.9 and 160.7 μg.ml-1 respectively. The derivative a6 could be considered as
the probability leads up to developing them as a novel anti-cancer effective agent, while compound b1b appears to be
active at a certain level of concentrations of HepG2 human cancer cell line. The studies suggested that derivative a6
has the potency to be employed up for further, especially against hepatocaricinoma.

INTRODUCTION
There were many challenges in the road to discover a novel anticancer chemotherapy especially for chemists
and oncologists as a part of the scientific community, where many intensive methods of screening carried out for
rectifying different natural or synthesized drugs.
Because of their wide spectrum of bioactivities, benzothiazole heterocyclic compounds are studied extensively,
especially the benzo[d]imidazo[2,1-b]thiazole [BIT] compounds for their pharmaceutical significance.
Several studies have been performed on [BIT] compounds as the screening for antitumor activities against
diversity of cancers involving hepatocellular carcinoma, cancer of colon, acute leukemia of bone marrow, breast
cancer, lung cancer etc. The statistics all over the world have demonstrated that hepatocaricinoma is the sixth most
prevalent malignant cancer which is severely resisting the chemotherapeutic treatment leading to raised death rates
[1-5].
Recently, many trials were conducted for modulating the benzothiazole [BT] core to enhance the antitumor
activity of former compounds. Modulations on the [BT] core have produced a large number of derivatives
possessing diversity of pharmaceutical activities including [BIT] compounds. Amongst them substituted [BIT]
derivatives and imidazo thiazole [IT] such as 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610) in (Fig.
1) exhibited an important and selective in vitro cytotoxicity for cancer cells in human carcinoma. The National
Cancer Institute (NCI) reported benzothiazole [BT] derivatives to exhibit a noteworthy antitumor activity toward
over 50 human malignant cancer cell lines [6, 7] (e.g., colon, and breast cancers). Amongst the [BIT] species, 2-
arylimidazo [2,1-b] [1,3]benzothiazole derivative (YM-201627) in (Fig. 1) was approved to be orally active and
potent antitumor agent especially for solid tumors treatment [8].
So that, synthesizing a new group of [BIT] compounds starting from substituted 2-aryl imidazo benzothiazole
derivatives represented a significant investment in the way of making new biologically active compounds.
Aminoalkylation of [BIT] compounds occur when it reacts with amines and formalin to provide Mannich base (N-
alkylation) [1, 9] is of a great significance for synthesizing and modulation of compounds which have biological
activity. Furthermore, the bridge head nitrogen may provide compound core with a promising biological activity
[10-12]. By the same manners of aminoalkylation we found a need for synthesizing new S-alkylated [BIT]
derivatives by the reaction with thionyl chloride and suitable thiol and O-alkylated [BIT] derivatives by same way
except replacing the thiol by a certain alcohol and screening the derivatives for their biological activity. Since
[BIT] compounds have a biological activity against cancers of different cell lines, also thioethers possess a similar

2nd International Conference on Materials Engineering & Science (IConMEAS 2019)

AIP Conf. Proc. 2213, 020108-1–020108-19; https://doi.org/10.1063/5.0000174
Published by AIP Publishing. 978-0-7354-1964-3/$30.00

020108-1
possibility [13-16], it worthwhile, from one hand, to extend the number of sulfur atoms (S-alkylation) in the [BIT]
molecule that may increase the biological activity by forming a new thioether bond, and from another hand forming
a new ether bond with oxygen [17] (O-alkylation) may increase it as well. Scheme 1 shows the protocol of
synthesis.

FIGURE 1. (1) 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX610), (2) N,N-Diethyl-2-(3-imidazo [2,1-b]

[1,3]benzothiazole-2-ylphenoxy)ethnamine dihydrochloride (YM-201627), (3) 2-([1,1'-biphenyl]-4-yl)-3-
((ethylthio)methyl)benzo[d]imidazo[2,1-b]thiazole(a6), (4)Mannich base of N-((2-(4-bromophenyl)benzo [d]imidazo[2,1-
b]thiazol-3-yl)methyl)-4-nitroaniline (b1b).

SCHEME 1. Total synthesis of formation S, N, O alkylated benzo[d]imidazo[2,1-b]thiazole derivatives.

020108-2
 EXPERIMENTAL
1- The FTIR 8300 Fourier transform infrared spectrophotometer manufactured by SHIMADZU Company which
been used to measure infra-red spectra for the prepared derivatives as a KBr disc in the wave number wave range
of (4000-400) cm-1, in College of Science, University of Baghdad.
2- NMR (1H-NMR &13C-NMR) spectral data were recorded on spectrophotometer of Bruker model Ultra shield
400 Mega Hertz using DMSO as solvent (Isfahan University of Technology (IUT), Iran) and Sharif University of
Technology (SUT).
3- Open capillary method with hot stage Gallen Kamp melting point apparatus were used to determine the melting
point (M.P.) which was uncorrected.

