Synthesis of novel pyrido[2,1-b]benzothiazole and N-substituted 2-pyridylbenzothiazole derivatives showing remarkable fluorescence and biological activities

Article abstract of DOI:10.1016/j.molstruc.2019.127194

The synthesis of novel pyrido[2,1-b]benzothiazole and pyrido[2,1-b]benzoimidazole derivatives was achieved via the reaction of N-aryl-2-cyano-3,3-bis(methylthio)acrylamide with benzothiazoleylacetonitrile and benzoimidazoleylacetonitrile, respectively, while N-substituted 2-pyridylbenzothiazole derivatives were synthesized by reacting 2-(benzo[d]thiazol-2-yl)-3-(dimethylamino)acrylonitrile with either cyanoacetamide, aryl cyanoacetamides or 2-cyano-N^’-(4-substituted benzylidene)acetohydrazide. Based on the steady state fluorescence measurements, the newly synthesized N-substituted 2-pyridylbenzothiazole derivatives exhibited remarkable fluorescence properties with considerably high quantum yields (0.25–0.29). In addition, the antimicrobial and antiviral activities were evaluated for all the newly synthesized derivatives. The antimicrobial examination revealed that triazolo-pyridone 21a had the highest potency among all tested compounds against Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus while pyrido[2,1-b]benzoimidazole derivatives 9a and 9b had the highest potency over other compounds against Candida albicans fungus.

Full text of DOI:10.1016/j.molstruc.2019.127194

Journal Pre-proof
Synthesis of novel pyrido[2,1-b]benzothiazole and N-substituted 2-
pyridylbenzothiazole derivatives showing remarkable fluorescence and biological
activities
Rasha A. Azzam, Galal H. Elgemeie, Rokia R. Osman
PII:
S0022-2860(19)31303-1
DOI:
https://doi.org/10.1016/j.molstruc.2019.127194
MOLSTR 127194
Reference:
To appear in: Journal of Molecular Structure
Received Date: 23 July 2019
Revised Date: 26 September 2019
Accepted Date: 7 October 2019
Please cite this article as: R.A. Azzam, G.H. Elgemeie, R.R. Osman, Synthesis of novel
pyrido[2,1-b]benzothiazole and N-substituted 2-pyridylbenzothiazole derivatives showing remarkable
fluorescence and biological activities, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.127194.
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Synthesis of novel pyrido[2,1-b]benzothiazole and N-substituted 2-
pyridylbenzothiazole derivatives showing remarkable fluorescence and
biological activities
Rasha A. Azzam, Galal H. Elgemeie,* Rokia R. Osman
Chemistry Department, Faculty of Science, Helwan University, Cairo 11785, Egypt
N
CN
S
N
O
N
NH
Ar
Ar
O
NH
H2N
N
CN
O
SMe
CN
N
S
S
N
CN
H2N
N
S
Ar

