MEDICINAL CHEMISTRY RESEARCH Med Chem Res DOI 10.1007/s00044-014-0962-8 ORIGINAL RESEARCH Synthesis, characterization, and biological activities of 4-imino-3arylazo-4H-pyrimido[2,1-b][1,3]benzothiazole-2-oles Selinay Erişkin • Nesrin Şener • Serkan Yavuz İzzet Şener • Received: 4 November 2013 / Accepted: 17 February 2014 Ó Springer Science+Business Media New York 2014 Abstract 4-Imino-3,4-dihydro-2H-pyrimido[2,1-b][1,3] benzothiazole-2-one (3) was synthesized by the reaction of 2-aminobenzothiazole with ethyl cyanoacetate in solvent free conditions at 150 °C. A series of pyrimido benzothiazole-based azo dyes 4(a–m) were obtained by the coupling of carbocyclic amine-based diazonium chloride with compound (3). The synthesized dyes were purified and characterized by elemental analysis, FT-IR, 1 H NMR, and high-resolution mass spectral data. The solvatochromic behaviors of dyes in various solvents were examined. All the azo dyes exhibited pronounced in vitro antibacterial activities against Gram-positive and Gram-negative bacteria, as well as fungi. The results revealed that most of the compounds exhibited good levels of antibacterial activity. Compounds 4d and 4h showed excellent levels of antimicrobial activity with MIC values of 8.25 lg/mL. Keywords Solvatochromism
Pyrimido benzothiazole
Diazo-coupling reaction
Azo dyes
Antimicrobial activity Electronic supplementary material The online version of this article (doi:10.1007/s00044-014-0962-8) contains supplementary material, which is available to authorized users. S. Erişkin
N. Şener
İ. Şener (&) Department of Chemistry, Faculty of Science-Arts, Pamukkale University, 20017 Denizli, Turkey e-mail: isener@pau.edu.tr; isener@pamukkale.edu.tr S. Yavuz Department of Chemistry, Faculty of Science, Gazi University, 06500 Ankara, Turkey Introduction Fused heterocyclic compounds are very important compounds partially because of their pharmacogical properties which include wide applications in medicinal chemistry (Ellis, 2008). Pyrimido benzothiazoles are very important class of heterocyclic because of their wide range of biological pharmacological properties. Some of which have antiviral, antitumor, anti-inflammatory, antihypertensive, and anti-allergy (Kappe, 1993; Atwal et al., 1989, 1991; Rovnyak et al., 1992; El-Sherbeny, 2000; Glennon et al., 1981; Bartovic et al., 1995). Pyrimido benzothiazole derivatives have also been known for their antimicrobial activities (Wade et al., 1983; Alaimo, 1973; Gupta and Rawat, 2010; Lanjewar et al., 2009; Heravi et al., 2008; Chang et al., 2008; Shah et al., 2009; Kumar et al., 2009; Chaitanya et al., 2010; Sahu et al., 2012). As a continuation of our previous work (Karcı et al., 2006, 2009; Öztürk et al., 2012; Şener et al., 2006; Yavuz and Yildirim, 2013). 4-imino-3,4-dihydro-2Hpyrimido[2,1-b][1,3]benzothiazole-2-one have been reported in the literature, by the cyclocondesation of benzothiazole with ethyl cyanoacetate in the presence of sodium ethoxide/ethanol, or phosphoric acid (Tadakazu, 1991). We wish to give new report the synthesis of 4-imino-3,4-dihydro-2H-pyrimido[2,1-b][1,3]benzothiazole-2-one by the reaction of 2-aminobenzothiazole with ethyl cyanoacetate in solvent free conditions at 150 °C. Although a number of paper have been published concerning the synthesis of pyrimidine and azopyrimidine derivatives, those containing an azo pyrimidobenzothiazole system have not yet been reported. In the present study, we reported the synthesis of 4-imino-3,4-dihydro-2H-pyrimido[2,1-b][1,3]benzothiazole-2one and some 4-imino-3-arylazo-4H-pyrimido[2,1-b][1,3]benzothiazole-2-ol derivatives. The synthesized azo dyes were evaluated biological and antimicrobial activities. 123 Med Chem Res O S S S CN NH2 + EtO 150 oC EtO- NH N N N N O O NC 1 NH 3 2 Scheme 1 Synthesis of compound (3) 13 S N NH2 O + N 8 12 7 S NaNO2 / HCl N 3 11 X 1 2 9 6 N 10 4 5 NH NH 3 14 OH 16 N 15 N 18 17 4 (a-m) 19 22 X 21 a; b; c; d; X: H X: p-NO2 X: p-OCH3 X: p-Cl e; f; g; h; X: p-CH3 X: m-NO2 X: m-OCH3 X: m-Cl i; j; k; l; X: m-CH3 X: o-NO2 X: o-OCH3 X: o-Cl 20 m; X: o-CH3 Scheme 2 Synthesis of compounds 4(a–m) Results and discussion Chemistry In this study, 4-imino-3-arylazo-4H-pyrimido[2,1-b][1,3] benzothiazole-2-ol derivatives have been reported. The structures of the synthesized dyes were determined by analysis of their FT-IR, NMR, high-resolution mass spectra for the molecular weights and elemental analysis. As shown in Scheme 1, firstly, 4-imino-3,4-dihydro-2H-pyrimido[2,1-b][1,3]benzothiazole-2-one was prepared by the reaction of 2-aminobenzothiazole with ethyl cyanoacetate in solvent free conditions at 150 °C. Secondly, aniline derivatives were diazotized using sodium nitrite in hydrochloric acid in ice bath at between 0 and 5 °C. The synthesized carbocyclic diazonium salts were then coupled with 4-imino-3,4-dihydro-2H-pyrimido[2,1-b][1,3]benzothiazole-2-one to give corresponding 4-imino-3-arylazo-4H-pyrimido[2,1-b][1,3]benzothiazole2-ol derivatives. This reaction route is depicted in Scheme 2. By the purification of the reaction mixture, the thirteen novel azo pyrimido[2,1-b][1,3]benzothiazole derivatives have been obtained. The obtained product 4(a–m) can exist in possible four tautomeric