Synthesis of Substituted Benzo[d]imidazo[2,1-b]thiazole[BIT] Derivatives

Two methods have been used for preparation sakes for both (a1) and (b1).
a- Microwave Methods (General Procedure)
The 2-aryl-imidazo[2,1-b]benzothiazole derivative (a1) was prepared by condensation reaction of substituted
2-aminobenzothiazoles (1.8 g, 0.012 mol) with the appropriate 2-bromoacetophenone (2.388 g, 0.012 mol) under
microwave irradiation conditions at 180 W for 4 min., ater that reaction mixture was left to cool at room
temperature. The formed pale yellow precipitate was filtered on Buchner funnel and air-dried to give 2-
phenylimidazo[2,1-b]benzothiazole hydrobromide and Et3N (5 mL) was added, and the mixture refluxed for 5
min. the resulting solution was left at room temperature first then poured into (50 mL) of ice water, the resulting
pale yellow/yellow precipitate was filtered, washed with ethyl acetate and air dried to obtain (a1/b1) .[1]
b- Thermal Method (General Procedure)

2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazole (a1)

2-aminobenzothiazole (1.8 g, 0.012 mol) and α-bromoacetophenone compound (2.4 g, 0.012 mol) in ethanol
(250 mL) was heated under reflux for 4 hrs. After that the solution was cooled and concentrated in vacuo, then the
residual solution diluted with water. Then solution was basified with sodium carbonate and extracted with CH2Cl2
(DCM). The residual organic layer were vaporized to dryness to obtain (a1) [18] : IR (KBr, ν, cm-1): 3134, 3056
(Ar-H), 1689 (C=N), 1600, 1546, 1488 (C=C), and 719 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 8.85-7.41 (m, Ar-
H), 13CNMR (DMSO, 400MHz) δ: 167.51 (3 C=N), 148.81 (S-C-N), 140.8 -121.8 (C=C), and 138.10 (C=S).

2-(4-bromophenyl)benzo[d]imidazo[2,1-b]thiazole (b1)

IR (KBr, ν, cm-1): 3132, 3056 (Ar-H), 1685 (C=N), 1583, 1535, 1492 (C=C), 833, 748, 682 (p-bromophenyl),
and 727 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 8.83-7.41 (m, Ar-H), 13CNMR (DMSO, 400MHz) δ: 148.50 (S-
C-N), 140.22-107.41 (3 C=N), 140.5 - 121.4 (C=C), and 138.05 (C=S).

Table 1. Physical properties of compounds a1 and b1.

Com.No. Symbol R M.F. M.P.(℃) Color Yield(%)
1 a1 C21H14N2S 140-145 Pale yellow 70
2 b1 -Br C15H9BrN2S 132-135 Dark Yellow powder 97

Synthesis of N-alkylated Compounds Named as Mannich Bases (General Procedure)

N-((2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl)-4-methylaniline (a1a)

A slurry consisting of the (a1) (1.6 g, 0.005 mol), (2 mL) of formalin (37 %) and ethanol (5 mL) was made.
Para toluidine (0.54 g, 0.005 mol) was added slowly to the slurry which been cooled and mixed thoroughly. The
reaction mixture was left standing at room temperature for 1 h with occasional stirring, then warmed on a water
bath for 15 min. Finally, the solution was cooled and the obtained compound was recrystallized from petroleum
ether–chloroform to obtain compound (a1a) [19]: IR (KBr, ν, cm-1): 3134 (NH), 3056, 3033 (Ar-H), 2979, 2914

020108-3
(CH3), 2825 (CH2), 1689 (C=N), 1600, 1546, 1492 (C=C aromatic), 842, 798, 692 (biphenyl), and 719 (C-S-C).
1
HNMR (DMSO, 400MHz) δ: 6.28 (s, 1H, NH), 4.33 (s, 1H, CH2), 2.32 (s, 1H, CH3) and 6.47-8.30 (m, Ar-H),
13
CNMR (DMSO, 400MHz) δ: 146.48 (S-C-N), 146.3-134.8 (4 C=N), 140.8 - 113.4 (2 C=C), 135.86 (C=S), 36.4
(C-N), and 21.3 (C-H).

N-((2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl)-3-nitroaniline (a1b)

IR (KBr, ν, cm-1): 3134 (NH), 3056 (Ar-H), 2975, 2912 (CH aliphatic), 2873 (C-H aliphatic CH2), 1689 (C=N),
1645,1622 (C=C aromatic), 1529 (NO2), 842, 798, 692 (biphenyl), and 740 (C-S-C). 1HNMR (DMSO, 400MHz)
δ: 6.99 (s, 1H, NH), 4.63(s, 1H, CH2) and 6.97-8.03 (m, Ar-H). 13CNMR ((DMSO, 400MHz)) δ: 148.48 (S-C-N),
146.45-129.67, (5 C=N), 144.15-113.29 (C=C), and 37.53 (C-N).

N-((2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl)-2,5-dimethylaniline (a1c)

IR (KBr, ν, cm-1): 2975, 2918 (C-H, CH3), 2825 (C-H, CH2), 3134 (NH), 3060 (Ar-H), 1677 (C=N), 1643,
1627, 1550 (C=C aromatic), 835, 742, 694 (biphenyl), and 719 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 6.81 (s,
1H, NH), 4.33 (s, 1H, CH2), 2.17, 2.01 (s, 1H, 2CH3) and 6.70-8.30 (m, Ar-H), 13CNMR (400MHz, DMSO) δ:
146.4-134.8 (4 C=N), 146.48 (S-C-N) , 40.8-113.1 (C-C), 36.7 (CH2), 17.6 (CH3), and 135.86 (C=S).