Synthesis of novel pyrido[2,1-b]benzothiazole and N-substituted 2-
pyridylbenzothiazole derivatives showing remarkable fluorescence and
biological activities
Rasha A. Azzam, Galal H. Elgemeie*, Rokia R. Osman
Chemistry Department, Faculty of Science, Helwan University, Cairo 11785, Egypt
Keywords: 2-Pyridylbenzothiazoles, Fluorescence, Antiviral, Antimicrobial
Abstract
The synthesis of novel pyrido[2,1-b]benzothiazole and
pyrido[2,1-b]benzoimidazole
derivatives was achieved via the reaction of N-aryl-2-cyano-3,3-bis(methylthio)acrylamide
with benzothiazoleylacetonitrile and benzoimidazoleylacetonitrile, respectively, while N-
substituted 2-pyridylbenzothiazole derivatives were synthesized by reacting 2-
(benzo[d]thiazol-2-yl)-3-(dimethylamino)acrylonitrile with either cyanoacetamide, aryl
cyanoacetamides or 2-cyano-N'-(4-substituted benzylidene)acetohydrazide. Based on the
steady state fluorescence measurements, the newly synthesized N-substituted 2-
pyridylbenzothiazole derivatives exhibited remarkable fluorescence properties with
considerably high quantum yields (0.25-0.29). In addition, the antimicrobial and antiviral
activities were evaluated for all the newly synthesized derivatives. The antimicrobial
examination revealed that triazolo-pyridone 21a had the highest potency among all tested
compounds against Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus while
pyrido[2,1-b]benzoimidazole derivatives 9a and 9b had the highest potency over other
compounds against Candida albicans fungus.
1. Introduction
Organic compounds containing pyridine scaffold as a core unit exhibit potent
pharmacological activities such as anti-hypertensive [1], anti-histamine [2], anticoagulant [3],
anti-inflammatory [4], antibacterial [5], antifungal [6], antiviral [7], anti-tubercular [8] and
anti-malarial [9]. It is interesting to note that the replacement of the substituents on the
pyridine nucleus with others has widened both its biological activities and the spectrum of its
biological targets from microbes to viruses and a variety of cancerous cells [10]. This class of
compounds and their derivatives are valuable synthetic target compounds and their syntheses
1

have been extensively reviewed [11,12]. Inasmuch, compounds containing benzothiazole
moieties were also found to exhibit a number of biological activities such as anticancer [13],
antitumor [14], antimicrobial [15] and antiviral [16,17] that demonstrated special significance
in the field of medicinal chemistry due to their remarkable pharmacological potentialities. It
is interesting to note that different pyridine and benzothiazole heterocyclic compounds may
show biological activities while others may possess some fluorescence properties in which
case the latter may be utilized as a fluorescent probe [18-21]. In specific compounds bearing
pyridine or benzothiazole rings, a significant increase in quantum yield, dual fluorescence
and solid stat fluorescence [22,23] as well as high Stokes shift, [24-26] were observed. It is
worth noting that combining two or more types of different heterocycle rings into one
structure may lead to novel substances with enhanced bioactivity [27,28]. For example,
pyridines bearing benzothiazole moieties exhibited enhanced antibacterial and antifungal
activity [29]. Moreover, dihydropyridino benzothiazole derivatives have been developed as
Pr-set7 inhibitors for therapeutic prevention of cancer [30].
To achieve this interesting target, a variety of new methods has been developed to
synthesize pyridine bearing benzothiazole moiety as well as pyrido[2,1-b]benzothiazole
derivatives by our research group [31-35]. The attractive biological profiles and fluorescence
properties of pyridine and benzothiazole derivatives prompted us to develop efficient
methods and direct routes for the synthesis of new derivatives that encompasses both the
pyridine and benzothiazole moieties. We report here on the synthesis of novel pyrido[2,1-
b]benzothiazole derivatives through the reaction of benzothiazoleylacetonitrile with N-aryl-2-
cyano-3,3-bis(methylthio)acrylamide and the synthesis of novel 2-
through the reaction of 2-(benzo[d]thiazol-2-yl)-3-(dimethylamino)acrylonitrile with
cyanoacetamide, aryl cyanoacetamides or
2-cyano-N'-(4-substituted
pyridylbenzothiazole
benzylidene)acetohydrazide. The study is further extended to evaluate the fluorescence
properties of these newly synthesized compounds as well as their antiviral and antimicrobial
activities.
2. Material and Methods
2.1 Measurements
All melting points were measured using a SMP3 melting point apparatus. The 1H and
13C NMR spectra were recorded on a Bruker Avance (III)-400 Spectrometer (400 and 100
MHz, respectively) in DMSO-d6 using Si(CH3)4 as an internal standard at Ain Shams
2