forms namely amino-azo-keto form T1, iminoazo-enol form T2, imino-hydrazo-keto form T3, iminoazo-keto form T4 as shown in Scheme 3. The FT-IR 123 spectra of synthesized compound (3) showed an intense (–OH) band at 3375 cm-1, and a band (=NH) located at 3167–3133 cm-1. The other band at 1697 cm-1assigned to (C=N). FT-IR spectra of compound (3) did not appear any band for (C=O) group. The 1H NMR spectra of compounds (3) exhibited singlet signal at d 4.15 ppm attributed to (–CH2) protons and broad peak at d 12.75 ppm, which was attributed to (=NH) proton. The IR spectra of dyes 4(a– m) showed characteristic hydroxyl (–OH) bands at 3211–3385 cm-1, but did not showed any bands for carbonyl (C=O) group. We can suggest that these compounds were only in imino-azo-enol form (T2) in solid state. 1H NMR spectra of synthesized azo pyrimido[2,1-b][1,3]benzothiazole derivatives 4(a–d) exhibit a broad peak at 12.01–12.28 ppm, which was attributed to =NH protons, a broad peak at 12.58–13.25 ppm which was attributed hydrazo (–NH) protons and showed a broad peak at 13.50–14.22 ppm attributed to tautomeric hydroxyl (–OH). 1 H NMR spectra of dyes did not showed –NH2 protons except for 4j. 1H NMR spectra of 4g, 4k, 4l, and 4m showed only =NH proton, but did not showed hydrazo –NH protons. 1H NMR spectra of 4e, 4f, 4h, and 4i showed only =NH proton, and hydrazo –NH protons. 1H NMR spectra of 4j showed only –NH2 protons. According to the 1 H NMR results, suggest that dyes 4(a–d) a mixture of predominantly in imino-azo-enol form (T2) and iminohydrazo-keto form (T3), the dyes of 4e, 4f, 4h, and 4i have Med Chem Res S N S N N OH N NH N O N H NH imino-azo-enol T2 N N X KT KT X S imino-hydrazo-keto N T3 N O N NH2 N X amino-azo-keto T1 KT S N N NH imino-azo-keto O N N X T4 Scheme 3 The tautomeric form of azo pyrimido[2,1-b][1,3] benzothiazole derivatives only in imino-hydrazo-keto form (T3), the dyes of 4g, 4k, 4l, and 4m have only in imino-azo-enol form (T2). The dye of 4j has in amino-azo-keto (T1). All the synthesized dyes did not have in imino-azo-keto (T4) tautomeric forms in DMSO-d6 as showed in Scheme 3. The mass spectra of dyes have molecular ion peak which corresponds to molecular weight of respective compounds. Absorption spectra The UV–Vis absorption spectra of the dyes 4(a–m) were recorded over the range of k between 350 and 700 nm, using variety of solvents in concentration 10-6 to 10-8 M. The spectral data of the synthesized dyes are depicted in Table 1. The visible absorption spectra of the dyes did not correlate with the polarity of solvent. The dyes showed color ranging from dark red (kmax 357 nm) to yellow (kmax 592 nm). It was observed that kmax of p-substitute dyes 4(a,c,d) shifted bathochromically in chloroform with respect to the kmax in dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) except for 4b. For example, for the dye 4a kmax is 385 nm in DMSO, 394 nm in chloroform. For the dye 4d kmax is 391 nm in DMSO 396 in chloroform. The explanation for this irregular behavior may be due to the presence of nonbonding electron pairs of carbonyl, oxygen, and nitrogen atoms in the molecule ring (Harikrishnan and Menon, 2008). General observation made is that most of the substituted dyes have higher absorption maxima when compared to their unsubstituted analogs. The absorption spectra of dye 4j showed two absorbance in all solvent except for acetic acid. The other spectra of the dyes showed single absorption peak with a shoulder or two absorption peak with a shoulder. It can be suggested that all dyes have a mixture of tautomer forms in various solvent. The spectral shifts of the dye 4b in various solvents are shown in Fig. 1. The effects of the acid and base on the absorption spectra of the dyes were investigated and these results are given in Table 2. 123 Med Chem Res Table 1 Influence of solvent on kmax (nm) of dyes Dye no. DMSO DMF 4a 385,325a 387 a Acetonitrile Methanol 403 409,331a Acetic acid 392 Chloroform 394 a 422,334a 411,333a 414,552a 416,341a 426,338a 408,325a 405 a a a 396 4b 417,592,332 4c 403,319a 4d 391,320 a 393,496 413,336 396,323 4e 389,328a 395 385 398,335a 381,320a 385 4f 392,322a 393 387 389,330a 383,322a 387 4g 386,320a 391 380 390,328a 378,319a,478a 380 4h 399,333a,487a 407,332a 392,325 394 4i 387,320a 386,330a 378,318a 382 a 357,414 420,590 419,584 401 a 396,491a 390 4j 365,425 363,416 4k 4l 416,336a 405,327a 413 405,491a 4m 408,330a a 407 404,330 403,485a 381 365,422 359,420 409,345 420 405 420,338 405,334a 410,328a 389,328a 414 401 411 414,341a 400,324a 405 Shoulder The absorption spectra of the dyes were sensitive in methanol to the addition of acid and base. With the exception of dyes 4c, 4k and 4m, when small amount of potassium hydroxide (0.1 M) was added to methanol solution, the kmax values showed bathochromic shift. When hydrochloric acid (0.1 M) was added in methanol hypsochromic shifts were detected except for 4j. The spectral shifts of 4b in acidic and basic solution are shown in Fig. 2. Antimicrobial activity In the present study, a total of thirteen diazo derivatives 4(a–m) was evaluated for their in vitro antibacterial and antifungal activities at 100 lg/mL concentration against microbial strains such as two Gram-positive bacteria (Staphylococcus aureus ATCC 29213 and Bacillus subtilis ATCC 6633), two Gram-negative bacteria (Klebseilla pneumonia ATCC13883 and Escherichia coli ATCC 25922), and two fungi (Saccharomyces cerevisiae and Candida albicans NRRL Y-477). Agar-diffusion method was used for the determination of the preliminary antibacterial and antifungal activity. Ciprofloxacin and Ketoconazole were used as standard antibacterial and antifungal reference, respectively. