Novel benzothiazole containing 4H-pyrimido[2,1-b]benzothiazoles … · 2017-03-02 · ESI-MS and Single crystal X-ray analysis (4a). All the ﬁnal scaffolds have been screened for

Arabian Journal of Chemistry (2016) xxx, xxx–xxx

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sawww.sciencedirect.com

ORIGINAL ARTICLE

Novel benzothiazole containing

4H-pyrimido[2,1-b]benzothiazoles derivatives:One pot, solvent-free microwave assisted

synthesis and their biological evaluation

* Corresponding authors at: Department of Chemistry, School of

Sciences, Gujarat University, Ahmedabad, Gujarat, India. Tel.: +91

079 26300969; fax: +91 079 26308545.

E-mail addresses: [email protected] (M.N. Bhoi), mayuri.

[email protected] (M.A. Borad), [email protected]

(E.A. Pithawala), [email protected] (H.D. Patel).

Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.arabjc.2016.01.0121878-5352 � 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Bhoi, M.N. et al., Novel benzothiazole containing 4H-pyrimido[2,1-b]benzothiazoles derivatives: One pot, solvent-free miassisted synthesis and their biological evaluation. Arabian Journal of Chemistry (2016), http://dx.doi.org/10.1016/j.arabjc.2016.01.012

Manoj N. Bhoi a,*, Mayuri A. Borad a, Edwin A. Pithawala b, Hitesh D. Patel a,*

aDepartment of Chemistry, School of Sciences, Gujarat University, Ahmedabad, IndiabDepartment of Life Sciences, School of Sciences, Gujarat University, Ahmedabad, India

Received 26 November 2015; accepted 28 January 2016

KEYWORDS

Antioxidant;

2-Amino benzothiazole;

Microwave irradiation;

4H-pyrimido[2,1-b]benzoth-

iazoles;

Solvent-free reaction;

Mycobacterium tuberculosis

Abstract Synthesis of new and desired compounds has an everlasting demand. The present work

emphasizes on the one pot, three component microwave assisted synthesis of novel ethyl 2-methyl-

4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate derivatives by the reaction of

2-aminobenzothiazole derivatives with Pyridine 2-aldehyde and Ethyl acetoacetate in the presence

of PdCl2 as an expeditious catalyst under solvent-free condition. The salient features of this

approach are operational simplicity, convergence, short reaction time, high atom economy, easy

workup, mild reaction conditions and environmentally benign conditions. All the newly synthesized

diverse poly-functionalized tri-heterocyclic benzothiazole derivatives have been characterized by

elemental analysis and various spectroscopic methods such as FT-IR, 1H NMR, 13C NMR,

ESI-MS and Single crystal X-ray analysis (4a). All the final scaffolds have been screened for

antibacterial and antioxidant activities. Also their antitubercular activity against Mycobacterium

tuberculosis H37RV was screened.� 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is

an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Search on efficient organic synthesis approach that substantiates the

rapid generation of complex organic molecules from simple and readily

accessible starting materials is in vogue during recent epochs and the

interests have been engrossed a current position (Chow and Shea,

2005; de Graaff et al., 2012; Jiang et al., 2010; Shiri, 2012). The impor-

tant medicinal compounds, which are synthesized through steadfast

reactions are receiving more fascinating attention. Literature shows

that scientists have also eventually facilitated the rapid development

of new chemical entities which are available for biological evaluation.

crowave

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2 M.N. Bhoi et al.

Non-traditional conditions and their approach to implement new

synthetic organic reactions have gained popularity, primarily to cir-

cumvent growing environmental concerns (Pillai et al., 2002;

Raghavendra et al., 2007). Microwave assisted reaction is an invalu-

able technology for synthesis of organic compounds with respect to

time as it decelerates reaction time with enhanced yields and selectivity.

Microwave reaction under solvent free conditions is consensus in offer-

ing reduced pollution with ease in processing and handling (Bejan

et al., 2012; de la Hoz et al., 2005; Desai et al., 2006; Hayes, 2002,

2004; Kappe, 2000, 2004, 2008; Kappe et al., 2012; Khabazzadeh

et al., 2008; Perreux and Loupy, 2002; Stadler et al., 2002, 2003).

The chemistry of fused heterocycles is known to fascinate field of

investigation in therapeutic chemistry, since they have been found to

display improved biological activity. Heterocyclic compounds show

significant role in both medicinal chemistry and their constitution in

natural products. Among them, benzothiazole derivatives have

received substantial attention in synthetic organic chemistry with

numerous significant and valuable applications in the pharmaceutical

industry. From the current literature survey, benzothiazole derivatives

have shown various biological activities (Bhoi et al., 2015a, 2015b,

2014; Boer and Gekeler, 1995; Borad et al., 2015; Bretzel et al.,

1993; Hutchinson et al., 2001; Klusa, 1995; Pande et al., 1982; Singh

et al., 1975; Singh and Vaid, 1986; Huang and Yang, 2006; Jiang

et al., 2009, 2011; Wu et al., 2014a, 2014b; Yang et al., 2013; Zuo

et al., 2012) used in material science and in other industrial applica-

tions (Choudhary et al., 2013; Hopper, 1993; Zhang et al., 2001).

The important and pharmacological properties of benzothiazole

derivatives along with their improved synthetic methods allow a facile

access to these heterocyclic compounds. Recently, many efforts have

been dedicated to improve new and highly effective synthetic protocols

such as [3 + 2] cycloadditions, transition metal catalyzed cyclization

and multicomponent coupling reactions for the synthesis of derivatives

of benzothiazole (Bastug et al., 2012; Jaseer et al., 2010; Karle et al.,

2012; Noolvi et al., 2012; Sahu et al., 2012a; Tale, 2002; Trapani

et al., 1996; Xue et al., 2013). Typically, multi-component reactions

(MCRs) are an influential tool which attract much more focus in syn-

thetic organic reactions owing to the complex molecules with varied

range of complexity where the starting materials are easily available

(Benetti et al., 1995; Domling, 2006; Kappe, 2002; Langer, 2001;

Nair et al., 2003; Ramon and Yus, 2005; Simon et al., 2004). Thus,

MCRs are influenced by microwave and solvent free condition as a

powerful green alternative over the conventional synthesis (Devineni

et al., 2013; Eynde et al., 2001; Kappe, 2000, 2002; Kappe and

Stadler, 2004; Leadbeater et al., 2006; Oliver Kappe, 1993; Saxena

and Chandra, 2011).

The Biginelli reaction is recognized multicomponent reaction for

the synthesis of 4H-pyrimido[2,1-b]benzothiazoles and associated poly-

heterocycles (Chebanov et al., 2010; Ranu et al., 2000; Sedash et al.,

2012). Since 1893, a one pot, three-component condensation reaction

between aldehyde, b-ketoesters and urea was first time reported by

Biginelli (Biginelli and Gazz, 1893) to produce 3,4-dihydropyrimidin-

2-(1H)one. Compound 2-amino benzothioazoles and

2-aminobenzimidazoles derivatives are used in place of urea

(Alajarin et al., 1995; Shaabani et al., 2005). The Biginelli reaction

can be carried out by heating as well as by acid and base catalysts.

Recently, catalysts such as FeF3 (Atar and Jeong, 2014), Kaolin

(Sahu et al., 2013), N,N-dichlorobis(2,4,6-trichlorophenyl)urea (Rao

et al., 2011), hydrotalcite (Sahu et al., 2012b), TBAHS (Nagarapu

et al., 2013), AlCl3 (Sahu et al., 2012c), and Zn(ClO4)2�6H2O(Kaur

et al., 2015; Singh et al., 2013) have shown to be effective for the syn-

thesis of 4H-pyrimido[2,1-b]benzothiazoles. Although these methods

afford good results, there is yet a great demand for quick and eco-

friendly catalytic MCRs conditions. Our aim is to find out the best

environmental friendly catalytic system for a one-pot reaction through

microwave assisted synthesis under solvent free condition.