university, Cairo, Egypt. The mass spectra were run in Al-Azhar university. Progress of the
reactions was monitored by thin-layer chromatography (TLC) using aluminum sheets coated
with silica gel F254 (Merck), and UV lamp. Optical absorption and fluorescence
measurements were carried out using JASCO spectrophotometer (V-780) and JASCO
spectrofluorometer (model FP-8300), respectively.
2.2. Synthesis procedures
2.2.1. General procedures for the synthesis of 7a-f
To a stirred solution of 2-cyanomethylbenzothiazole 4 (10 mmol) in dry 1,4-dioxane
(30 ml) containing potassium hydroxide (10 mmol), aryl-2-cyano-3,3-bis(methylthio)-
acrylamide derivatives 3a-f (10 mmol) were added and the reaction mixture was stirred
further at room temperature for an additional one hour. The solid precipitate in each case was
filtered off and was then recrystallized from ethanol.
2.2.1.1.
4-Cyano-1-imino-3-methylsulfanyl-benzo[4,5]thiazo[1,2-a]pyridine-2-carboxylic
acid phenylamide 7a
Yellow needles; (DMF); Yield 83%; m.p 290-292 oC; IR (KBr, cm−1): υ 3443, 3252 (=NH, -
1
NH), 2204 (CN), 1647 (C=O) and 1597 (C=N). H NMR (400 MHz, DMSO-d6): δ 2.51 (s,
3H, SCH3), 7.15 (t, 1H, J=8 Hz, benzothiazole-H), 7.38-7.62 (m, 4H, Ar-H & benzothiazole-
H), 7.71 (d, 2H, J=8 Hz, Ar-H ), 8.13 (d, 1H, J=8 Hz, benzothiazole-H), 8.25 (br, 1H, NH),
9.50 (d, 1H, J=8 Hz, benzothiazole-H), 10.66 (br, 1H, NH) ); 13C NMR (100 MHz, DMSO-
d6): δ 13.5 (SCH3), 81.2 (Ar-C), 117.1 (CN), 121.4, 122.0, 124.4, 124.0, 126.0, 128.0, 129.1,
129.9, 132.4, 135.1, 136.5, 138.5, 150.1, 155.3 (Ar-C), 159.5 (C=O). Anal.Calcd.For.
C20H14N4OS2 (390.48): C% 61.52; H% 3.61; N% 14.35. Found: C% 61.82; H% 3.56; N%
14.10.
2.2.1.2. 4-Cyano-1-imino-3-methylsulfanyl-benzo[4,5]thiazo[1,2-a]pyridine-2-carboxylic
acid (4-chlorophenyl)amide 7b
Yellow needles; (DMF); Yield 85%; m.p 248-250 oC; IR (KBr, cm−1): υ 3439, 3260 (=NH, -
1
NH), 3115 (Ar C), 2201 (CN), 1644 (C=O), and 1589 (C=N). H NMR (400 MHz, DMSO-
d6): δ 2.54 (s, 3H, SCH3), 7.44 (d, 2H, J=8 Hz, Ar-H), 7.56–7.63 (m, 2H, benzothiazole-H),
7.73 (d, 2H, J=8 Hz, Ar-H), 8.13 (d, 1H, J=8 Hz, benzothiazole-H), 8.27 (br, 1H, NH), 9.52
(d, 1H, J=8 Hz, benzothiazole-H), 10.79 (br, 1H, NH); 13C NMR (100 MHz, DMSO-d6): δ
3