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the disks in mm. Most of the newly synthesized compounds showed excellent antimicrobial activities with respect to the control drugs. The results in Table 3 revealed that the majority of the synthesized compounds showed variable inhibition activities against the tested strains. Data in Table 3 revealed that the most of tested compounds displayed 123 variable inhibitory effects on the growth of the tested Gram-positive and Gram-negative bacterial strains, and also against antifungal strains. In general, the results revealed that para phenyl substituted compounds exhibited better activities than ortho and meta phenyl substituted compounds. Furthermore, compound 4d having chlorine substituent at para and 4h meta positions of the phenyl ring exhibited potent antimicrobial activities against Grampositive and Gram-negative bacteria, as well as fungi. Most of pyrimido benzothiazole derivatives have superior significant antibacterial potency than antifungal potency. One of the tested compound 4d exhibited good antimicrobial activity against all tested organisms with respect to reference drugs. Compound 4d inhibited the growth of S. aureus ATCC 29213, B. subtilis ATCC6633, and C. albicans NRRL Y-477 with IZs 30, 32, and 31 mm, respectively. Also compound 4h showed excellent activity against S. aureus ATCC 29213 and E. coli ATCC 25922 with IZ 31 and 30 mm, respectively. The minimum inhibitory concentration (MIC) of the synthesized compounds against highly inhibited organisms is reported in Table 4. Compound 4d exhibited low MIC (8.25 lg/mL) against S. aureus ATCC 29213, B. subtilis ATCC 6633, K. pneumonia ATCC13883, S. cerevisiae, and C. albicans NRRL Y-477. In addition, compound 4h showed MIC 8.25 lg/mL against S. aureus ATCC 29213, K. pneumonia ATCC13883, E. coli ATCC 25922, S. cerevisiae, and C. albicans NRRL Y-477. Additionally, compounds 4f and 4l exhibited MIC 8.25 lg/mL against S. aureus ATCC 29213 and also compounds 4b and 4j showed good inhibitory activities against B. subtilis ATCC 6633 (MIC 8.25 lg/mL). Med Chem Res DMSO DMF Acetonitrile Methanol Acetic acid Chloroform Absorbance (Normalized) 1,0 0,8 0,6 0,4 0,2 0,0 400 500 600 MS) instruments (Waters Corporation, Milford MA, USA). Elemental analyses were done on a Leco CHNS-932 analyzer (USA). UV–Vis absorption spectra were recorded on an ATI (UK) Unicam UV-100 spectrophotometer over the range of k between 300 and 700 nm. The wavelengths of maximum absorption (kmax) were investigated in various solvents such as DMSO, DMF, acetonitrile, methanol, acetic acid, and chloroform at various concentrations (1 9 10-6 M). Change of (kmax) was also investigated when 0.1 mL of base (potassium hydroxide, 0.1 M) or 0.1 mL of acid (hydrochloric acid, 0.1 M) was added to 1 mL of the dye solution in methanol. 700 Wavelength (nm) Fig. 1 Absorption spectra of dye 4b in various solvents Synthesis Preparation of 4-imino-3,4-dihydro-2H-pyrimido[2,1b][1,3]benzothiazole-2-one (3) Table 2 Absorption maxima of dyes in acidic and basic solutions Dye no. Methanol Methanol ? KOH Methanol ? HCl 4a 409,331a 410 392 4b 422,334a 408a, 542 415 4c 426,338a 413 415 4d 413,336a 430 396 4e 398, 335a 406 388 4f 389,330a 410 361a, 386 4g 390,328 a 416 381 4h 407,332a 445, 498a 391 a 4i 386,330 406 385 4j 359,420 365a, 466 360a, 418 4k 4l 420,338 405,334a 411 424 416 404 4m 414,341a 402 408 a Shoulder Experimental General All chemicals were purchased and were used without further purification. Solvents were of spectroscopic grade. Melting points of the synthesis dyes were determined using Stuart smp 30 melting point apparatus (UK). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker (Germany) Spectrospin Avance DPX 400 UltraShield spectrometer at room temperature by using tetramethylsilane (TMS) as the internal standard. Chemical shifts were (d) given in ppm. FT-IR spectra were recorded on a Perkin Elmer (USA) spectrometer. MS analyses were recorded on from Waters LCT Premier XE LTOF (TOF The starting compounds were synthesized by the reaction of 2-aminobenzothiazole with ethyl cyanoacetate. 2-aminobenzothiazle 0.2 g (1.33 mmol) reacted with ethyl cyanoacetate 0.7 mL (6.65 mmol) in solvent free conditions. The reaction mixture was heated at 150 °C for 2 h, and then cooled at room temperature and upon dilution with 100 mL ethanol:water mixture (1:1 by volume). The precipitated yellow products separated were filtered off, washed with water several times, and dried. The obtained product was crystalized from DMF:water mixture as yellow crystals, yield (90 %), mp: 236–238 °C; IR (cm-1) tmax: 3375 (OH), 3167–3133 (=NH), 3055 (Ar–H), 2944 (Aliphatic C–H), 1697 (C=N); 1H NMR (DMSO-d6) d (ppm): 4.15 (s, 2H, CH2), 7.31 (d, 1H, J = 7.47 Hz, H-10), 7.45 (1H, J = 7.47 Hz, H-13), 7.78 (1H, t, J = 7.47 Hz, H-11), 8.05 (1H, t, J = 7.47, H-12), 12.75 (b, 1H, pyrimidone =NH); 13C NMR (DMSO-d6) d: 40.09 (CH2, C-5), 110.49 (CH, C-10), 122.05 (CH, C-12), 122.68 (CH, C-13), 124.8 (C, C-8), 127.11 (CH, C-11), 147.6 (C, C-2), 147.82 (C, C-9), 166.9 (C, C-6), 166.96 (C, C-2); HR-MS: 217.2482 [M?H]?, calcd. 