Encouraged by the auspicious biological activity of the structurally

diverse heterocycles with fused heterocyclic systems and our

continuing research plan on the synthesis of therapeutically interesting

Please cite this article in press as: Bhoi, M.N. et al., Novel benzothiazole containingassisted synthesis and their biological evaluation. Arabian Journal of Chemistry (20

heterocycles, we have developed new structural motifs with promising

bioactivity. An efficient, one pot three component synthesis of ethyl

2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a] pyrimidine-3-

carboxylate derivatives by reaction between various derivatives of

2-amino-benzothiazole with Pyridine 2-aldehyde and Ethyl acetoac-

etate using PdCl2 as an efficient catalyst under microwave assisted

solvent-free conditions, with better yield and short reaction time is

introduced to develop new structural motifs with promising bioactiv-

ity. We have evaluated their antibacterial activity against gram +ve

and gram �ve bacterial strains and antitubercular activity against

Mycobacterium tuberculosis H37RV and also screened antioxidant

activity.

2. Material and methods

2.1. Chemistry

All chemicals and solvents were acquired from commercialsources (Aldrich-Sigma and Merck chemical suppliers). The

reactions were scrutinized by thin layer chromatography(TLC) and judged by the consumption of starting material.TLC was performed on silica gel G 60 F254 (Merck) plate. Itwas visualized by UV radiation, exposure to iodine vapor

and spray reagent also. Column chromatography was con-ducted over silica gel 60 (60–120 lm). CEM Discover micro-wave system (model No.: 908010; make up CEM Matthews.

Inc, USA) was used for synthesis with external IR temperaturecontrol. The melting points recorded on optimelt automatedmelting point system were uncorrected. IR spectra were

recorded on a Perkin–Elmer 377 spectrophotometer in KBrwith absorption in cm�1. 1H NMR and 13C NMR spectra wererecorded on Bruker AV 400 and 100 MHz using DMSO-d6 as

solvent and TMS as an internal standard. Mass spectra wererecorded on Advion Expression CMS, USA, using Metha-nol:Water:Formic acid (80:20:0.1) as mobile phase. Elementalanalysis was performed on vario MICRO cube, elementar

CHNS analyser serial No.: 15084053. Single crystal X-raydiffraction intensity data of the compound 4a were collectedon an Oxford XCALIBUR-S CCD diffractometer with Cu

Ka radiation (k = 1.54184 A) at 195(100) K.

2.2. Synthesis of ethyl-(substituted)-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a] pyrimidine-3-carboxylate(4a–4l)

2.2.1. Method A (microwave assisted method)

Mixture of pyridine 2-aldehydes 1 (4.668 mmol), b-diketonelike Ethyl acetoacetate 2 (4.668 mmol) and various derivativesof 2-amino benzothiazole 3a–3l (4.668 mmol) in 10 ml micro-

wave open vessel glass tube was placed in the cavity of amicrowave oven (CEM Discover microwave) and irradiatedfor 2–5 min at 90 �C under solvent-free conditions by using

PdCl2 (10 mol%) as a catalyst with continuous monitoringof the reaction by TLC (30% Ethyl acetate: hexane). Aftercompletion of the reaction, the reaction mixture was cooled

to room temperature and then poured into ice-water by solu-bilizing in ethanol solvent. The precipitated solid was filtered,and subjected to column chromatography using 20% (v/v)Ethyl acetate : hexane mixture as an eluent to afford ethyl-(s

ubstituted)-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5] thiazolo[3,2-a]pyrimidine-3-carboxylate (4a–4l) as pure product with77–88% yield.

4H-pyrimido[2,1-b]benzothiazoles derivatives: One pot, solvent-free microwave16), http://dx.doi.org/10.1016/j.arabjc.2016.01.012


Novel benzothiazole containing 4H-pyrimido[2,1-b]benzothiazoles derivatives 3

2.2.2. Method B (conventional method)

As mentioned in above method same chemical ratio was taken

in a 25 ml round bottom flask. Resulting mixture was placed inoil bath for 20–70 min at 90 �C under solvent-free conditionsusing PdCl2 (10 mol%) as a catalyst. The time taken by differ-

ent derivatives of 2-amino benzothiazole in the reaction isshown in Table 3. After confirmation of each reaction usingTLC (30% Ethyl acetate: hexane), the reaction mixture was

allowed to cool at room temperature and poured in cold waterafter solubilizing in ethanol solvent with a requisite volume.The mixture was filtered continuously giving a wash of water.The solid crude product was simply purified by column chro-

matography over silica gel using solvent system (20% ethylacetate: hexane) as an eluent to obtain subsequent pure pro-duct (4a–4l) with 69–80% yield.

2.2.3. Ethyl-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate (4a)

Yellow solid, mp 175–176 �C; Anal. Calcd for C19H17N3O2S:

C, 64.94; H, 4.88; N, 11.96; O, 9.11; S, 9.12%; found C,64.81; H, 4.75; N, 11.79; S, 8.95%; IR (KBr) (tmax, cm

�1):3072 (CAHstr), 2943 (CAHstr), 1745 (C‚Ostr), 1677 (C‚Cstr),

1688 (C‚Nstr), 1352 (CANstr), 1240 (CAOstr);1H NMR

(400 MHz, DMSO) d 8.59 (1H, dd, J = 7.5, 1.3 Hz, C6AH),7.74 (2H, dtd, J = 9.3, 7.5, 1.6 Hz, C8&9AH), 7.42–7.08 (2H,

m, C7&4AH), 7.04 (1H, td, J= 7.5, 1.4 Hz, C2AH), 6.73(2H, dd, J = 11.8, 4.5 Hz, C1&3AH), 6.11 (1H, s, C5AH),4.25 (2H, q, J= 5.9 Hz, ACH2), 2.23 (3H, s, ACH3), 1.36

(3H, t, J= 5.9 Hz, ACH3);13C NMR (100 MHz, DMSO-d6)

d 166.42, 166.12, 160.08, 157.22, 146.82, 141.40, 139.47,127.91, 127.44, 127.15, 124.57, 121.61, 114.02, 107.69, 61.50,55.90, 18.12, 14.70; ESI-MS: m/z calculated 351.42, found

[M+H]+ 352.4.

2.2.4. Ethyl-8-bromo-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]

thiazolo[3,2-a]pyrimidine-3-carboxylate (4b)

Yellow solid, mp 165 �C; Anal. Calcd for C19H16BrN3O2S: C,53.03; H, 3.75; Br, 18.57; N, 9.76; O, 7.44; S, 7.45%; found C,53.22; H, 3.55; N, 9.58; S, 7.66%; IR (KBr) (tmax, cm

�1): 3042

(CAHstr), 2933 (CAHstr), 1749 (C‚Ostr), 1656 (C‚Cstr), 1675(C‚Nstr), 1277 (CANstr), 1170 (CAOstr);

1H NMR (400 MHz,DMSO) d 8.67 (1H, dd, J = 7.5, 1.4 Hz, C6AH), 7.77 (1H, td,

J = 7.4, 1.4 Hz, C8AH), 7.72–7.62 (2H, m, C9&4AH), 7.33(1H, td, J = 7.4, 1.5 Hz, C7AH), 7.19 (1H, dd, J = 7.5,1.6 Hz, C2AH), 6.47 (1H, d, J = 7.5 Hz, C1AH), 5.74 (1H, s,

C5AH), 4.25 (2H, q, J = 5.9 Hz, ACH2), 2.21 (3H, s,ACH3), 1.36 (3H, t, J = 5.9 Hz, ACH3);

13C NMR(100 MHz, DMSO-d6) d 166.42, 166.12, 160.08, 157.22,146.82, 143.49, 139.47, 128.82, 127.91, 126.10, 124.22, 123.54,

121.56, 115.45, 107.69, 61.50, 55.90, 18.12, 14.70; ESI-MS:m/z calculated 430.32, found [M+H]+ 431.3.