12.7 (SCH3), 83.0 (Ar-C), 113.0 (CN), 120.1, 121.2, 123.4, 124.8, 125.3, 127.2, 127.5, 129.7,
133.7, 136.5, 137.8, 139.6, 155.2, 157.8 (Ar-C), 158.1 (C=O). MS: m/z = 424.
Anal.Calcd.For. C20H13ClN4OS2 (424.93): C% 56.53; H% 3.08; N% 13.19. Found: C%
56.76; H% 2.90; N% 13.45.
2.2.1.3.
4-Cyano-1-imino-3-methylsulfanyl-benzo[4,5]thiazo[1,2-a]pyridine-2-carboxylic
acid (3-chlorophenyl)amide 7c
1
Buff crystals; (DMF); Yield 77 %; m.p 338-340 oC; H NMR (400 MHz, DMSO-d6): δ 2.91
(s, 3H, SCH3), 7.29 (s, 1H, C6H4), 7.40 (t, 1H, J=8 Hz, benzothiazole-H), 7.48 (t, 1H, J=8 Hz,
benzothiazole-H), 7.52-7.74 (m, 3H, Ar-H), 8.27 (d, 1H, J=8 Hz, benzothiazole-H), 9.01 (d,
1H, J=8 Hz, benzothiazole-H), 9.46 (br, 1H, NH) ); 13C NMR (100 MHz, DMSO-d6): δ 14.5
(SCH3), 82.6 (Ar-C), 111.5 (CN), 121.9, 122.6, 123.7, 124.9, 125.9, 126.7, 127.9, 128.4,
133.0, 136.0, 137.2, 139.0, 153.9, 156.5 (Ar-C), 160.0 (C=O). Anal.Calcd.For.
C20H13ClN4OS2 (424.93): C% 56.53; H% 3.08; N% 13.19. Found: C% 56.21; H% 3.35; N%
13.06.
2.2.1.4.
4-Cyano-1-imino-3-methylsulfanyl-benzo[4,5]thiazo[1,2-a]pyridine-2-carboxylic
acid p-tolylamide 7d
Yellow needles; (DMF); Yield 87%; m.p 271-273 oC; IR (KBr, cm−1): υ 3436, 3261 (=NH, -
1
NH), 3119 (Ar C), 2204 (CN), 1641 (C=O) and 1590 (C=N). H NMR (400 MHz, DMSO-
d6): δ 2.29 (s, 3H, CH3), 2.54 (s, 3H, SCH3), 7.18 (d, 2H, J=8 Hz, Ar-H), 7.58–7.60 (m, 4H,
2Ar-H & 2benzothiazole-H), 8.13 (d, 1H, J=8 Hz, benzothiazole-H), 8.18 (br, 1H, NH), 9.50
13
(d, 1H , J=8 Hz, benzothiazole-H), 10.57 (br, 1H, NH); C NMR (100 MHz, DMSO-d6): δ
18.2 (CH3), 21.0 (SCH3), 83.0 (Ar-C), 116.8 (CN), 120.1, 121.2, 123.4, 124.8, 125.3, 127.2,
127.5, 129.7, 133.7, 136.5, 137.8, 139.6, 155.2, 157.8 (Ar-C), 162.6 (C=O). Anal.Calcd.For.
C21H16N4OS2 (404.51): C% 62.35; H% 3.99; N% 13.85. Found: C% 62.45; H% 3.91; N%
14.05.
2.2.1.5. 4-Cyano-1-imino-3-methylsulfanyl-benzo[4,5]thiazo[1,2-a]pyridine-2-carboxylic
acid m-tolylamide 7e
Orange crystals; (DMF); Yield 85%; m.p 235-237 oC; IR (KBr, cm−1): υ 3442, 3258 (C=NH,
1
C-NH), 3068 (Ar CH), 2202 (CN), 1644 (C=O) and 1588 (C=N). H NMR (400 MHz,
DMSO-d6): δ 2.32 (s, 3H, CH3), 2.55 (s, 3H, SCH3), 6.97 (d, 1H, J=8 Hz, Ar-H), 7.25 (t, 1H,
4