217.2485. Anal. Cald. for C10H7N3OS: C, 55.2 %; H, 3.25 %; N, 19.34 %. Found: C, 55.28 %; H, 3.26 %; N, 19.34 %. General procedure for the synthesis of azo dyes 4(a–m) Preparation of 4-imino-3-phenylazo-4H-pyrimido[2.1b][1,3]benzothiazole-2-ole (4a) Aniline 0.93 g (10 mmol) was dissolved in hydrochloric acid (7.5 mL) and water (15 mL) was added. The solution was cooled to 0–5 °C. Sodium nitrite 0.69 g (10 mmol) in water (10 mL) was then added to this cold solution drop 123 Med Chem Res Methanol Methanol + HCl Methanol + KOH Absorbance (Normalized) 1,0 0,8 0,6 0,4 0,2 0,0 400 500 600 700 Wavelength (nm) Fig. 2 Absorption spectra of dye 4b in acidic and basic solutions wise with vigorous stirring over 1 h. Excess nitrous acid was destroyed by the addition of the urea. The resulting diazonium solution was then added in portion over 30 min. to a vigorously stirred solution of 4-imino-3, 4-dihydro-2H-pyrimido [2,1-b][1,3]benzothiazole-2-one 2.17 g (10 mmol), which was dissolved in potassium hydroxide (20 mmol) and water (20 mL). The solution was stirred at 0–5 °C for 2 h and the pH of the reaction mixture was maintained at 6–7 by the simultaneous addition of sodium acetate solutions. The precipitated product separated upon dilution with water (50 mL) was filtered, washed with water several times, dried and crystallized from DMF:water (2:3 by volume) to give 4-imino-3phenylazo-4H-pyrimido[2.1-b][1,3]benzothiazole-2-ole as a yellow crystals, yield (88 %), mp: 249–250 °C. IR (cm-1) tmax: 3385 (–OH), 3167–3132 (=NH), 3039 (Ar– H), 2924 (Aliphatic C–H), 1641 (C=N), 1510, 1453 (N=N); 1 H NMR (400 MHz, DMSO-d6) d (ppm): 7.16 (1H, t, J = 7.24 Hz, H-20), 7.45 (2H, t, J = 7.71 Hz, H-19, H-21), 7.51 (2H, J = 6.95 Hz, H-11, H-12), 7.51 (1H, d, J = 7.45 Hz, H-10), 7.57 (1H, d, J = 7.43 Hz, H-13), 8.03 (2H, d, J = 9.24 Hz, H-18, H-22), 12.25 (1H, b, =NH), 12.72 (1H, b, hydrazo-NH), 14.22 (s, tautomeric –OH); 13C NMR (100 MHz, DMSO-d6) d: 104.90 (CH, C-5), 108.71 (CH– C-10), 112.19 (CH, C-18, C-22), 118.22 (CH, C-20), 122.20 (CH, C-12), 123.27 (C, C-8), 125.18 (CH, C-13), 127.46 (CH, C-11), 129.97 (CH, C-19, C-21), 131.57 (C, C-4), 151.72 (C, C-6), 152.37 (C, C-9), 153.59 (C, C-17), 168 (C, C-2); HR-MS: 321.3576 [M?H]?, calcd. 321.3577. Anal. Cald. for C15H11N5OS: C, 59.80 %; H, 3.45 %; N, 21.79 %; Found: C, 59.81 %; H, 3.46 %; N, 21.78 %. The above procedure was also used to synthesize dye 4(b–g). The general route of synthesized dyes is outlined in Scheme 1. 123 Preparation of 4-imino-3-(40 -nitrophenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4b) Brown solid crystals, (82 %), mp: 232–234 °C. IR (cm-1) tmax: 3211 (–OH), 3104 (=NH), 3088 (Ar–H), 2987 (Aliphatic C–H), 1661 (C=N), 1598–1455 (N=N), 1514, 1336 (NO2); 1H NMR (400 MHz, DMSO-d6) d (ppm): 7.37 (1H, t, J = 8.1 Hz, H-12), 7.54 (1H, t, J = 8.1 Hz, H-11), 7.62 (1H, d, J = 8.74 Hz, H-13), 7.70 (1H, d, J = 8.75 Hz, H-10), 8.01 (1H, d, J = 7.24 Hz, H-22), 8.04 (1H, d, J = 7.24 Hz, H-18), 8.30 (2H, d, J = 8.74 Hz, H-19, H-21), 12.01 (1H, b, =NH), 13.25 (1H, b, hydrazo-NH), 14.13 (s, tautomeric –OH). 13C NMR (100 MHz, DMSOd6) d: 104.90 (C, C-5), 108.71 (CH, C-10), 122.2 (CH, C-12), 122.9 (CH, C-18, C-22), 123.27 (C, C-8), 125.18 (CH, C-13), 126.01 (CH, C-21), 127.46 (CH, C-11), 131.57 (C, C-4), 149.65 (C, C-20), 151.72 (C, C-6), 152.37 (C, C-9), 155.28 (C, C-17), 168 (C, C-2); HR-MS: 366.3552 [M?H]?, calcd. 366.3553. Anal. Calcd. For C16H10N6O3S: C, 52.45 %; H, 2.75 %; N, 22.94 %; Found: C, 52.46 %; H, 2.76 %; N, 22.93 %. Preparation of 4-imino-3-(40 -methoxyphenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4c) Orange solid crystals, (79 %), mp: 216–218 °C. IR (cm-1) tmax: 3251 (–OH), 3128 (=NH), 3028 (Ar–H), 2995 (Aliphatic C–H), 1637 (C=N), 1509–1455 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 3.79 (3H, s, –OCH3), 7.35 (1H, t, J = 7.79 Hz, H-12); 7.39 (1H, t, J = 7.79 Hz, H-11); 7.47 (1H, d, J = 8.27 Hz, H-13); 7.64 (1H, d, J = 7.79 Hz, H-10), 7.78 (1H, d, J = 7.79 Hz, H-21), 7.87 (2H, d, J = 7.79 Hz, H-18, H-22), 8.03 (1H, d, J = 7.79 Hz, H-19), 12.20 (1H, b, =NH), 12.58 (1H, b, hydrazo-NH), 14.06 (s, tautomeric –OH). 13C NMR (100 MHz, DMSO-d6) d: 55.46 (CH3, C-25), 104.90 (C, C-5), 108.71 (CH– C-10), 115.31 (CH, C-19, C-21), 122.2 (CH, C-12), 123.27 (C, C-8), 124.16 (CH, C-18, C-22), 125.18 (CH, C-13), 127.46 (CH, C-11), 131.57 (C, C-4), 147.46 (C, C-17), 151.72 (C, C-6), 152.37 (C, C-9), 160.04 (C, C-20), 168 (C, C-2); HR-MS: 351.3836 [M?H]?, calcd. 