N-((2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-yl) methyl) aniline (a1d)

IR (KBr, ν, cm-1): 3250 (NH), 2968 (C-H aliphatic), 2858 (CH2), 3058 (Ar-H), 1685, 1668 (C=N), 1602, 1541,
11490 (C=C aromatic), 844, 763, 696 (biphenyl) , and 748 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 6.67-8.30 (m,
Ar-H), 6.28 (s, 1H, NH) and 4.33 (s, 1H, CH2). 13CNMR (400MHz, DMSO) δ: 149.3-134.8 (4 C=N), 146.48 (S-
C-N), 135.75 (C=S), 140.8 - 113.4 (C=C), and 36.4 (C-N).

4-((2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl) morpholine (a1e)

IR (KBr, ν, cm-1): 3110 (NH), 3055 (Ar-H), 2962, 2856 (C-H aliphatic), 1689, 1650, 1600 (C=N), 1541, 1490
(C=C aromatic), 842, 761, 694 (biphenyl) , and 736 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.42-8.83 (m, Ar-H),
3.86 (s, 1H, CH2), 3.54 (s, 4H, H-C-O) and 2.42 (s, 4H, CH2). 13CNMR (DMSO, 400MHz) δ: 146.51 (S-C-N),
145.45-134.12, (3 C=N), 139.19-124 (C=C), 138.27 (C=S), 65.62 (C-O), 38.86-38.53 (3C-N), and 38.53 (C-C).

Table 2. Physical properties of compounds (a1a – a1e).

Com.No. Symbol R M.F. M.P. (℃) Color Yield(%)
3 a1a C29H23N3S 125-128 Yellow powder 80

4 a1b C28H20N4O2S 160-162 Bright yellow 71

powder

5 a1c C30H25N3S 140-142 Pale yellow 52

powder

6 a1d C28H21N3S 120-123 Yellow powder 98

7 a1e C26H23N3OS 86-88 White powder 50

020108-4
FIGURE 2. FT-IR Spectrum of compound [7].

FIGURE 3. 1HNMR Spectrum of compound [7].

020108-5
 FIGURE 4. 13CNMR Spectrum of compound [7].

N-((2-(4-bromophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl)-4-methylaniline (b1a)

IR (KBr, ν, cm-1): 3213 (NH), 3055, 3028 (Ar-H), 2972, 2918 (C-H aliphatic), 2864 (CH2), , 1614 (C=N),
1595, 1515, 1492 (C=C aromatic), 810, 750, 680 (biphenyl), and 723 (C-S-C). 1HNMR (DMSO, 400MHz) δ:
6.28 (s, 1H, NH), 6.47-8.2 (m, Ar-H), 4.33(s, 1H, CH2) and 2.32 (s, 1H, CH3). 13CNMR (DMSO, 400MHz) δ:
146.48 (S-C-N), 146.3-134.8 (4 C=N), 135.86 (C=S), 132-113.4 (C=C), 36.4 (CH2) and 21.3 (CH3).

N-((2-(4-bromophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl)-4-nitroaniline (b1b)

IR (KBr, ν, cm-1): 3134 (NH), 3056 (Ar-H), 2975, 2912 (C-H aliphatic), 2873 (CH2), 1622 (C=N), 1575, 1535,
1494 (C=C aromatic), 804, 746, 663 (biphenyl), and 734 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 6.95-8.01 (m,
Ar-H), 6.85 (s, 1H, NH), and 4.23 (s, 1H, CH2). 13CNMR (DMSO, 400MHz) δ: 148.37 (S-C-N), 144.33-131.95
(5 C=N), 144.09-119.27 (C=C), 130.94 (C=S), and 36.20 (C-N).

N-((2-(4-bromophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl)-2,5-dimethylaniline (b1c)

IR (KBr, ν, cm-1): 3199 (NH), 3028 (Ar-H), 2968 (C-H aliphatic), 2893 (CH2), 1681 (C=N), 1596, 1535,
1492 (C=C aromatic), 804, 800, 680 (biphenyl) and 723 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 6.75 (s, 1H, NH),
6.71-8.30 (m, Ar-H), 4.28 (s, 1H, CH2), and 2.15, 2.01 (s, 1H, 2CH3), 13CNMR (DMSO, 400MHz) δ: 146.47 (S-
C-N), 146.3-134.6 (4 C=N), 140.5-113.09 (C=C), 135.84 (C=S),36.5 (CH2), and 21.1, 17.5 (CH3).

N-((2-(4-bromophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl) aniline (b1d)

IR (KBr, ν, cm-1): 3136 (NH), 3055 (Ar-H), 2974, 2893 (C-H aliphatic), 1677 (C=N), 1595, 1535, 1492 (C=C
aromatic), 810, 746, 682 (biphenyl), and 723 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 6.67-8.02 (m, Ar-H), 6.28
(s, 1H, NH), and 4.33 (s, 1H, CH2). 13CNMR (DMSO, 400MHz) δ: 149.3-134.8 (4 C=N), 146.48 (S-C-N), 135.75
(C=S), 132-113.4 (C=C), and 36.4 (C-H).