2.2.5. Ethyl-2,8-dimethyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate (4c)

Yellow solid, mp 148 �C; Anal. Calcd for C20H19N3O2S: C,65.73; H, 5.24; N, 11.50; O, 8.76; S, 8.77%; found C, 65.55;

H, 5.41; N, 11.69; S, 8.56%; IR (KBr) (tmax, cm�1): 3046(CAHstr), 2946 (CAHstr), 1741 (C‚Ostr), 1651 (C‚Cstr),1610 (C‚Nstr), 1343 (CANstr), 1230 (CAOstr);

1H NMR


(400 MHz, DMSO) d 8.60 (1H, dd, J = 7.5, 1.4 Hz, C6AH),7.74 (2H, dtd, J = 9.1, 7.5, 1.5 Hz, C8&9AH), 7.44–7.28 (2H,m, C4&7AH), 6.89 (1H, dd, J= 7.5, 1.4 Hz, C2AH), 6.56

(1H, d, J = 7.5 Hz, C1AH), 6.08 (1H, s, C5AH), 4.25 (2H,q, J = 5.9 Hz, ACH2), 2.34 (3H, s, ACH3), 2.23 (3H, s,C3ACH3), 1.36 (3H, t, J = 5.9 Hz, ACH3);

13C NMR

(100 MHz, DMSO-d6) d 166.42, 166.12, 160.08, 157.22,146.82, 140.75, 139.47, 135.27, 128.24, 127.91, 124.00, 122.43,121.56, 112.72, 107.69, 61.50, 55.90, 21.21, 18.12, 14.70; ESI-

MS: m/z calculated 365.45, found [M+H]+ 366.4.

2.2.6. Ethyl-2,6-dimethyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate (4d)

Off white solid, mp 260 �C; Anal. Calcd for C20H19N3O2S: C,65.73; H, 5.24; N, 11.50; O, 8.76; S, 8.77%; found C, 65.48; H,5.14; N, 11.35; S, 8.55%; IR (KBr) (tmax, cm�1): 3089

(CAHstr), 2923 (CAHstr), 1744 (C‚Ostr), 1625 (C‚Cstr),1593 (C‚Nstr), 1289 (CANstr), 1249 (CAOstr);

1H NMR(400 MHz, DMSO) d 8.61 (1H, dd, J = 7.5, 1.4 Hz, C6AH),7.71 (2H, dtd, J = 8.9, 7.5, 1.4 Hz, C8&9AH), 7.43–7.22 (2H,

m, C4&7AH), 6.88 (1H, dd, J= 7.5, 1.4 Hz, C2AH), 6.67(1H, t, J = 7.5 Hz, C3AH), 5.94 (1H, s, C5AH), 4.24 (2H, q,J= 5.9 Hz, ACH2), 2.38 (3H, s, C1-CH3), 2.11 (3H, s,

ACH3), 1.37 (3H, t, J = 5.9 Hz, ACH3);13C NMR

(100 MHz, DMSO-d6) d 166.11, 160.08, 157.22, 146.82,139.50, 130.19, 128.61, 127.91, 127.02, 121.56, 118.62, 118.08,

107.69, 61.5, 55.58, 18.07, 14.70; ESI-MS: m/z calculated365.45, found [M+H]+ 366.4.

2.2.7. Ethyl-2-methyl-8-nitro-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate (4e)

Yellow solid, mp 174–178 �C; Anal. Calcd for C19H16N4O4S:C, 57.57; H, 4.07; N, 14.13; O, 16.14; S, 8.09%; found C,

57.37; H, 4.27; N, 14.31; S, 8.22%; IR (KBr) (tmax, cm�1):

3050 (CAHstr), 2933 (CAHstr), 1737 (C‚Ostr), 1632 (C‚Cstr),1597 (C‚Nstr), 1313 (CANstr), 1256 (CAOstr);

1H NMR(400 MHz, DMSO) d 8.61 (1H, dd, J = 7.5, 1.4 Hz, C6AH),

8.41 (1H, d, J = 1.4 Hz, C4AH), 7.97 (1H, dd, J = 7.5,1.4 Hz, C2AH), 7.77 (1H, td, J = 7.5, 1.4 Hz, C8AH), 7.59(1H, dd, J = 7.5, 1.6 Hz, C9AH), 7.33 (1H, td, J = 7.5,

1.4 Hz, C7AH), 6.85 (1H, d, J = 7.5 Hz, C1AH), 5.57 (1H, s,C5AH), 4.23 (2H, q, J = 5.9 Hz, ACH2), 2.18 (3H, s,ACH3), 1.37 (3H, t, J = 5.9 Hz, ACH3);

13C NMR

(100 MHz, DMSO-d6) d 166.42, 166.12, 160.08, 157.22,147.15, 146.82, 145.82, 139.47, 133.29, 127.91, 123.63, 121.56,115.53, 112.10, 107.69, 61.50, 55.90, 18.12, 14.70; ESI-MS:

m/z calculated 396.42, found [M+H]+ 397.4.

2.2.8. Ethyl-8-chloro-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate (4f)

Fluorescent Yellow solid, mp 177–178 �C; Anal. Calcd forC19H16ClN3O2S: C, 59.14; H, 4.18; Cl, 9.19; N, 10.89; O,8.29; S, 8.31%; found C, 59.32; H, 4.29; N, 10.65; S, 8.13%;

IR (KBr) (tmax, cm�1): 3075 (CAHstr), 2831 (CAHstr), 1741

(C‚Ostr), 1642 (C‚Cstr), 1610 (C‚Nstr), 1329 (CANstr),1287 (CAOstr);

1H NMR (400 MHz, DMSO) d 8.67 (1H, dd,J= 7.5, 1.4 Hz, C6AH), 7.77 (1H, td, J = 7.4, 1.4 Hz,

C8AH), 7.68 (1H, dd, J = 7.5, 1.4 Hz, C9AH), 7.49 (1H, d,J= 1.6 Hz, C4AH), 7.33 (1H, td, J = 7.4, 1.5 Hz, C7AH),7.03 (1H, dd, J = 7.5, 1.4 Hz, C2AH), 6.52 (1H, d,



4 M.N. Bhoi et al.

J= 7.5 Hz, C2AH), 5.75 (1H, s, C5AH), 4.25 (2H, q,J= 5.9 Hz, ACH2), 2.21 (3H, s, ACH3), 1.36 (3H, t,J= 5.9 Hz, ACH3);

13C NMR (100 MHz, DMSO-d6) d166.42, 166.12, 160.08, 157.22, 146.82, 141.00, 139.47, 132.27,129.15, 127.91, 127.13, 121.56, 120.29, 117.30, 107.69, 61.50,55.90, 18.12, 14.70; ESI-MS: m/z calculated 385.87, found

[M+H]+ 386.8.

2.2.9. Ethyl-6-chloro-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]

thiazolo[3,2-a]pyrimidine-3-carboxylate (4g)

Pink solid, mp 195 �C; Anal. Calcd for C19H16ClN3O2S: C,59.14; H, 4.18; Cl, 9.19; N, 10.89; O, 8.29; S, 8.31%; foundC, 59.33; H, 4.39; N, 10.65; S, 8.24%; IR (KBr) (tmax,

cm�1): 3012 (CAHstr), 2846 (CAHstr), 1740 (C‚Ostr), 1643(C‚Cstr), 1669 (C‚Nstr), 1290 (CANstr), 1283 (CAOstr);

1HNMR (400 MHz, DMSO) d 8.70 (1H, dd, J = 7.5, 1.4 Hz,

C6AH), 7.72 (2H, dtd, J= 9.1, 7.5, 1.5 Hz, C8&9AH), 7.44–7.25 (2H, m, C4&7AH), 7.04 (1H, dd, J = 7.5, 1.4 Hz,C2AH), 6.67 (1H, t, J = 7.5 Hz, C3AH), 6.29 (1H, s,C5AH), 4.23 (2H, q, J = 5.9 Hz, ACH2), 2.40 (3H, s,

ACH3), 1.36 (3H, t, J = 5.9 Hz, ACH3);13C NMR

(100 MHz, DMSO-d6) d 166.11, 160.08, 157.22, 146.82,139.47, 137.37, 129.08, 128.21, 127.91, 125.88, 123.72, 121.56,

121.02, 107.69, 61.50, 55.58, 18.12, 14.70; ESI-MS: m/z calcu-lated 385.87, found [M+H]+ 386.8.