351.3837. Anal. Calcd. for C17H13N5O2S: C, 58.11 %; H, 3.73 %; N, 19.93 %; Found: C, 58.12 %; H, 32.74 %; N, 19.92 %. Preparation of 4-imino-3-(40 -chlorophenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4d) Dark orange solid crystals, (81 %), mp: 248–250 °C. IR (cm-1) tmax: 3260 (–OH), 3186 (=NH), 3062 (Ar–H), 2940 (Aliphatic C–H), 1637 (C=N), 1495–1454 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 7.41 (1H, t, J = 7.47 Hz, H-12), 7.54 (1H, t, J = 7.47 Hz, H-11), 7.63 (1H, d, Med Chem Res Table 3 Antimicrobial activity expressed as inhibition diameter zones in millimeters (mm) of chemical compounds against the pathological strains based on well-diffusion assay Compounds Zone of inhibition (mm) Bacteria Fungi Gram-positive S. aureus Gram-negative B. subtilis K. pneumoniae E. coli S. cerevisiae C. albicans 4a 22 ± 0.5 19 ± 1.7 16 ± 1.1 18 ± 0.7 14 ± 1.2 15 ± 1.6 4b 28 ± 1.8 32 ± 1.3 26 ± 0.6 16 ± 0.9 14 ± 1.4 18 ± 1.4 4c 22 ± 1.2 22 ± 0.8 25 ± 1.3 28 ± 2.0 16 ± 1.1 16 ± 1.2 4d 30 ± 1.6 32 ± 0.9 29 ± 0.8 28 ± 1.9 29 ± 0.3 31 ± 0.3 4e 24 ± 0.7 22 ± 1.5 24 ± 1.6 20 ± 1.3 16 ± 0.6 15 ± 2.1 4f 32 ± 0.9 28 ± 1.4 24 ± 1.3 25 ± 1.5 21 ± 1.1 19 ± 1.4 4g 25 ± 2.1 27 ± 1.5 30 ± 0.9 31 ± 1.7 24 ± 1.4 28 ± 1.6 4h 31 ± 0.4 28 ± 0.9 29 ± 0.8 30 ± 1.8 23 ± 1.2 29 ± 0.8 4i 18 ± 0.7 16 ± 0.3 20 ± 1.6 19 ± 0.8 12 ± 0.5 13 ± 0.7 4j 4k 28 ± 1.2 22 ± 1.6 30 ± 1.3 24 ± 2.4 24 ± 1.4 24 ± 1.2 22 ± 1.3 26 ± 0.6 18 ± 0.7 29 ± 1.2 18 ± 1.5 28 ± 0.6 4l 30 ± 1.0 24 ± 1.8 18 ± 0.5 22 ± 1.4 19 ± 1.6 23 ± 1.2 4m 19 ± 1.4 18 ± 1.4 12 ± 1.7 12 ± 1.1 12 ± 1.4 14 ± 2.2 Ciprofloxacin 28 ± 1.2 29 ± 0.6 29 ± 1.3 24 ± 0.7 NT NT Ketoconazole NT NT NT NT 28 ± 1.2 28 ± 1.4 Zone of inhibition values are presented as the mean ± SEM from at least three separated experiments NT not tested J = 7.47 Hz, H-13), 7.65 (1H, d, J = 7.47 Hz, H-10), 7.85 (1H, d, J = 8.18 Hz, H-21), 8.97 (2H, d, J = 7.44 Hz, H-22, H-18), 8.02 (1H, J = 7.43 Hz, H-19), 12.28 (1H, b, = NH), 12.79 (1H, b, hydrazo-NH), 13.50 (b, tautomeric –OH); 13C NMR (100 MHz, DMSO-d6) d: 104.90 (C, C-5), 108.71 (CH– C-10), 122.2 (CH, C-12), 123.27 (C, C-8), 123.75 (CH, C-18, C-22), 125.18 (CH, C-13), 127.46 (CH, C-11), 130.4 (CH, C-21, C-19), 131.57 (C, C-4), 133.63 (C, C-20), 151.72 (C, C-6), 152.28 (C, C-17), 152.37 (C, C-9), 168 (C, C-2); HR-MS: 355.8024 [M?H]?, calcd. 355.8023. Anal. Calcd. For C16H10ClN5OS: C, 54.01 %; H, 2.83 %; N, 19.68 %; Found: C, 54.01 %; H, 2.84 %; N, 19.67 %. hydrazo-NH); 13C NMR (100 MHz, DMSO-d6) d: 20.65 (CH3, C-25), 108.98 (CH, C-10), 117.05 (CH– C-18, C-22), 123.18 (CH, C-12), 123.49 (CH, C-13), 124.3 (C, C-8), 127.92 (CH, C-11), 130.51 (CH, C-19, C-21), 133.88 (C, C-4), 136.95 (C, C-20), 140.04 (C, C-17), 141.96 (C, C-5), 147.11 (C, C-9), 153.35 (C, C-6), 168.9 (C, C-2); HR-MS: 335.3842 [M?H]?, calcd. 335.3841. Anal. Calcd. for C17H13N5OS: C, 60.88 %; H, 3.91 %; N, 20.88 %; Found: C, 60.89 %; H, 3.92 %; N, 20.87 %. Preparation of 4-imino-3-(40 -methylphenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4e) Burgundy red solid crystals, (79 %), mp: 250–251 °C. IR (cm-1) tmax: 3290 (–OH), 3160 (=NH), 3065 (Ar–H), 2985 (Aliphatic C–H), 1665 (C=N), 1520–1455 (N=N), 1510, 1332 (NO2); 1H NMR (400 MHz, DMSO-d6) d (ppm): 7.23 (1H, t, J = 6.52 Hz, H-21), 7.35 (1H, t, J = 7.73 Hz, H-12), 7.39 (1H, t, J = 7.73 Hz, H-11), 7.47 (1H, d, J = 7.32 Hz, H-13), 7.79 (1H, d, J = 7.33 Hz, H-10), 7.81 (1H, d, J = 7.33 Hz, H-22), 7.97 (1H, d, J = 7.33 Hz, H-20), 8.03 (1H, s, H-18), 12.18 (1H, b, =NH), 12.63 (1H, b, hydrazo-NH); 13C NMR (100 MHz, DMSO-d6) d: 109.98 (CH, C-10), 113.73 (CH, C-22), 115.14 (CH, C-20), 120.6 (CH, C18), 123.18 (CH, C-12), 123.48 (CH, C-13), Orange solid crystals, (83 %), mp: 251–252 °C. IR (cm-1) tmax: 3285 (–OH), 3168 (=NH), 3055 (Ar–H), 2930 (Aliphatic C–H), 1683 (C=N), 1428–1487 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.32 (3H, s, –CH3), 7.27 (2H, d, J = 7.08 Hz, H-19, H-21), 7.35 (1H, t, J = 7.08 Hz, H-12), 7.57 (1H, t, J = 7.09 Hz, H-11), 7.79 (1H, d, J = 7.08 Hz, H-13), 7.81 (1H, d, J = 7.09 Hz, H-10), 7.97 (1H, d, J = 7.09 Hz, H-22), 8.03 (1H, d, J = 7.09 Hz, H-18), 12.18 (1H, b, =NH), 12.62 (1H, b, Preparation of 4-imino-3-(30 -nitrophenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4f) 123 Med Chem Res Table 4 MIC (lg/mL) against the pathological strains based on two fold serial dilution technique Compounds MIC in lg/mL Bacteria Fungi Gram-positive Gram-negative S. aureus B. subtilis K. pneumoniae E. coli S. cerevisiae C. albicans 4a 33 66 132 66 132 132 4b 16.5 8.25 33 132 132 66 4c 33 33 33 16.5 132 132 4d 8.25 8.25 8.25 16.5 8.25 8.25 4e 33 66 33 66 132 132 4f 8.25 16.5 33 33 66 66 4g 33 16.5 8.25 8.25 33 16.5 4h 8.25 16.5 8.25 8.25 33 8.25 4i 66 132 66 66 132 132 4j 16.5 8.25 33 33 132 132 4k 33 33 33 16.5 16.5 16.5 4l 8.25 33 66 33 66 33 4m 66 66 132 132 132 132 Ciprofloxacin Ketoconazole 8.25 NT 8.25 NT 8.25 NT 16.5 NT NT 8.25 NT 16.5 NT not tested 124.3 (C, C-8), 127.92 (CH, C-11), 130.42 (CH, C-19), 133.88 (C, C-4), 141.96 (C, C-5), 146.37 (C, C-17), 147.11 (C, C-9), 149.6 (C, C-21), 153.35 (C, C-6), 168.9 (C, C-2); HR-MS: 366.3552 [M?H]?, calcd. 