020108-6
 4-((2-(4-bromophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methyl) morpholine (b1e)

IR (KBr, ν, cm-1): 3105 (Ar-H), 2945, 2885, 2802 (C-H aliphatic), 1681 (C=N), 1641, 1558, 1492 (C=C
aromatic), 829, 748, 682 (biphenyl), and 725 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.42-8.83 (m, Ar-H), 3.45
(s, 1H, CH2), 3.27, (s, 2H, H-C-O), and 2.21 (s, 4H, CH2).13CNMR (DMSO,400MHz) δ: 146.51 (S-C-N) , 145.22,
138.20, 134.07 (3 C=N), 139.18-124.05 (C=C), 138.20 (C=S), 65.33 (C-O), 38.86, 38.53 (3C-N), and 38.28 (C-
C).

Table 3. Physical properties of compounds (b1a-b1e).

Com. No. Symbol R M.F. M.P. (℃) Color Yield(%)
8 b1a C23H18BrN3S 78-80 Dark Yellow powder 80

9 b1b C22H15BrN4 173-175 Bright yellow powder 71

O2S

10 b1c C24H20BrN3S 73-70 Dark yellow powder 52

11 b1d C22H16BrN3S 100-105 Dark Yellow powder 98

12 b1e C20H18BrN3 138-140 Yellow powder 50

OS

Synthesis of Carbaldehydes Vilsmeier-Haak Reaction (General Procedure)

2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazole-3-carbaldehyde (a2)

To an ice-cold solution of DMF. (1mL, 0.012 mol) in chloroform (5 mL, 0.060 mol) POCl3 (2mL, 0.021 mol)
was added drop by drop and the temperature maintained at (10 oC). To the resulting mixture an ice-cold solution
of compound (a1) (≈1g, 0.0036 mol) in chloroform (3 mL) was added slowly. After completion of addition, the
mixture was refluxed in water bath for 2 hrs. After refluxing been stopped the resulting solution was left to cool,
filtered, and the crystals washed with ice water. At last the solid powder obtained, so that, a mixture of solvents
(acetone: ethanol) (1:1) was used to recrystallize produced derivative (a2)[20]: IR (KBr, ν, cm-1): 3080,3022 (Ar-
H), 1697 (C=O carbaldehyde), 1647 (C=N), 1585, 1541, 1485 (C=C aromatic), 823, 754, 669 (biphenyl), and
710 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 9.83 (s, 1H, CHO) and 8.43 -7.40 (m, Ar-H). 13CNMR (DMSO,
400MHz) δ: 189.88 (C=O), 167.51 (C=N), 148.51 (S-C-N), 140.65 -121.64 (C=C), 138.75, 138.61 (2 C=N), and
138.05 (C=S).

2-(4-bromophenyl) benzo [d]imidazo [2,1-b] thiazole-3-carbaldehyde (b2)

IR (KBr, ν, cm-1): 3066 (Ar-H), 1697 (C=O carbaldehyde), 1668, 1656 (2C=N), 1585, 1521, 1485 (C=C
aromatic), 825, 752, 680 (bromophenyl), and 713 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 9.75 (s, 2H, CHO) and
7.43-8.02 (m, Ar-H). 13CNMR (DMSO, 400MHz) δ: 188.9 (C=O), 167.20, 138.35, 138.55 (3 C=N), 148.30 (S-C-
N), 140.24-121.37 (C=C) and 137.45 (C=S).

020108-7
 TABLE 4. Physical properties of compounds a2 and b2.
Com.No. Symbol R M.F. M.P. (℃) Color Yield(%)
13 a2 C22H14N2OS 160-165 Dark Yellow powder 68
14 b2 -Br C16H9BrN2OS 245-250 Dark Yellow powder 96

FIGURE 5. FT-IR Spectrum of compound [13].

FIGURE 6. 1HNMR Spectrum of compound [13].

020108-8
 FIGURE 7. 13CNMR Spectrum of compound [13].

Synthesis of Alcohol Derivatives (General Procedure)

(2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methanol (a5)

To a solution of an aldehyde (a2) (7.44 g, 0.021 mol) in methanol (100 ml) cooled to (5 oC), NaBH4 (19 g,
0.503 mol) was added drop-wise. After that, the mixture was heated under reflux for (3 hrs.). Finally, the solution
was acidified to pH = 1 with (HCl), basified with ammonia, concentrated in vacuo, extracted with dichloromethane
and evaporated to obtain the sold derivative (a5)[18]: IR (KBr, ν, cm-1): 3471-3434 (OH broad), 3026 (Ar-H),
2848, 2810 (CH2), 1647 (C=N), 1598, 1546, 1490 (C=C aromatic), 823, 752, 694 (biphenyl), and 738 (C-S-C).
1
HNMR (DMSO, 400MHz) δ: 7.22-8.04 (m, Ar-H), 4.50 (s, 2H, O-H), and 1.20 (s, 1H, CH2). 13CNMR (DMSO,
400MHz) δ: 151.31 (S-C-N), 142.96, 132.7, 133.3 (3C=N), 131.9 (C=S), 128.86-120.25 (C=C), and 51.5 (C-O).