2.2.10. Ethyl-8-fluoro-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a] pyrimidine-3-carboxylate (4h)

Greenish Yellow solid, mp 160 �C; Anal. Calcd forC19H16FN3O2S: C, 61.77; H, 4.37; F, 5.14; N, 11.37; O, 8.66;

S, 8.68%; found C, 61.51; H, 4.25; N, 11.52; S, 8.83%; IR(KBr) (tmax, cm�1): 3077 (CAHstr), 2941 (CAHstr), 1743(C‚Ostr), 1636 (C‚Cstr), 1641 (C‚Nstr), 1370 (CANstr),

1183 (CAOstr);1H NMR (400 MHz, DMSO) d 8.65 (1H, dd,

J= 7.5, 1.4 Hz, C6AH), 7.74 (1H, td, J= 7.5, 1.5 Hz,C8AH), 7.62 (1H, dd, J= 7.5, 1.4 Hz, C9AH), 7.33 (1H, td,J= 7.5, 1.5 Hz, C7AH), 7.20 (1H, dd, J= 8.1, 1.4 Hz,

C4AH), 6.78 (1H, td, J = 7.9, 1.5 Hz, C2AH), 6.61 (1H, dd,J= 7.5, 5.1 Hz, C1AH), 6.00 (1H, s, C5AH), 4.23 (2H, q,J= 5.9 Hz, ACH2), 2.44 (3H, s, ACH3), 1.36 (3H, t,

J= 5.9 Hz, ACH3);13C NMR (100 MHz, DMSO-d6) d

166.42, 166.12, 160.08, 159.48, 157.22, 146.82, 139.47, 138.13,128.01–127.80, 121.56, 117.98, 115.88, 110.25–110.05, 107.69,

61.50, 55.90, 18.12, 14.70; ESI-MS: m/z calculated 369.09,found [M+H]+ 370.0.

2.2.11. Ethyl-8-methoxy-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate (4i)

Yellow solid, mp 150 �C; Anal. Calcd for C20H19N3O3S: C,62.97; H, 5.02; N, 11.02; O, 12.58; S, 8.41%; found C, 62.75;

H, 5.29; N, 11.27; S, 8.25%; IR (KBr) (tmax, cm�1): 3082(CAHstr), 2932 (CAHstr), 1739 (C‚Ostr), 1629 (C‚Cstr),1655 (C‚Nstr), 1269 (CANstr), 1181 (CAOstr);

1H NMR

(400 MHz, DMSO) d 8.60 (1H, dd, J= 7.5, 1.4 Hz, C6AH),7.77 (1H, td, J = 7.5, 1.5 Hz, C8AH), 7.68 (1H, dd, J = 7.5,1.4 Hz, C9AH), 7.33 (1H, td, J = 7.5, 1.6 Hz, C7AH), 7.06(1H, d, J = 1.4 Hz, C4AH), 6.62 (1H, dd, J = 7.5, 1.4 Hz,

C2AH), 6.52 (1H, d, J = 7.5 Hz, C1AH), 5.73 (1H, s,C5AH), 4.23 (2H, q, J = 5.9 Hz, ACH2), 3.80 (3H, s,AOCH3), 2.58 (3H, s, ACH3), 1.36 (3H, t, J = 5.9 Hz,


ACH3);13C NMR (100 MHz, DMSO-d6) d 166.42, 166.12,

160.08, 157.59, 157.22, 146.82, 139.47, 136.72, 127.91, 127.49,121.56, 118.44, 114.13, 111.15, 107.69, 61.50, 55.97, 18.12,

14.70; ESI-MS: m/z calculated 381.11, found [M+H]+ 382.1.

2.2.12. Ethyl-8-ethoxy-2-methyl-4-(pyridin-2-yl)-4H-benzo

[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate (4j)

Greenish Yellow solid, mp 141 �C; Anal. Calcd for C21H21

N3O3S: C, 63.78; H, 5.35; N, 10.63; O, 12.14; S, 8.11%; foundC, 63.57; H, 5.15; N, 10.41; S, 8.29%; IR (KBr) (tmax, cm

�1):


1H NMR(400 MHz, DMSO) d 8.66 (1H, dd, J= 7.5, 1.4 Hz, C6AH),

7.76 (1H, td, J= 7.5, 1.4 Hz, C8AH), 7.62 (1H, dd, J = 7.5,1.4 Hz, C9AH), 7.33 (1H, td, J= 7.4, 1.5 Hz, C7AH), 7.06(1H, d, J = 1.4 Hz, C4AH), 6.62 (1H, dd, J= 7.5, 1.4 Hz,

C2AH), 6.53 (1H, d, J = 7.4 Hz, C1AH), 5.63 (1H, s,C5AH), 4.21 (2H, q, J= 5.9 Hz, ACH2), 4.03 (2H, q,J= 5.9 Hz, AOCH2), 2.41 (3H, s, ACH3), 1.35 (6H, dt,J= 31.7, 5.9 Hz, -2CH3);

13C NMR (100 MHz, DMSO-d6) d166.42, 166.12, 160.08, 157.29, 146.82, 139.47, 136.46, 127.91,127.11, 121.56, 118.40, 115.70, 112.52, 107.69, 63.99, 61.50,55.90, 18.12, 14.70, 13.83; ESI-MS: m/z calculated 395.47,

found [M+H]+ 396.4.

2.2.13. Ethyl-2-methyl-4-(pyridin-2-yl)-8-(trifluoromethoxy)-

4H-benzo[4,5]thiazolo[3,2-a] pyrimidine-3-carboxylate (4k)

Yellow solid, mp 177–180 �C; Anal. Calcd for C20H16F3N3

O3S: C, 55.17; H, 3.70; F, 13.09; N, 9.65; O, 11.02; S,7.36%; found C, 55.35; H, 3.54; N, 9.48; S, 7.12%; IR (KBr)

(tmax, cm�1): 3052 (CAHstr), 2925 (CAHstr), 1742 (C‚Ostr),

1657 (C‚Cstr), 1654 (C‚Nstr), 1370 (CANstr), 1170 (CAOstr);1H NMR (400 MHz, DMSO) d 8.60 (1H, dd, J= 7.5, 1.4 Hz,

C6AH), 7.75 (2H, dtd, J = 9.1, 7.5, 1.5 Hz, C8&9AH), 7.42–7.23 (1H, m, C7AH), 7.09 (1H, d, J = 1.4 Hz, C4AH), 6.74(1H, dd, J= 7.5, 1.6 Hz, C2AH), 6.59 (1H, d, J = 7.5 Hz,C1AH), 5.63 (1H, s, C5AH), 4.25 (2H, q, J = 5.9 Hz,

ACH2), 2.58 (3H, s, ACH3), 1.36 (3H, t, J = 5.9 Hz,ACH3);

13C NMR (100 MHz, DMSO-d6) d 166.42, 166.12,160.08, 157.22, 149.85, 146.82, 139.47, 131.49, 127.91, 125.37,

122.75, 122.34, 121.56, 114.16, 107.69, 61.50, 55.90, 18.12,14.70; ESI-MS: m/z calculated 435.42, found [M+H]+ 436.4.

2.2.14. Ethyl-8-hydroxy-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate (4l)

Yellow solid, mp 177–178 �C; Anal. Calcd for C19H17N3O3S:C, 62.11; H, 4.66; N, 11.44; O, 13.06; S, 8.73%; found C,

62.39; H, 4.48; N, 11.68; S, 8.61%; IR (KBr) (tmax, cm�1):


1H NMR

(400 MHz, DMSO) d 8.59 (1H, dd, J= 7.5, 1.4 Hz, C6AH),7.77 (1H, td, J= 7.4, 1.4 Hz, C8AH), 7.69 (1H, dd, J= 7.5,1.6 Hz, C9AH), 7.33 (1H, td, J= 7.5, 1.5 Hz, C7AH), 6.95

(1H, d, J= 1.4 Hz, C4AH), 6.59–6.49 (2H, m, C1&2AH), 5.97(1H, s, C5AH), 4.81 (1H, s, AOH), 4.24 (2H, q, J = 5.9 Hz,ACH2), 2.23 (3H, s, ACH3), 1.35 (3H, t, J= 5.9 Hz, ACH3);13C NMR (100 MHz, DMSO-d6) d 166.42, 166.12, 160.08,

157.22, 154.78, 146.82, 139.47, 134.83, 127.91, 126.04, 121.56,118.57, 113.55, 111.39, 107.69, 61.50, 55.90, 18.12, 14.70;ESI-MS: m/z calculated 367.10, found [M+H]+ 368.1.