366.3551. Anal. Calcd. for C16H10N6O3S: C, 52.45 %; H, 2.75 %; N, 22.94 %; Found: C, 52.43 %; H, 2.75 % N, 22.94 %. Preparation of 4-imino-3-(30 -methoxyphenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4g) Yellow solid crystals, (83 %), mp: 219–221 °C. IR (cm-1) tmax: 3311 (–OH), 3175 (=NH), 3062 (Ar–H), 2993 (Aliphatic C–H), 1644 (C=N), 1514–1454 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 3.82 (3H, s, –OCH3), 6.76 (1H, t, J = 6.15 Hz, H-21), 7.08 (2H, t, J = 6.15 Hz, H-11, H-12), 7.34 (1H, d, J = 6.77 Hz, H-13), 7.40 (1H, d, J = 6.77 Hz, H-10), 7.62 (1H, d, J = 6.77 Hz, H-22), 7.97 (1H, d, J = 6.77 Hz, H-20), 8.00 (1H, s, H-18), 14.01 (1H, b, =NH), 14.16 (1H, b, –OH); 13C NMR (100 MHz, DMSO-d6) d: 55.71 (CH3, C-25), 104.9 (C, C-5), 107 (CH, C-18), 108.71 (CH, C-10), 109.62 (CH, C-20), 111.72 (CH, C-22), 122.2 (CH, C-12), 123.27 (C, C-8), 125.18 (CH, C-13), 127.48 (CH, C-11), 131.57 (C, C-4), 133.32 (CH, C-21), 151.72 (C, C-6), 152.15 (C, C-17), 152.37 (C, C-9), 162.88 (C, C-19), 168 (C, C-2); HR-MS: 351.3836 [M?H]?, calcd. 351.3837. Anal. Calcd. for C17H13N5O2S: C, 58.11 %; H, 3.73 %; N, 19.93 %; Found: C, 58.10 %; H, 32.74 %; N, 19.91 %. 123 Preparation of 4-imino-3-(30 -chlorophenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4h) Light orange solid crystals, (84 %), mp: 215–217 °C. IR (cm-1) tmax: 3212 (–OH), 3074 (=NH), 3006 (Ar–H), 2922 (Aliphatic C–H), 1639 (C=N), 1515–1456 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 7.42 (1H, t, J = 7.85 Hz, H-21), 7.52 (2H, t, J = 7.85 Hz, H-11, H-12), 7.75 (1H, d, J = 7.43 Hz, H-13), 7.83 (1H, d, J = 7.44 Hz, H-10), 7.97 (2H, d, J = 7.43 Hz, H-20, H-22), 9.49 (1H, s, H-18), 10.79 (1H, s, =NH), 11.93 (1H, b, hydrazo-NH,); 13C NMR (100 MHz, DMSO-d6) d: 104.9 (C, C-5), 108.71 (CH, C-10), 114.78 (CH, C-22), 115.02 (CH, C-18), 122.2 (CH, C-12), 122.93 (CH, C-20), 123.27 (C, C-8), 125.18 (CH, C-13), 127.46 (CH, C-11), 131.57 (C, C4), 131.67 (CH, C21), 138.84 (C, C-19), 151.72 (C, C-6), 152.37 (C, C-9), 152.59 (C, C-17), 168 (C, C-2); HR-MS: 355.8024 [M?H]?, calcd. 355.8023. Anal. Calcd. for C16H10ClN5OS: C, 54.01 %; H, 2.83 %; N, 19.68 %; Found: C, 54.02 %; H, 2.84 %; N, 19.69 %. Preparation of 4-imino-3-(30 -methylphenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4i) Pink solid crystals, (81 %), mp: 242–243 °C. IR (cm-1) tmax: 3221 (–OH), 3168 (=NH), 3035 (Ar–H), 2960 (Aliphatic C–H), 1683 (C=N), 1508–1489 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.38 (3H, s, –CH3), 7.01 Med Chem Res (1H, d, J = 7.47 Hz, H-20), 7.31 (1H, t, J = 7.47 Hz, H-12), 7.36 (1H, t, J = 7.47 Hz, H-11), 7.49 (1H, t, J = 7.47 Hz, H-21), 7.68 (1H, d, J = 7.47 Hz, H-13), 7.80 (1H, d, J = 7.47 Hz, H-10), 7.96 (1H, s, H-18), 8.03 (1H, d, J = 7.47 Hz, H-22), 12.15 (1H, b, 1H, =NH), 12.56 (1H, b, hydrazo-NH); 13C NMR (100 MHz, DMSO-d6) d: 20.94 (CH3, C-24), 109.98 (CH, C-10), 113.19 (CH, C-18), 118.68 (CH, C-22), 123.18 (CH, C-12), 123.49 (CH, C-13), 124.3 (C, C-8), 126.67 (CH, C-20), 127.92 (CH, C-11), 129.04 (CH, C-19), 133.88 (C, C-4), 140.9 (C, C-21), 141.96 (C, C-5), 147.11 (C, C-9), 147.28 (C, C-17), 153.59 (C, C-6), 168.9 (C, C-2); HR-MS: 335.3842 [M?H]?, calcd. 335.3841. Anal. Calcd. for C17H13N5OS: C, 60.88 %; H, 3.91 %; N, 20.88 %; Found: C, 60.88 %; H, 3.93 %; N, 20.89 %. Preparation of 4-imino-3-(20 -nitrophenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4j) Red solid crystals, (81 %), mp: 264–265 °C. IR (cm-1) tmax: 3260 (–OH), 3150 (=NH), 3076 (Ar–H), 2935 (Aliphatic C–H), 1641 (C=N), 1506–1453 (N=N), 1515, 1337 (NO2); 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.90 (2H, s, –NH2), 7.35 (1H, t, J = 7.73 Hz, H-12), 7.43 (1H, t, J = 7.73 Hz, H-11), 7.58 (2H, t, J = 7.73 Hz, H-21, H-20), 7.86 (1H, d, J = 7.32 Hz, H-13), 7.97 (1H, d, J = 7.32 Hz, H-10), 8.03 (1H, d, J = 7.32 Hz, H-22), 8.27 (1H, d, J = 7.32 Hz, H-19); 13C NMR (100 MHz, DMSOd6) d: 109.86 (CH, C-9), 116.68 (C, C-12), 119.98 (C, C-5), 121.53 (CH, C-6), 122.98 (CH, C-20), 124.71 (CH, C-7), 125.3 (CH, C-22), 125.88 (CH, C-19), 128.62 (CH, C-8), 133.3 (CH, C-21), 136.98 (C, C-18), 143.7 (C, C-4), 143.79 (C, C-17), 147.67 (C, C-11), 162.83 (C, C-2), 175.22 (C, C-13); HR-MS: 366.3552 [M?H]?, calcd. 366.3551. Anal. Calcd. for C16H10N6O3S: C, 52.45 %; H, 2.75 %; N, 22.94 %; Found: C, 52.44 %; H, 2.76 %; N, 22.95 %. Preparation of 4-imino-3-(20 -methoxyphenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4k) Dark red solid crystals, (81 %), mp: 163–165 °C. IR (cm-1) tmax: 3210 (–OH); 3132 (=NH), 3042 (Ar–H), 2963 (Aliphatic C–H), 1651 (C=N), 1506–1454 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 3.98 (3H, s, –OCH3), 7.05 (1H, t, J = 7.66 Hz, H-12), 7.17 (1H, t, J = 7.66 Hz, H-11), 7.19 (1H, t, J = 7.66 Hz, H-20), 7.40 (1H, t, J = 7.95 Hz, H-21), 7.63 (1H, d, J = 7.66 Hz, H-13),7.67 (1H, d, J = 7.66 Hz, H-10), 7.98 (1H, d, J = 7.65 Hz, H-19, H-22), 14.12 (1H, b, =NH), 14.25 (1H, b, –OH); 13C NMR (100 MHz, DMSO-d6) d: 56.1 (CH3, C-25), 104.9 (C, C-5), 108.71 (CH, C-10), 112.58 (CH, C-19), 117.72 (CH, C-22), 121.96 (CH, C-20), 122.2 (CH, C-12), 123.27 (C, C-8), 124.68 (CH, C-21), 125.18 (CH, C-13), 127.46 (CH, C-11), 131.57 (C, C-4), 138.03 (C, C-17), 150.91 (C, C-6), 151.37 (C, C-8), 152.37 (C, C-9), 168 (C, C-2); HRMS: 351.3836 [M?H]?, calcd. 