(2-(4-bromophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methanol (b5)

IR (KBr, ν, cm-1): 3417-3436 (OH broad), 3018 (Ar-H), 2964, 2810 (CH2), 1662 (C=N), 1589, 1515, 1479
(C=C aromatic), 829, 748, 682 (bromophenyl), and 730 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.52-8.06 (m,
Ar-H), 4.96 (s, 2H, O-H) and 1.37 (s, 1H, CH2). 13CNMR (DMSO, 400MHz) δ: 152.41 (S-C-N), 143.0, 134.8,
135.5 (3C=N), 132.12 (C=S), 129.95-120.50 (C=C), and 52.4 (C-O).

020108-9
 TABLE 5. Physical properties of compounds a5 and b5.
Com. No. Symbol R M.F. M.P. (℃) Color Yield(%)
15 a5 C22H16N2OS 155-157 Yellow powder 78

16 b5 -Br C16H11BrN2OS 260 Dark Yellow powder 89

decomposition

FIGURE 8. FT-IR Spectrum of compound [16].

FIGURE 9. 1HNMR Spectrum of compound [16].

Synthesis of Ethers and Thioethers (O-alkylated and S-alkylated compounds)

Williamson Reaction (General Procedure)
2-([1,1'-biphenyl]-4-yl)-3-((ethylthio)methyl)benzo[d]imidazo[2,1-b]thiazole (a6)

020108-10
 The [BIT] derivatives (a5) (1.07g, 0.003 mol) were dissolved portion-wise under stirring in freshly distilled
thionyl chloride (5 mL) cooled to 0oC. The solution was refluxed for 2h and after cooling evaporated in vacuo.
The residual solution was dissolved in distilled acetonitrile (10 mL), pyridine (1.5 ml) and the mercaptan (0.19g,
0.003 mol) was added, then mixture was heated under reflux for 3h. After that, the residue was cooled, evaporated
to dryness and treated with water (10 ml). The solution was basified with Na2CO3 and extracted with CH2Cl2
(DCM). The organic layer were separated and dried over CaCl2, concentrated in vacuo to obtain derivatives [18]:
IR (KBr, ν, cm-1): 3028 (Ar-H), 2866 (CH2), 1668 (C=N), 1595, 1556, 1479 (C=C aromatic), 844, 765, 696
(biphenyl), 746, and 732 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.46-8.33 (m, Ar-H), 1.17(t, 2H, CH3), 2.50 (m,
3H, CH2) and 3.66 (s, 1H, CH2). 13CNMR (DMSO, 400MHz) δ: 148.47 (S-C-N), 143.2, 135.6,137.03 (3 C=N),
140.85-121.2 (C=C), 135.5 (C=S), and 25.4, 26.3 (2C-S).

2-((2-([1,1'-biphenyl]-4-yl)benzo[d]imidazo[2,1-b]thiazol-3-yl)methoxy)ethan-1-ol (a7)

IR (KBr, ν, cm-1):, 3060, 3031 (Ar-H), 2954, 2920, 2852 (CH2), 1629 (C=N), 1600, 1539, 1481 (C=C aromatic),
846, 763, 698 (biphenyl), and 750 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.45-8.06 (m, Ar-H), 5.5 (s, 1H, OH),
4.04 (s, 1H, CH2O), 3.75 (t, 1H, CH2O), and 3.57 (t, 1H, CH2O). 13CNMR (DMSO, 400MHz) δ: 148.50 (S-C-N),
143.2, 136.8,137 (3 C=N), 140.86-121 (C=C), 135.12 (C=S), and 72.2, 62.5, 61.3 (3C-O).

3-(((1H-benzo[d]imidazol-2-yl)thio)methyl)-2-([1,1'-biphenyl]-4-yl) benzo [d] imidazo [2,1-b]thiazole (a9)

IR (KBr, ν, cm-1):, 3155, 3116 (NH), 3060 (Ar-H), 2981, 2881 (CH2), 1652 (C=N), 1564, 1537, 1488 (C=C
aromatic), 844, 786, 698 (biphenyl) and 744 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 12.55 (s, 1H, NH), 7.12-8.29
(m, Ar-H) and 4.91 (s, 1H, CH2). 13CNMR (DMSO, 400MHz) δ: 138.22 - 129.41 (4C=N), 52.62, 38.86 (2C-N),
38.50 (C-S), 149.04, 167.62 (2S-C-N), 127.46 (C=S), and 126.95-109.25 (C=C).

2-([1,1'-biphenyl]-4-yl)-3-((benzo[d]thiazol-2-ylthio) methyl) benzo [d] imidazo [2,1-b] thiazole (a10)

IR (KBr, ν, cm-1): 3155 (NH), 3060 (Ar-H), 2981, 2881 (CH2), 1620 (C=N) , 1600, 1512, 1488 (C=C aromatic),
844, 744, 698 (biphenyl), and 744 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.41-8.30 (m, Ar-H) and 5.13 (s, 1H,
S-C-H), 13CNMR (400MHz, DMSO) δ: 164.7 (S-C-S), 163.62 (S-C-N) , 145.5-134.8 (4C=N), 140.8-109.25
(C=C), 135.1 (2C=S) , and 29 (C-S).

TABLE 6. Physical properties of compounds (a6-a10).

Com. No. Symbol R M.F. M.P. (℃) Color Yield(%)
17 a6 -S-CH2CH3 C24H20N2S2 130-133 Yellow powder 55
18 a7 -O-CH2CH2OH C24H20N2O2S 120-122 Dark brown powder 56
19 a9 C29H20N4S2 190-192 Brown powder 96

20 a10 C29H19N3S3 88-90 Dark Yellow 56

powder

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FIGURE 10. FT-IR Spectrum of compound [19].