2.3. Determination of antibacterial activity

Antibacterial activities of 4a–4l were carried out in Nutrient-agar plates by well diffusion assay. Cultures were activatedin Nutrient broth. Isolates were inoculated in Nutrient broth

and incubated at 37 �C for 24 h for activation of culturesand then centrifuged at 3000 rpm for 15 min and the super-natant was collected to study antibacterial activity.

Using in vitro agar well diffusion method, antimicrobial

activity experiments were carried out. The activity of 4a–4l

against test microorganisms of activated test cultures (24 hold culture) (2 Gram negative and 2 Gram positive; viz. Enter-

obacter aerogens MTCC No. 8558, Escherichia coli MTCCNo. 1610, Micrococcus luteus MTCC No. 11948 and Bacilluscereus MTCC No. 8557) was inoculated in molten agar and

poured into sterile plates. The volume of test culture was0.1 ml whose optical density is equal to 0.235 at 750 nm andwas allowed to solidify. Wells with 5 mm diameter were pre-

pared at equal distance in solidified agar plates usingcup-borer. Various derivatives 4a–4l with 1000 lg/ml concen-tration were inoculated in the wells of nutrient agar whereastest microorganisms were inoculated by pour plate technique.

The plates were incubated at 37 �C for 24 h. The inhibitionzones were measured at the end of the incubation period andwere compared and envisaged with positive and negative con-

trols where streptomycin (1000 lg/ml) is a standard taken andthe drug was dissolved in 95% DMF.

2.4. Antioxidant assay protocol

Each sample was dissolved in 95% DMF to make a concentra-tion of 1 mg/ml and then diluted to prepare a series of concen-trations for antioxidant assays. Reference chemicals were used

for comparison in all assays.

2.4.1. DPPH radical scavenging activity assay

The free radical scavenging activity of the 4a–4l was measuredin vitro by 2,20-diphenyl-1-picrylhydrazyl (DPPH) assayaccording to the method described by Brand-Williams et al.(1995). The stock solution was prepared by dissolving 24 mg

DPPH with 100 ml methanol and stored at 20 �C as required.The working solution was obtained by diluting DPPH solutionwith DMF to attain an absorbance of about 0.98 ± 0.02 at

517 nm using the spectrophotometer. A 3 ml aliquot of thissolution was mixed with 100 ll of the sample at various con-centrations (0–600 lg/ml) as shown in Table 5. The reaction

mixture was shaken well and incubated in the dark for15 min at room temperature. Then the absorbance was mea-sured at 517 nm.

The control was prepared as aforesaid without any sample.The scavenging activity was estimated based on the percentageof DPPH radical scavenged as per the following equation:

Scavenging effect ð%Þ¼ Control absorbance� Sample absorbance

Control absorbance� 100

2.4.2. Superoxide radical scavenging activity

The superoxide radical scavenging activity was assayed

according to the reported method as shown by Nishikimi


et al. (1972), Beauchamp and Fridovich (1971), andPithawala and Jain (2014). Superoxide anions were generatedin a phenazine methosulfate–nicotinamide adenine dinu-

cleotide (PMS–NADH) system through the reaction ofPMS–NADH and oxygen. It was assayed by the reductionof nitroblue tetrazolium (NBT). All the solutions used in this

experiment were prepared in phosphate buffer (pH 7.4). 1 mlof NBT (156 lM), 1 ml of NADH (468 lM) and 1 ml of com-pound (0–600 lg/ml) were mixed. The reaction was initiated by

adding 1 ml of PMS (60 lM) and the mixture was incubated at25 �C for 5 min followed by measurement of absorbance at560 nm spectrophotometrically. The decreased absorbance ofthe reaction mixture indicated increased superoxide anion

scavenging activity. The percentage inhibition was calculatedfrom the formula,

Inhibition ð%Þ ¼ Control absorbance� Sample absorbance

Control absorbance

� 100

A percent inhibition versus concentration curve was plottedand the concentration of 4a–4l required for 50% inhibitionwas determined and expressed as IC50 value. The lower IC50

value indicates high antioxidant capacity (Raghavendraet al., 2013).

2.4.3. ABTS+ radical scavenging activity

The 2,20-azinobis (3-ethylbenzthiazoline-6-sulfonic acid), com-monly called ABTS+ cation scavenging activity was per-formed (Re et al., 1999). Briefly, ABTS solution (7 mM) was

reacted with potassium persulfate (2.45 mM) solution and keptfor overnight in the dark to yield a dark colored solution con-taining ABTS+ radical cations. Prior to use in the assay, theABTS+ radical cation was diluted with 50% DMF for an ini-

tial absorbance of about 0.70 ± 0.02 at 745 nm, with temper-ature control set at 30 �C. Free radical scavenging activity wasassessed by mixing 300 ll of test sample with 3.0 ml of ABTS

working standard in a micro cuvette. Each concentration ofsample was taken in range of 0–600 lg/ml as shown in Table 5.The decrease in absorbance was measured exactly one minute

after mixing the solution, and then up to 6 min. The percentageinhibition was calculated according to the formula:

Scavenging effect ð%Þ¼ Control absorbance� Sample absorbance

Control absorbance� 100

The antioxidant capacity of test samples was expressed as EC50

(anti-radical activity), the concentration necessary for 50%reduction of ABTS+ (Gulcin et al., 2011).

2.5. In vitro anti-mycobacterial activity

Here we have performed in vitro anti-mycobacterial activity of

all synthesized compounds. Antimicrobial assays performed inLowenstein Jensen (L-J) medium. Determination of Colonyforming units (c.f.u) on Lowenstein-Jensen (L-J) – The ten-fold dilution of standard 1 mg/ml M. tuberculosis suspension

(Canetti et al., 1969) was streaked on L-J medium for deter-mining c.f.u in the presence and absence of plant extracts.An M. tuberculosis suspension of 1 mg/ml is equivalent to

MacFarland standard (Kent and Kubica, 1985). One loopful(6 ll) of this suspension was streaked on the L-J slants using



6 M.N. Bhoi et al.

3 mm external diameter loop. Reagents of L-J media includedpotassium di hydrogen phosphate anhydrous (Qualigens),magnesium sulfate anhydrous (Qualigens), magnesium citrate

(Loba Chemie), L-asparagine (Hi-media, Mumbai), glycerol(Fisher Scientific, Mumbai), and malachite green (Hi-Media,Mumbai). The drug was incorporated in the medium at con-

centration of 2 percent v/v and 4 percent v/v of drug was dis-solved into 100 ml of culture medium prior to inspissation. Themedium set inoculated with the standard bacterial suspension

and incubated at 37 �C for 42 days. Reading was taken weekly.For comparison, drug free control slants were used. Suscepti-bility testing of MDR isolates was also performed against stan-dard drugs such as rifampicin and isoniazid in the same batch

of media for comparison of cfu on drug free controls. Each testwas done in duplicate. Percentage inhibition was calculated bymean reduction in number of colonies on drug containing as

compared to drug free controls.

Table 1 Optimization of reaction by catalysts and solvents for

the synthesis of 4a by conventional method.