351.3837. Anal. Calcd. for C17H13N5O2S: C, 58.11 %; H, 3.73 %; N, 19.93 %; Found: C, 58.12 %; H, 32.72 %; N, 19.92 %. Preparation of 4-imino-3-(20 -chlorophenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4l) Orange solid crystals, (80 %), mp: 159–162 °C. IR (cm-1) tmax: 3223 (–OH), 3123 (=NH), 3062 (Ar–H), 2979 (Aliphatic C–H), 1644 (C=N), 1500–1452 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm) 7.20 (1H, t, J = 7.50 Hz, H-12), 7.41 (1H, t, J = 7.52 Hz, H-11), 7.45 (1H, t, J = 7.52 Hz, H-20), 7.55 (1H, t, J = 7.50 Hz, H-21), 7.59 (1H, d, J = 7.50 Hz, H-10), 7.71 (1H, d, J = 7.50 Hz, H-13), 7.97 (1H, d, J = 7.50 Hz, H-22), 8.00 (1H, d, J = 7.50 Hz, H-19), 14.21 (1H, b, =NH), 14.48 (1H, b, –OH); 13C NMR (100 MHz, DMSO-d6) d: 104.9 (C, C-5), 108.71 (CH, C-10), 117.26 (CH, C-22), 117.91 (C, C-18), 120.86 (CH, C-20), 122.2 (CH, C-12), 123.27 (C, C-8), 125.18 (CH, C-13), 127.46 (CH, C-11), 128.12 (CH, C-21), 130.82 (CH, C-19), 131.57 (C, C-4), 143.20 (C, C-17), 151.69 (C, C-6), 152.37 (C, C-9), 168 (C, C-2); HR-MS: 355.8024 [M?H]?, calcd. 355.8023. Anal. Calcd. for C16H10ClN5OS: C, 54.01 %; H, 2.83 %; N, 19.68 %; Found: C, 54.03 %; H, 2.82 %; N, 19.67 %. Preparation of 4-imino-3-(20 -methylphenylazo)-4Hpyrimido[2.1-b][1,3]benzothiazole-2-ole (4m) Light red solid crystals, (79 %), mp: 222–224 °C. IR (cm-1) tmax: 3228 (–OH), 3120 (=NH), 3037 (Ar–H), 2974 (Aliphatic C–H), 1661 (C=N), 1497–1453 (N=N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.40 (3H, s, –CH3), 7.31 (1H, t, J = 7.22 Hz, H-12), 7.35 (1H, t, J = 7.22 Hz, H-11), 7.40 (1H, t, J = 6.5 Hz, H-20), 7.54 (1H, t, J = 6.5 Hz, H-21), 7.58 (1H, d, J = 7.22 Hz, H-13), 7.97 (1H, d, J = 7.21 Hz, H-10), 7.99 (1H, d, J = 7.22 Hz, H-22), 14.21 (1H, b, 1H, =NH), 14.34 (1H, b, –OH); 13C NMR (100 MHz, DMSO-d6) d: 16.7 (CH3, C-24), 104.9 (C, C-5), 108.71 (CH, C-10), 111.15 (CH, C-22), 121.22 (CH, C-20), 122.2 (CH, C-12), 123.27 (C, C-8), 125.18 (CH, C-13), 127.07 (CH, C-21), 127.46 (CH, C-11), 128.78 (C, C-18), 131.57 (C, C-4), 132.07 (C, C-19), 150.32 (C, C-17), 152.17 (C, C-6), 152.37 (C, C-9), 168 (C, C-2); HR-MS: 335.3842 [M?H]?, calcd. 335.3841. Anal. Calcd. for C17H13N5OS: C, 60.88 %; H, 3.91 %; N, 20.88 %; Found: C, 60.87 %; H, 3.92 %; N, 20.87 %. Antimicrobial evaluation Newly synthesized compounds 4(a–m) were individually tested against a panel of Gram-positive and Gram-negative bacterial pathogens, yeast, and fungi. The antimicrobial 123 Med Chem Res activity of synthesized compounds was evaluated by the agar well-diffusion method (Yavuz and Yildirim, 2013; Scott, 1989) using 100 lL of suspension containing 1 9 106 CFU/mL of pathological tested bacteria and 1 9 106/mL of yeast spread on nutrient agar (NA) and Sabourand dextrose agar (SDA) respectively. After the media had cooled and solidified, wells (10 mm in diameter) were made in the solidified agar and loaded with 100 lL of tested compound solution prepared by dissolving 100 mg of the chemical compound in 1 mL of DMSO. The inculcated plates were then incubated for 24 h at 37 °C for bacteria and 48 h at 28 °C for fungi. Negative controls were prepared using dimethyl sulphoxide employed for dissolving the tested compound. Ciprofloxacin (50 lg/mL) and Ketoconazole (50 lg/mL) were used as standard for antibacterial and antifungal activity, respectively. After incubation time, antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms and compared with that of the standard. Antimicrobial activities were expressed as inhibition diameter zones in millimeters (mm). The experiment was carried out in triplicate and the average zone of inhibition was calculated. compounds were evaluated and compared with standard drugs. The results revealed that the most of the compounds exhibited good levels of antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, as well as fungi. In particular, compounds 4d and 4h showed excellent levels of antimicrobial activity with MIC values of 8.25 lg/mL. Many of the synthesized motifs, possessing electron withdrawing atom/group such as chlorine and nitro were identified as the most potent antimicrobial activity. The mechanism of action of the compounds tested in this study currently remains unknown. Thus, further studies of related compounds in the context of their structure– activity relationship, toxicity, and other biological effects might be helpful in designing new antimicrobials for therapeutic use. Determination of minimal inhibitory concentration (MIC) Alaimo RJ (1973) Synthesis of some 4H-pyrimido[2,1-b]benzothiazol-4-ones. J Heterocyclic Chem 10:769–772 Atwal KS, Rovnyak GC, O’Reilly BC, Schwartz J (1989) Substituted 1,4-dihydropyrimidines. 3. Synthesis of selectively functionalized 2-hetero-1,4-dihydropyrimidines. J Org Chem 54:5898–5907 Atwal KS, Swanson BN, Unger SE, Floyd DM, Moreland S, Hedberg A, O’Reilly BC (1991) ihydropyrimidine calcium channel blockers. 3. 