FIGURE 11. 1HNMR Spectrum of compound [19].

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 FIGURE 12. 13CNMR Spectrum of compound [19].

2-(4-bromophenyl)-3-((ethylthio)methyl)benzo[d]imidazo[2,1-b]thiazole(b6)

IR (KBr, ν, cm-1): 3060, 3035 (Ar-H), 2956, 2925 (CH2), 1677 (C=N), 1591, 1541, 1487 (C=C aromatic), 837,
752, 678 (biphenyl) and 729, 702 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.45-8.32 (m, Ar-H), 1.15(t, 2H, CH3),
2.49 (m, 3H, CH2) and 3.64 (s, 1H, CH2). 13CNMR (DMSO, 400MHz) δ: 148.45 (S-C-N), 143.09, 135.5,137.01
(3 C=N), 140.83-121.1 (C=C), 135.09 (C=S), and 25.2, 26.09 (2C-S).

2-((2-(4-bromophenyl)benzo[d]imidazo [2,1-b]thiazol-3-yl) methoxy) ethan-1-ol (b7)

IR (KBr, ν, cm-1):, 3028, 3130 (Ar-H), 2877 (CH2), 1660, 1654 (2C=N), 1560, 1541, 1483 (C=C aromatic),
831, 790, 667 (biphenyl) and 748 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.43-8.03 (m, Ar-H), 5.4 (s, 1H, OH),
4.02 (s, 1H, CH2O), 3.73 (t, 1H, CH2O), and 3.56 (t, 1H, CH2O). 13CNMR (DMSO, 400MHz) δ: 148.48 (S-C-N),
143.09, 136.5,137 (3 C=N), 140.82-120 (C=C), 135.10 (C=S), and 72.1, 62.3, 61.1 (3C-O).

3-(((1H-benzo[d]imidazol-2-yl)thio)methyl)-2-(4-bromophenyl)benzo [d] imidazo [2,1-b] thiazole (b9)

IR (KBr, ν, cm-1):, 3085 (Ar-H), 2972, 2927 (CH2 aliphatic), 1618 (C=N), 1585, 1512, 1473 (C=C aromatic),
829, 744, 682 (biphenyl) and 705 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 12.56 (s, 1H, NH), 7.13-8.02 (m, Ar-
H), and 4.77 (s, 1H, CH2). 13CNMR (DMSO, 400MHz) δ: 163.62, 148.98, (2S-C-N), 145.5- 134.8 (4C=N), 128.3-
115.2 (C=C), 127.20 (C=S), and 29 (CH2).

3-((benzo[d]thiazol-2-ylthio)methyl)-2-(4-bromophenyl) benzo [d] imidazo[2,1-b]thiazole (b10)

IR (KBr, ν, cm-1): 3062, 3060 (Ar-H), 2968, 2821 (CH2), 1662 (C=N), 1585, 1533, 1479 (C=C aromatic), 829,
748, 682 (biphenyl) and 723 (C-S-C). 1HNMR (DMSO, 400MHz) δ: 7.43-8.02 (m, Ar-H) and 5.13 (s, 1H, CH2).
13
CNMR (DMSO, 400MHz) δ: 164.7 (S-C-S), 163.62 (S-C-N),145.5-134.8 (4C=N), 135.1 (2C=S) 132.1-121.82
(C=C) and 29 (CH2).

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 TABLE 7. Physical properties of compounds (b6-b10).
Com. No. Symbol R M.F. M.P. (℃) Color Yield(%)
21 b6 -S-CH2CH3 C18H15BrN2S2 150-153 Dark Yellow 56
powder
22 b7 -O- C18H15BrN2O2S 285 Dark Brown 85
CH2CH2OH decomposition powder
23 b9 C23H15BrN4S2 115-117 Dark Yellow 51
powder

24 b10 C23H14BrN3S3 gummy brown gum 54

RESULT AND DISCUSSION

As been shown in protocols of synthesis proposed in Scheme 1 the [BIT] derivatives produced in a good yield
by reacting substituted 2-aminobenzothiazole with bromoacetophenone to produce derivatives (a1, b1). 2-
sbstituted derivative was treated with Vilsmeier-Haack reagent to form the 3-substituted carbaldehyde. Structures
of the title derivatives were approved by FT-IR and 1HNMR spectral data.
All analogue derivatives showed relative ranges of spectra values for both IR and NMR except the differences
between compounds, weather the substituent was 4-phenyl or 4-bromo substituted compound.
The mechanism of a1 and b1 is showen in Scheme 2.

SCHEME 2. Mechanism of reaction 2-substituted [BIT].