No. Time (h) Catalyst Solvent Temp. (�C) Yielda (%)

1 0.75 PdCl2 Neat 90 75.6

2 0.95 Zn(NO3)2 Neat 90 30.7

3 1.17 Na2WO4 Neat 90 63.0

4 1.25 InCl3 Neat 90 73.2

5 1.30 Cr(NO3)2 Neat 90 51.3

6 1.33 Pb(NO3)2 Neat 90 59.7

7 1.40 Mg

(NO3)2

Neat 90 44.6

8 1.42 Cu(NO3)2 Neat 90 43.5

9 1.42 CoCl2 Neat 90 67.2

10 1.58 PdCl2 DMF 90 64.8

11 1.58 BaCl2 Neat 90 67.8

12 1.60 AlCl3 Neat 90 59.3

13 1.60 LaCl3 Neat 90 72.0

14 1.65 CaCl2 Neat 90 45.6

15 1.67 ZnCl2 Neat 90 56.6

16 1.72 Co(NO3)2 Neat 90 50.6

17 1.75 MgCl2 Neat 90 43.1

18 1.77 NiCl2 Neat 90 62.8

19 1.80 Cd(NO3)2 Neat 90 58.6

20 1.82 TiCl3 Neat 90 64.6

21 1.83 Ca(NO3)2 Neat 90 47.7

22 1.83 Sr(NO3)2 Neat 90 54.7

23 1.83 CAN Neat 90 71.8

24 1.87 CrCl3 Neat 90 65.4

25 1.92 Ba(NO3)2 Neat 90 62.7

26 2.00 NH4VO3 Neat 90 61.1

27 2.00 Bi(NO3)2 Neat 90 71.6

28 2.33 MnCl2 Neat 90 52.3

29 2.50 FeCl3 Neat 90 51.2

30 2.50 ZnOCl2 Neat 90 63.1

31 2.67 SnCl2 Neat 90 65.7

32 48.0 – DMF 90 36.7

33 72.0 – ACN 90 39.3

34 96.0 – Methanol 65 46.2

35 120 – THF 70 40.2

36 240 – DCM 45 20.5

Reaction conditions: Pyridine 2-aldehyde (1 mmol) 1, ethyl ace-

toacetate (1 mmol) 2, 2-aminobenzothiazole (1 mmol) 4a, various

catalysts (10 mol%), solvent/solvent-free conditions, D.a Isolated yield.


% Inhibition ¼ Cc � Ct

Cc

� 100

wherec = control,

t = test.

2.6. Statistical analysis

The determination assay for each antioxidant parameter was

carried out in thrice to minimize the percentage of error.Statistical analysis was performed using the Statistical Packagefor Social Science (IBM SPSS Statistics 20 for Windows, SPSS

Inc., Chicago, IL, USA).

3. Result and discussion

In the present study it has been interpreted and observed thatthe catalysts have a major role in the organic synthesis, byeither enhancing the rate of reaction or any other aspects in

pertaining to the reaction. The same has been implementedbut however it may not divulge any enhancement in the

Figure 1 Optimization study of PdCl2 as catalyst in the synthesis

of 4a.

Table 2 Optimization study of PdCl2 for the synthesis of 4a

by conventional method.

No. Catalyst (%) Time (h) Yielda (%)

1 No catalyst 10.0 45.5

2 1 2.87 58.2

3 2 2.58 62.1

4 5 2.17 68.3

5 10 0.75 75.6

6 15 0.75 76.2

7 20 0.75 74.1

Reaction conditions: Pyridine 2-aldehyde (1 mmol) 1, ethyl ace-

toacetate (1 mmol) 2, 2-aminobenzothiazole (1 mmol) 4a, various

catalysts (mol%), solvent-free conditions, 90 �C, D.a Isolated yield.



Table 3 Comparison of yield and reaction time for the synthesis of 4a–4l.

Entry Benzothiazole Product D MW

Time (min) Yielda (%) Time (min) Yielda (%)

1 45 75.6 3 85.1

2 51 74.7 3 82.3

3 47 78.2 3 87.4

4 55 71.6 5 80.6

5 69 70.6 5 80.1

6 53 72.3 3 82.6

7 44 70.3 3 81.4

8 40 75.6 3 85.3

9 57 73.3 3 82.5

10 20 79.4 3 87.2

(continued on next page)


Please cite this article in press as: Bhoi, M.N. et al., Novel benzothiazole containing 4H-pyrimido[2,1-b]benzothiazoles derivatives: One pot, solvent-free microwaveassisted synthesis and their biological evaluation. Arabian Journal of Chemistry (2016), http://dx.doi.org/10.1016/j.arabjc.2016.01.012


Scheme 1 Synthetic route for the synthesis of title compounds under solvent-free condition.

Table 3 (continued)

Entry Benzothiazole Product D MW

Time (min) Yielda (%) Time (min) Yielda (%)

11 52 71.2 4 80.8

12 58 69.3 4 77.8

Reaction conditions: Pyridine 2-aldehyde (1 mmol) 1, ethyl acetoacetate (1 mmol) 2, various derivatives of 2-amino benzothiazole (1 mmol) 3a–

3l, PdCl2 catalyst (10 mol%), solvent-free conditions, 90 �C, D & 90 �C, MW.a Isolated yield.

8 M.N. Bhoi et al.

reaction which could give necessary prediction or their reac-tions as well.

Based on promising result as shown in Table 1, we have

subsequently studied the effect of five solvents DMF(N,N-Dimethylformamide), ACN (Acetonitrile), Methanol,THF (Tetrahydrofuran), DCM (Dichloromethane) and vari-

ous catalysts (30) in the reaction for the synthesis of 4a from2-amino benzothiazole 3a as a basic structural configuratoralong with pyridine 2-aldehyde 1 and ethyl acetoacetate 2 by

conventional method. Methanol was used as a solvent for thisreaction at 65 �C temperature and it gave 46.2% yield in 96 h(entry 34), while the relative yield of 4a is 36.7% in 48 h by

using DMF as solvent at 90 �C temperature (entry 32). But,to our surprise, we observed that the reaction did not proceedefficiently (Table 1, entries 33, 35–36) in other remaining sol-vents. The yield and time of product 4a could be improved to

64.8% (1.58 h) when the reaction was performed in DMF at90 �C temperature by using PdCl2 as catalyst (entry 10).But, the reaction proceeds efficiently when it was carried out

under neat condition by conventionally heating for 0.75 h inthe presence of PdCl2 catalyst to afford compound 4a in75.6% yield (entry 1). InCl3 is known to have a second cata-

lyst (with 73.2% yield at 1.3 h) of interest from rest of the cat-alysts used (entry 4). Thus, we conclude that PdCl2 catalystand solvent-free condition is the optimized condition for thistransformation.

The selected catalyst (PdCl2), was then optimized to workout the maximum yield with minimized reaction time by con-ventional method. Catalyst loading of 0, 1, 2, 5, 10, 15, and


20 (PdCl2 mol%) was used. Out of all these varying concentra-tions, the maximum efficient value was obtained with 10 mol%, as shown in Fig. 1 with maximum yield of 75.6% in

0.75 h of reaction time. The value of yield increases in a grad-ual fashion, but is known to decrease its value in higher con-centrations of catalysts beyond 15 mol%. This might be due

to the interference of catalyst. However, it was also noticedto have a fixed reaction time (0.75 h) for the completion ofthe 4a product as revealed in Table 2.

By following the same criteria of conventional method, wehave carried out the reaction under the CEM discover micro-wave reactor using an open vessel technique (Dallinger and

Kappe, 2007; Stadler and Kappe, 2000; Stadler et al., 2002)for the synthesis of 4a as well as other derivatives (seeScheme 1). In Table 3, we have shown the comparison of reac-tion time and yield of product by both conventional and MW

methods, comparative aspects with reference to yield and rateof reaction. MW method was found to be better than the con-ventional one as there is a remarkable reduction in reaction

rate. MW is superior in terms of reaction rate, where there isa decrease from 12 to 15 folds and an increase in yield of pro-duct from 8% to 10%.

The compound 4a was formed with 85.1% yield in 3 min ofreaction time, which was confirmed by different spectroscopictechniques such as IR, 1H NMR, 13C NMR, Mass and elemen-tal analysis and X-ray analysis. The IR spectrum of 4a exhib-

ited seven main peaks located at 3072, 2943, 1745, 1677, 1688,1352, 1240 cm�1, which were assigned to the (CAHstr) aro-matic, (CAHstr) alkanes, (C‚Ostr) ester, (C‚Cstr) alkene,




(C‚Nstr) imine, (CANstr) aromatic amine, and (CAOstr) esterrespectively. The mass spectrum of 4a showed a molecular ionpeak at ESI-MS: m/z calculated 351.42, found [M+H]+ 352.4

which indicates the existence of 4a. The 1H NMR spectrum of4a revealed pyridine ring hydrogen (PyAH) peaks shown at8.59 (1H, dd, J= 7.5, 1.3 Hz, C6AH), 7.74 (2H, dtd,

J = 9.3, 7.5, 1.6 Hz, C8&9AH), 7.42–7.08 (1H, C7AH) d value.Chiral center hydrogen resonated at 6.11 d and side chainof ester group contains ACH2, ACH3 shows peak at 4.25,

Figure 2 ORTEP diagram of compound 4a with the atom

numbering.