3-Carbamoyl-4-aryl-1,2,3,4-tetrahydro-6-methyl-5pyrimidinecarboxylic acid esters as orally effective antihypertensive agents. J Med Chem 34:806–811 Bartovic A, Ilavski D, Simo O, Zalibera L, Belicová A, Seman M (1995) Synthesis of nitro-substituted 4-oxo-4H-pyrimido[2,1b]benzothiazole-3-carboxylic acids and their spectral characteristics. Collect Czech Chem C 60:583–593 Chaitanya MS, Negendrappa G, Vaidya VP (2010) Synthesis, biological and pharmacological activities of 2-methyl-4H-pyrimido[2,1-b][1,3]benzothiazoles. J Chem Pharm Res 2:206–213 Chang S, Shun-Jun J, Yu L (2008) A novel, simple and efficient synthesis of 3-amino-benzo[d]imidazo[2,1-b]thiazole derivatives via a multicomponent procedure. J Chin Chem Soc 55:292–296 Ellis GP (2008) Chemistry of heterocyclic compounds: synthesis of fused heterocycles, vol 47. Wiley, New York El-Sherbeny MA (2000) Synthesis of certain pyrimido[2,1-b]benzothiazole and benzothiazolo[2,3-b]quinazoline derivatives for in vitro antitumor and antiviral activities. Arzneimittel-Forsch 50:848–853 Glennon RA, Gaines JJ, Rogers ME (1981) Benz-fused mesoionic xanthine analogs as inhibitors of cyclic-AMP phosphodiesterase. J Med Chem 24:766–769 Gupta A, Rawat S (2010) Synthesis and anti-inflammatory study of novel fluorobenzothiazole derivatives. J Chem Pharm Res 2:244–258 Harikrishnan U, Menon SK (2008) The synthesis, characterization and spectral properties of crown ether based disazo dyes. Dyes Pigment 77:462–468 Heravi MM, Ranjbar L, Derikvand F, Alimadadi B, Oskooie HA, Bamoharram FF (2008) A three component one-pot procedure MIC is the lowest concentration of an antimicrobial compound that will inhibit the visible growth of a microorganism after overnight incubation. The micro dilution susceptibility test in MüllereHinton Broth (Oxoid) was used for the determination of antibacterial activity and Sabouraud Liquid Medium (Oxoid) was used for the determination of antifungal activity. Stock solutions of the tested compounds, Ciprofloxacin and Ketoconazole were prepared in DMF at concentration of 1,000 lg/mL. Twofold serial dilutions of the tested compounds solutions were prepared using the proper nutrient broth. The final concentration of the solutions was 132, 66, 33, 16.5, and 8.25 lg/mL. The tubes were then inoculated with the test organisms, grown in their suitable broth at 37 °C for 24 h for bacteria (about 1 9 106 CFU/ mL), each 5 mL received 0.1 mL of the above inoculum and incubated at 37 °C for 24 h. The lowest concentration showing no growth was taken as the MIC. Control experiments with DMF and uninoculated media were run parallel to the test compounds under the same conditions. The MIC (lg/mL) values are recorded in Table 4. Conclusion In summary, based on our previous work, we have synthesized new series of azo pyrimido[2,1-b][1,3]benzothiazole derivatives. The antimicrobial activities of these 123 Acknowledgments The authors are grateful to the Scientific Research Projects Council of Pamukkale University (PAU.BAP, 2012FBE066, 2011FBE069). References Med Chem Res for the synthesis of [1,2,4]triazolo/benzimidazolo-quinazolinone derivatives in the presence of H6P2W18O62.18H2O as a green and reusable catalyst. Mol Divers 12:181–185 Kappe CO (1993) 100 years of the Biginelli dihydropyrimidine synthesis. Tetrahedron 49:6937–6963 Karcı F, Demirçalı A, Şener İ, Tilki T (2006) Synthesis of 4-amino-1Hbenzo[4,5]imidazo[1,2-a]pyrimidin-2-one and its disperse azo dyes. Part 1. Phenylazo derivatives. Dyes Pigments 71:90–96 Karcı F, Şener N, Yamaç M, Şener İ, Demirçalı A (2009) Synthesis, antimicrobial activity and absorption characteristics of some novel heterocyclic disazo dyes. Dyes Pigments 80:47–52 Kumar R, Malik S, Chandra R (2009) Synthesis and antimicrobial activity of 4-[5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl]-dihydropyridines and 4-[5-chloro-3-methyl-1-phenyl-1H-pyrazol4-yl]-3,4-dihydropyrimidin-2-ones. Indian J Chem B 48B: 718–724 Lanjewar KR, Rahatgaonkar AM, Chorghade MS, Saraf BD (2009) Synthesis and antimicrobial activity of 5-(2-aminothiazol-4-yl)3,4-dihydro-4-phenylpyrimidin-2(1H)-one. Indian J Chem B 48B:1732–1737 Öztürk F, Açık L, Şener İ, Karcı F, Kılıç E (2012) Antimicrobial properties and DNA interaction studies of 3-hetarylazoquinoline2,4-diol compounds. Turk J Chem 36:293–302 Rovnyak GC, Atwal KS, Hedberg A, Kimball SD, Moreland S, Gougoutas JZ, O’Reillly BC, Schwartz J, Malley MF (1992) Dihydropyrimidine calcium channel blockers. 4. Basic 3-substi- tuted-4-aryl-1,4-dihydropyrimidine-5-carboxylic acid esters. Potent antihypertensive agents. J Med Chem 35:3254–3263 Sahu PK, Sahu PK, Gupta SK, Thavaselvam D, Agarwal DD (2012) Synthesis and evaluation of antimicrobial activity of 4Hpyrimido[2,1-b]benzothiazole, pyrazole and benzylidene derivatives of curcumin. Eur J Med Chem 54:366–378 Scott AC (1989) Laboratory control of antimicrobial therapy. In: Collee JG, Duguid JP, Duguid JP, Fraser AG, Marmion BP (eds) Mackie and Mac-Cartney practical medical microbiology, vol 13. Churchill Livingstone, Edinburgh, pp 161–181 Şener İ, Karcı F, Ertan N, Kılıç E (2006) Synthesis and investigations of the absorption spectra of hetarylazo disperse dyes derived from 2,4-quinolinediol. Dyes Pigments 70:143–148 Shah NK, Patel MP, Patel RG (2009) One-pot, multicomponent condensation reaction in neutral conditions: Synthesis, characterization, and biological studies of fused thiazolo[2,3-b]quinazolinone derivatives. Phosphorus Sulfur 184:2704–2719 Tadakazu T (1991) Synthesis and reactions of [1,3-4]thiadiazole[3,2a]pyrimidinone derivatives. J Heterocyclic Chem 28:489–492 Wade JJ, Toso CB, Matson CJ, Stelzer VL (1983) Synthesis and antiallergic activity of some acidic derivatives of 4H-pyrimido[2,1-b]benzazol-4-ones. J Med Chem 26:608–611 Yavuz S, Yıldırım H (2013) Ferrocene derivatives carrying urea, thiourea and sulfonamide moieties: synthesis and evaluation of antibacterial and antifungal activities. J Chem 149693:1–7 123