The formation of (a1) was approved by its infra-red spectrum (C=N) 1689 cm-1 which is resembles the IR
bands showen by its para bromophenyl analogue (b1) viewed in literature. While the formation of (a2, b2) was
approved by its infra-red spectra by the occurrence of new band of aldehyde (C=O) at 1697 cm-1 as referred to in
literature, the NMR spectrum displayed δ 9.75 (s, 2H, aldehyde) and 7.43-8.02 (m, Ar-H) and 13CNMR δ of 188.9
(C=O) of aldehyde.
After that, employing reduction reaction from another hand changes infra-red spectrum by the disappearance of
aldehyde band at 1697 cm-1of (C=O) with alternative appearance of hydroxyl (OH) group broad band at 3471-
3434 cm-1 for both compounds in (a5, b5), NMR spectrum showed the (OH) proton δ of 4.50 (s, 2H, O-H), also

020108-14
1.20 (s, 1H, CH2) signals. The reaction mechanism of formation of alcohols, thiols and Mannich bases was also
demonstrated in Scheme 3

SCHEME 3. Synthesis of alcohols, thioethers and Mannich bases.

The next step was the formation of (a6, b6) by SN2 reaction to form thiol derivatives these derivatives were
confirmed their structures by FT-IR spectra, that is the band of (O-H) at 3479 cm-1 disappeared and two new bands
appeared at 746, 732 cm-1 belongs to (C-S-C) to thioether and 1HNMR signals at 2.50 of (m, 3H, CH2) and 3.64
of (s, 1H, CH2) and 13CNMR signals at 25.4, 26.3 of (2C-S). As well as (a7, b7) which were confirmed by the
appearance of (C-O) at 1230 and 1022 cm-1 of alkyl aryl ether, also in 1HNMR δ 4.05 of (s, 1H, ether), 3.54, 3.70
(s, 2H, H-C-O) and 13CNMR showed signals δ of 62.5 (C-O ) of ether. Similarly, the synthesis of (a9, b9) were
confirmed by the appearance of (C-S-C) band 727 cm-1and (C-H) at 2889 cm-1 in IR spectra , also the appearance
of 1HNMR signal at 4.91 of (s, 1H, CH2) and 13CNMR at 38.50 of (C-S), and 149.04, 167.62 of (S-C-N) as two
separated signals instead of one. By the same manner's compounds (a10, b10) IR spectra demonstrates the
appearance of (C-S-C) band 744 cm-1 and 2981, 2881 cm-1 of (C-H), also appeared by 1HNMR spectra which
displays δ of 4.91 (s, 1H, CH2) and for 13CNMR showed δ 38.50 of (C-S).
Mannich bases started with (a1a, b1a) were proved by the appearance of new bands of IR at 2825 of (C-H)
and 3134 cm-1 of (N-H), also 1HNMR spectra displayed δ at 6.28 of (s, 1H, N-H) and 4.33 of (s, 1H, CH2) and

020108-15
13
CNMR showed δ equal to 36.4 of (C-N). Also (a1b, b1b) were approved similarly by the appearance of new
bands at 2873 of (C-H), 3134 cm-1 of (N-H) and 1HNMR spectra displayed δ at 6.99 of (s, 1H, N-H) and 4.63 of
(s, 1H, CH2) and 13CNMR showed δ equal to 37.53 (C-N). Also, (a1c, b1c) formation was approved by the
appearance of new bands of IR spectrum at 2975 and 2918 cm-1 of (C-H) and 3134 cm-1 of (N-H) also as seen in
NMR spectra of 1HNMR spectra displayed δ equal to 6.81 of (s, 1H, NH) and δ 4.33(s, 1H, CH2) and 13CNMR δ
at 36.7 of (CH2). Similarly, (a1d, b1d) existence were proved by the appearance of new bands of IR spectra at
2858 of (C-H) and 3250 cm-1 of (N-H) and 1HNMR spectra displayed δ at 6.28 of (s, 1H, NH) and 4.33 of (s, 1H,
CH2) and 13CNMR showed δ equal to 149.3, of (C=C) and 36.4of (C-N). By the same way, (a1e, b1e) was
approved by the appearance of new bands at 2856, 2800 cm-1 of (C-H) and 3250 cm-1 of (N-H) in IR spectra as
well as spectra of 1HNMR which displayed δ at 3.45 of (s, 1H, CH2), 3.27, (s, 2H, H-C-O), 2.21 (s, 4H, CH2) and
13CNMR δ ppm of 38.86, 38.53 (3C-N aliphatic).

Cytotoxicity Effect of Three New Benzo[d]imidazo[2,1-b]thiazole derivatives [BIT] in vitro Using MTT Assay
(anticancer)

The test of 3-(dimethyltiazole-2-yl) 2,5-diphenyltetrazolium bromide (MTT) was accomplished to conclude

the cytotoxic effect of the new benzo[d]imidazo[2,1-b]thiazole [BIT] derivatives on liver cancer cell line (HepG2).
MTT assay was made to calculate the inhibition rate and cell viability on the tumor cell line by using different
concentrations of compounds. The viability percent was calculated for treated cells in a comparison with normal
cells WRL.
The cytotoxic effect of [BIT] derivative (a6) in concentration ranged from 2.65-400 μg.ml-1 on HepG2 cells
(Table 8) presented a decrease in cell viability in a dose-dependent pattern. The cell viability is reduced by increase
the concentration of the [BIT] derivative (a6). The decreasing in HepG2 cell viability (%) was noted by 400 μg.ml-
1
(52.62±2.655) while the highest HepG2 cell viability at 6.25 μg.ml -1 reached (95.68±0.5711).
TABLE 8. Cytotoxicity Effect of The New [BIT] derivatives on HepG2 and WRL cells after 24 hours incubation at 37 oC.
BITs concentrations μg.ml-1 Viable cell count of HepG2 cell Viable cell count of WRL cell
line Mean ±S.D. line Mean ±S.D.
400 52.26±2.655 85.03±1.037
200 61.96±1.270 91.26±2.205
100 73.65±6.108 92.21±2.778
50 90.20±5.950 96.49±1.291
25 96.80±0.9356 96.95±1.142
12.5 95.02±3.042 94.29±2.979
6.25 95.68±0.5711 96.07±0.1160