Scheme 2 Plausible mechanism for the synthe


1.36 d. Also aromatic hydrogens (ArAH) resonated at 7.04(J= 7.5, 1.4 Hz), 6.73 (J= 11.8, 4.5 Hz) d value.

The structure of compound 4a was also confirmed from

single-crystal X-ray diffraction studies. Fine crystals of com-pound 4a were obtained by slow evaporation of ethyl acetate:hexane (4:1, v/v) mixture with compound 4a. It revealed the

formation of pure 4a, which crystallizes as a monoclinic crystalsystem with a space group P21/n. The results showed theproper structure and configuration as 4a, shown in ORTEP

plot (CCDC No.: 1048059) along with the atom numberingin Fig. 2.

Taking into consideration the entire outcome, a plausiblemechanistic pathway for the synthesis of 4a is depicted in

Scheme 2.

3.1. Antibacterial activity

Antibacterial activity of 4a–4l was carried out on Nutrient-agar plates by well-diffusion assay against test culture.Cultures were activated in Nutrient broth. The zone of inhibi-

tion was measured in terms of zone diameter and with the helpof that zone index was calculated where streptomycin was usedas standard.

3.1.1. Determination of activity index

The activity index of the probiotic culture was calculated asfollows:

Activity index ðA:I:Þ¼ Mean of zone of inhibition of derivative

Zone of inhibition obtained for standard antibiotic drug

sis of 4H-pyrimido[2,1-b]benzothiazoles 4a.



Table 4 Antibacterial activity of 4a–4l.

Derivatives Enterobacter aerogens MTCC

No. 8558

Escherichia coli MTCC

No. 1610

Micrococcus luteus MTCC

No. 11948

Bacillus cereus MTCC

No. 8558

Mean value for

zone of inhibition

(mm)

Activity

index

(A.I.)

Mean value for

zone of inhibition

(mm)

Activity

index

(A.I.)

Mean value for

zone of inhibition

(mm)

Activity

index

(A.I.)

Mean value for

zone of inhibition

(mm)

Activity

index

(A.I.)

4a 22 ± 1.135 0.9166 21 ± 1.047 0.8750 19 ± 1.062 0.7916 18 ± 0.802 0.7500

4b 23 ± 1.112 0.9583 24 ± 1.124 1.0000 18 ± 1.045 0.7500 17 ± 0.812 0.7083

4c 34 ± 1.156 1.4166 31 ± 1.075 1.2910 27 ± 1.023 1.1250 19 ± 0.815 0.7916

4d 32 ± 1.142 1.3333 27 ± 1.072 1.1250 26 ± 1.047 1.0833 22 ± 0.842 0.9166

4e 30 ± 1.179 1.2500 30 ± 1.034 1.2500 28 ± 1.051 1.1666 24 ± 0.812 1.0000

4f 26 ± 1.120 1.0833 28 ± 1.159 1.1666 24 ± 1.078 1.0000 16 ± 0.823 0.6666

4g 27 ± 1.200 1.1250 25 ± 1.124 1.0416 29 ± 1.051 1.2083 19 ± 0.864 0.7916

4h 23 ± 1.141 0.9583 22 ± 1.118 0.9166 28 ± 1.023 1.1666 25 ± 0.846 1.0416

4i 24 ± 1.423 1.0000 21 ± 1.113 0.8750 21 ± 1.036 0.8750 22 ± 0.815 0.9166

4j 22 ± 1.231 0.9166 20 ± 1.076 0.8333 23 ± 1.061 0.9583 18 ± 0.841 0.7500

4k 26 ± 1.187 1.0833 25 ± 1.041 1.0416 27 ± 1.036 1.1250 21 ± 0.826 0.8750

4l 26 ± 1.154 1.0833 27 ± 1.023 1.1250 23 ± 1.078 0.9583 20 ± 0.831 0.8333

Std drug 24 – 24 – 24 – 24 –

Figure 3 Antibacterial activity of 4a–4l.

Figure 4 % DPPH radical scavenging activity assay at various

concentrations.

10 M.N. Bhoi et al.


Note: Standard drug used was streptomycin with 1000 lg/mlconcentration.

Table 4 shows that all synthesized derivatives 4a–4l havebeen screened with antimicrobial activity. Gram negative was

more inhibited as compared to Gram Positive bacteria. Also4c–4g were proved to have more antibacterial property as com-pared to the streptomycin while contrastingly 4a, 4b, 4j were

having a lesser activity index, as shown in Fig. 3; however, itcould be considered to have antimicrobial activity.

3.2. Antioxidant activity

Free radical scavenging activity was evaluated by performingin vitro DPPH assay. As shown in Fig. 4 and Table 5, %

DPPH activity was calculated and it is found that all deriva-tives 4a–4l have an antioxidant property.

Also the activity is dose dependent in order to scavenge theradical. The higher concentration indicates a greater amount

of scavenging of the DPPH radical. The colored DPPH solu-tion faded reducibly to half at an optimal concentration rangefrom 200 lg/ml to 400 lg/ml. This may be induced and influ-

enced due to electron transfer or the reduction of the DPPHmolecules or thought to be due to their hydrogen donatingability (Baumann et al., 1979).

As an indirect indication it may also be a time dependentscavenging activity. As obtained almost all of the derivativesfollowed a general rule of dose dependent scavenging activitydespite the degree of electron transfer. However 4l is known

to have a more efficient scavenging property and the leastknown 4a with a gradual antioxidant property. At higher con-centration 4h has a consistent reduction of DPPH scavenging

activity to 50% with an optimal concentration range and rela-tively lesser % scavenging activity rate, as compared to itslower concentration. 4e and 4f showed a constant % scaveng-

ing activity following an accuracy as a dose dependent antiox-idant reaction.

The scavenging of the ABTS+ radical by the derivatives

4a–4l was found to be higher than that of DPPH radical athigher concentration level, indicating that they may be usefulas therapeutic agents for treating radical-related pathological



Table 5 % DPPH radical scavenging activity assay.

Antioxidant assays*

% DPPH radical scavenging activity assay at various concentrations

Mean ± S.E.

Derivatives 0.00 lg/ml 200 lg/ml 400 lg/ml 600 lg/ml

4a 0.00 30.00 ± 1.434 43.11 ± 2.627 76.63 ± 1.083

4b 0.00 36.71 ± 1.421 57.68 ± 2.645 76.77 ± 1.082

4c 0.00 29.48 ± 1.415 53.69 ± 2.678 79.83 ± 1.074

4d 0.00 32.75 ± 1.399 59.03 ± 2.632 84.75 ± 1.062

4e 0.00 36.18 ± 1.423 63.95 ± 2.641 86.51 ± 1.087

4f 0.00 37.75 ± 1.420 63.73 ± 2.678 84.66 ± 1.079

4g 0.00 38.47 ± 1.478 70.01 ± 2.694 79.92 ± 1.069

4h 0.00 47.02 ± 1.456 58.86 ± 2.632 75.55 ± 1.082

4i 0.00 32.06 ± 1.406 57.84 ± 2.591 83.84 ± 1.076

4j 0.00 36.71 ± 1.478 56.55 ± 2.589 82.21 ± 1.080

4k 0.00 39.99 ± 1.495 53.70 ± 2.645 79.03 ± 1.082

4l 0.00 40.73 ± 1.435 79.83 ± 2.632 84.88 ± 1.023

* p value < 0.05, data significant.

Figure 5 % Superoxide anion scavenging activity assay at

various concentrations.

Table 6 % Superoxide anion scavenging activity assay.

Antioxidant assays*

% Superoxide anion scavenging activity assay at various concentrations

Mean ± S.E.