The [BIT] derivative (a6) exhibited significantly the most cytotoxic activity with IC50 value of 104.9 μg.ml -1.
However, an IC50 of 564.6 μg.ml -1 was obtained from the effect of [BIT] derivatives on WRL normal cell line
(Fig. 13)

FIGURE 13. Representation of the IC50 of HepG2 and WRL cell lines of compound (a6).

020108-16
 The cytotoxic effect of [BIT] derivative (b1b) in concentration ranged from 2.65-400 μg.ml-1 on HepG2 cells
(Table 9) presented a decrease in cell viability in a dose-dependent pattern. The cell viability is reduced by increase
the concentration of the [BIT] derivative (b1b) to affect WRL cell line in determined concentrations only.
TABLE 9. Cytotoxicity Effect of The New [BIT] derivatives on HepG2 and WRL cells after 24 hours incubation at 37 oC.
BITs concentrations μg.ml-1 Viable cell count of HepG2 cell line Viable cell count of WRL cell line
Mean±S.D. Mean±S.D.
400 65.93±2.791 69.98±2.906
200 75.00±3.614 86.81±1.514
100 89.20±2.696 94.29±1.831
50 95.22±2.151 95.83±0.3063
25 94.56±2.806 95.37±0.9039
12.5 96.88±0.9891 96.64±0.7041
6.25 96.10±1.706 95.68±0.4064
The [BIT] derivatives exhibited significantly the most cytotoxic activity with IC50 value of 160.7 μg.ml -1.
However, an IC50 of 298.7 μg.ml -1 was obtained from the effect of [BIT] derivatives on WRL normal cell line
(Fig. 14)

FIGURE 14. Representation of the IC50 of HepG2 and WRL cell lines of compound b1b.
The cytotoxic effect of [BIT] derivative (a11) in concentration ranged from 2.65-400 μg.ml-1 on HepG2 cells
(Table 10) presented a small decrease in cell viability in a dose-dependent pattern. The cell viability is reduced by
increase the concentration of the [BIT] derivative (a11) which indicate that the compound was killing both living
and cancer cells similarly so it considered to be un efficient in such treatment field.
TABLE 10. Cytotoxicity Effect of The New [BIT] derivatives on HepG2 and WRL cells after 24 hours incubation at 37 oC.
BITs concentrations μg.ml-1 Viable cell count of HepG2 cell line Viable cell count of WRL cell line
Mean±S.D. Mean±S.D.
400 73.38±4.421 63.71±5.126
200 80.67±1.978 80.42±5.906
100 93.60±3.902 93.60±2.100
50 94.29±0.8209 95.33±1.183
25 95.95±0.9043 95.22±0.8209
12.5 94.87±0.2914 95.95±1.028
6.25 94.83±0.7072 95.95±0.2003
The (BIT) derivatives exhibited significantly the most cytotoxic activity with IC50 value of 216.7 μg.ml -1.
However, an IC50 of 175.5 μg.ml -1 was obtained from the effect of (BIT) derivatives on WRL normal cell line
(Fig. 15)

020108-17
 FIGURE 15. Representation of the IC50 of HepG2 and WRL cell lines of compound a11.

CONCLUSION
A new species of 2-aryl imidazo[2,1-b]benzothiazole compounds (Mannich bases) were synthesized and
rectified for their antiproliferative activities in HepG2 cancer cell line. Due to MTT proliferation assay, it was
observed that derivative a6 is the most effective antiproliferative agent among the series derivatives against
(HepG2) cancer cells when tested at range of (400-6.25 μg.ml-1) concentration. Their better activity can be
attributed to the existence of various electron withdrawing groups substituted at position C-3 in the ring system.
The FACS analysis also showed more population in G2/M phase in compounds (a6, b1b) indicating that these
compounds caused G2/M cell cycle arrest with a6 being the most effective. From the results of the detailed
biological assays, drastic down regulation of cell cycle regulatory protein such as cyclin B as well up-regulation
DNA damage specific check point protein (Chk2) protein was observed, suggesting that these proteins are
responsible for cell cycle blockade at G2/M phase in compound a6. Moreover, caspase-3 protein level was
increased that indicate apoptotic inducing nature of the compounds a6 and b1b. Further downstream proteins wich
have potential role in cell cycle as well as proliferation such as MAPK family (p38 and p-JNK) and Jun family
proteins were down regulated. These results are strong backing to the significant cytotoxic and also cell cycle role
of regulation of these 2-arylimidazo[2,1-b]benzothiazoles compounds (Mannich bases). In this study a vision into
the cell cycle role of regulation and also apoptotic inducing ability of the compound a6 was explained. These
studies suggest that compound a6 has the potential to be taken up for further, particularly against
hepatocaricinoma.

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