4a 0.00 37.94 ± 0.799 55.90 ± 1.681 83.15 ± 1.294

4b 0.00 31.93 ± 0.785 52.49 ± 1.675 81.16 ± 1.285

4c 0.00 32.66 ± 0.789 53.33 ± 1.684 78.31 ± 1.279

4d 0.00 35.61 ± 0.794 59.60 ± 1.672 88.88 ± 1.286

4e 0.00 36.95 ± 0.779 55.10 ± 1.689 83.47 ± 1.284

4f 0.00 38.83 ± 0.784 61.11 ± 1.628 79.06 ± 1.296

4g 0.00 31.00 ± 0.796 50.88 ± 1.678 84.45 ± 1.276

4h 0.00 37.66 ± 0.769 69.04 ± 1.684 88.29 ± 1.263

4i 0.00 36.74 ± 0.782 62.20 ± 1.689 79.04 ± 1.284

4j 0.00 39.00 ± 0.788 65.90 ± 1.691 74.37 ± 1.275

4k 0.00 36.66 ± 0.784 58.36 ± 1.693 81.11 ± 1.263

4l 0.00 33.30 ± 0.783 65.65 ± 1.682 88.03 ± 1.290


Figure 6 % ABTS+ radical scavenging activity assay at various

concentrations.


Please cite this article in press as: Bhoi, M.N. et al., Novel benzothiazole containing 4H-pyrimido[2,1-b]benzothiazoles derivatives: One pot, solvent-free microwaveassisted synthesis and their biological evaluation. Arabian Journal of Chemistry (2016), http://dx.doi.org/10.1016/j.arabjc.2016.01.012


Table 7 % ABTS+ radical scavenging activity assay.

Antioxidant assays*

% ABTS+ radical scavenging activity assay at various concentrations

Mean ± S.E.


4a 0.00 12.33 ± 1.709 39.45 ± 1.227 64.93 ± 1.741

4b 0.00 22.83 ± 1.712 43.43 ± 1.233 66.83 ± 1.751

4c 0.00 25.76 ± 1.756 41.07 ± 1.2s45 68.33 ± 1.762

4d 0.00 13.94 ± 1.741 36.72 ± 1.245 70.84 ± 1.743

4e 0.00 18.42 ± 0.762 38.11 ± 1.224 63.63 ± 1.742

4f 0.00 11.63 ± 1.723 42.66 ± 1.278 68.92 ± 1.795

4g 0.00 19.20 ± 1.722 36.99 ± 1.235 73.94 ± 1.756

4h 0.00 26.39 ± 1.730 40.40 ± 1.212 76.76 ± 1.745

4i 0.00 28.23 ± 1.749 45.82 ± 1.254 84.69 ± 1.762

4j 0.00 27.46 ± 1.764 47.97 ± 1.252 65.92 ± 1.732

4k 0.00 17.30 ± 1.785 38.41 ± 1.274 75.41 ± 1.742

4l 0.00 22.73 ± 1.734 49.50 ± 1.262 68.43 ± 1.746


12 M.N. Bhoi et al.

damage. DPPH and ABTS+ radical scavenging activities arebased on the ability of antioxidants to donate a hydrogen atomor an electron to stabilize radicals by converting them to the

non-radical species (Binsan et al., 2008; Chandrasekara andShahidi, 2010). DPPH is a radical having an odd electronand reacts with hydrogen donated from antioxidant.

In case of ABTS+ radical scavenging activity, the effective-ness depends on the molecular weight, the number of aromaticrings and the nature of hydroxyl group’s substitution than

the specific functional groups (Hagerman et al., 1998). Super-oxide anion scavenging assay at various concentrations in therange of 0–600 lg/ml was evaluated to calculate the IC50

values of each of derivatives 4a–4l. The IC50 values were cor-

respondingly in between the range of 280–400 lg/ml. The val-ues correspond to 4a (332 lg/ml), 4b (372 lg/ml), 4c (368 lg/ml), 4d (316 lg/ml), 4e (344 lg/ml), 4f (300 lg/ml), 4g

(390 lg/ml), 4h (281 lg/ml), 4i (301 lg/ml), 4j (285 lg/ml), 4k(321 lg/ml), and 4l (301 lg/ml) respectively as shown inFig. 5 and Table 6. ABTS+ radical scavenging activity at var-

ious concentrations indicated the EC50 values as 4a (482 lg/

Table 8 In vitro anti-mycobacterial activity of newly synthe-

sized compound.

Sample code Mean c.f.u. on media Percentage

inhibition (%)Control Treatment

(100 lg/ml)

4a 155 50 67.74

4b 155 65 58.00

4c 155 42 72.90

4d 155 75 51.61

4e 155 46 70.32

4f 155 81 47.71

4g 155 49 68.38

4h 155 66 57.41

4i 155 71 54.19

4j 155 62 60.00

4k 155 55 64.51

4l 155 53 65.80


ml), 4b (455 lg/ml), 4c (468 lg/ml), 4d (479 lg/ml), 4e

(492 lg/ml), 4f (455 lg/ml), 4g (470 lg/ml), 4h (450 lg/ml), 4i(425 lg/ml), 4j (425 lg/ml), 4k (462 lg/ml), and 4l (405 lg/ml) respectively as shown in Fig. 6 and Table 7. This may sheda new horizon in pharmaceutical based drug development andan active drug moiety indicating the increased application of

the synthesized compounds.

3.3. In vitro anti-mycobacterial activity

In vitro anti-mycobacterial activity showed that all the com-pounds are bioactive against M. tuberculosis H37Rv. FromTable 8, Compounds 4c and 4e showed very good % inhibition

72.9% and 70.32% against M.tuberculosis H37RV. Also com-pounds 4a, 4g, 4j, 4k and 4l exhibited good antimycobacterialactivity (% inhibition 67.74%, 68.38%, 60%, 64.51% and65.80%) against M.tuberculosis H37RV. The compounds 4b,

4d and 4h showed moderate to good antimycobacterial activity(% inhibition 58%, 51.61% and 57.41%) against M.tuberculo-sis H37RV. Compounds 4f, displayed less antimycobacterial

activity (% inhibition 47.71%) toward M.tuberculosis H37RV.

4. Conclusion

In summary, we have described a novel, efficient, one pot, three

component reaction of some typical 2-aminobenzothiazole derivatives,

pyridine 2-aldehyde and ethyl acetoacetate to develop ethyl 2-methyl-

4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate

derivatives using PdCl2 as a prompt catalyst under microwave assisted

solvent-free conditions. The simple one-pot nature of the reaction

makes it an interesting approach. The main advantages of this current

methodology are novel with operational simplicity, good yields, simple

workup, superficial automation, without any anhydrous conditions.

The reactions were carried out under solvent free condition which

are considerably safer, nontoxic, and environmental friendly. Further-

more, the existence of three different important heterocyclic moieties

such as benzothiazole, pyridine, and pyrimidine rings in one molecule

is fantastic on account of the potential applications of these derivatives

in biological and pharmacological activities. The synthesized com-

pounds were evaluated in vitro antibacterial and antioxidant activities.

The antibacterial data revealed that the all synthesized compounds

proved to be active against the test organism, two gram negative and




two gram positive reference strains compared to standard drugs.

Moreover, all the synthesized compounds were evaluated as antioxi-

dants according to a DPPH radical scavenging activity, superoxide

anion scavenging assay and ABTS+ radical scavenging activity of

our synthesized compound. All compounds showed moderate to good

antioxidant activity. We have also screened antitubercular activity

against Mycobacterium tuberculosis H37RV strain. Compounds 4c

and 4e showed very good % inhibition 72.9% and 70.32% respectively

against M.tuberculosis H37RV and other compound showed moderate

to good antitubercular activity. Further optimization and pharmacoki-

netic characterization of this series are in progress in our laboratory.

Acknowledgment

The authors are thankful to UGC-Info net and INFLIBNETGujarat University for providing e-source facilities. BothManoj N. Bhoi and Mayuri A. Borad are thankful to UGC-BSR fellowship for financial assistance.

Appendix A. Supplementary material

Crystallographic data for the structures 4a have been depos-

ited with Cambridge Crystallo graphic Data Centre as supple-mentary CCDC 1048059, respectively. These data can beobtained from http://www.ccdc.cam.ac.uk/conts/retrieving.

html or from the Cambridge Crystallographic Data Centre,12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223336 033; or e-mail: [email protected]. Supplementary

data associated with this article can be found, in the onlineversion, at http://dx.doi.org/10.1016/j.arabjc.2016.01.012.

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