J=8 Hz, benzothiazole-H), 7.46–7.69 (m, 4H, 3Ar-H & benzothiazole-H), 7.85 (d, 1H, J=8
Hz, benzothiazole-H), 8.27 (br, 1H, NH), 8.60 (d, 1H, J=8 Hz, benzothiazole-H), 10.9 (br,
13
1H, NH); C NMR (100 MHz, DMSO-d6): δ 18.2 (CH3), 21.0 (SCH3), 83.0 (Ar-C), 116.8
(CN), 118.0, 120.1, 121.2, 123.4, 124.8, 125.3, 127.5, 129.7, 137.7, 138.5, 138.8, 139.6,
155.2, 157.8 (Ar-C), 162.6 (C=O). Anal.Calcd.For. C21H16N4OS2 (404.51): C% 62.35; H%
3.99; N% 13.85. Found: C% 62.72; H% 3.86; N% 13.91.
2.2.1.6. 4-Cyano-1-imino-3-methylsulfanyl-benzo[4,5]thiazo[1,2-a]pyridine-2-carboxylic
acid o-tolylamide 7f
o
Yellow crystals; (DMF); Yield 72%; m.p 263 C; IR (KBr, cm−1): υ 3435, 3234 (C=NH, C-
1
NH), 3057 (Ar C), 2202 (CN), 1635 (C=O) and 1582 (C=N). H NMR (400 MHz, DMSO-
d6): δ 2.32 (s, 3H, CH3), 2.61 (s, 3H, SCH3), 7.17 (t, 1H, J=8 Hz, benzothiazole-H), 7.23–
7.29 (m, 2H, Ar-H & benzothiazole-H), 7.56 -7.64 (m, 3H, Ar-H), 8.14 (d, 1H, J=8 Hz,
benzothiazole-H), 8.21 (br, 1H, NH), 9.53 (d, 1H, J=8 Hz, benzothiazole-H), 10.07 (br, 1H,
NH); 13C NMR (100 MHz, DMSO-d6): δ 17.0 (CH3), 20.0 (SCH3), 84.2 (Ar-C), 115.0 (CN),
121.4, 122.0, 123.6, 125.0, 126.1, 127.7, 128.0, 129.5, 131.5, 134.9, 136.0, 138.6, 154.0,
156.0 (Ar-C), 160.0 (C=O). Anal.Calcd.For. C21H16N4OS2 (404.51): C% 62.35; H% 3.99;
N% 13.85. Found: C% 62.54; H% 4.14; N% 13.98.
2.2.2. General procedures for the synthesis of 9a-c
To a stirred solution of 2-cyanomethylbenzimidazole 8 (10 mmol) in dry 1,4-dioxane
(30ml) containing potassium hydroxide (10mmol), aryl-2-cyano-3,3-bis(methylthio)-
acrylamide derivatives 3b,d,e (10 mmol) were added and the reaction mixture was stirred
further at room temperature for an additional one hour. The solid precipitate was filtered off
and then recrystallized from ethanol.
2.2.2.1. 1-Amino-4-cyano-3-methylsulfanyl-benzo[4,5]imidazo[1,2-a]pyridine-2-carboxylic
acid (4-chlorophenyl)amide 9a
o
Buff crystals; (DMF); Yield 73%; m.p 279-281 C; IR (KBr, cm−1 ): υ 3445, 3361 (NH,
NH2), 3098 (Ar CH), 2209 (CN), 1634 (C=O) and 1596 (C=N); 1H NMR (400 MHz, DMSO-
d6): δ 2.63 (s, 3H, SCH3), 7.39-7.90 (m, 9H, 4Ar-H, NH2 & 3benzimidazole-H), 8.50 (d, 1H,
J=8 Hz, benzimidazole-H), 10.51 (br, 1H, NH) ); 13C NMR (100 MHz, DMSO-d6): δ 21.0
(SCH3), 82.6 (Ar-C), 115.0 (CN), 119.5, 121.7, 122.3, 124.0, 125.0, 126.4, 127.0, 129.0,
5

135.5, 137.0, 138.0, 139.0, 154.9, 156.4 (Ar-C), 163.0 (C=O). Anal.Calcd.For.
C20H14ClN5OS (407.88): C% 58.89; H% 3.46; N% 17.17. Found: C% 58.65; H% 3.15; N%
17.34.
2.2.2.2. 1-Amino-4-cyano-3-methylsulfanyl-benzo[4,5]imidazo[1,2-a]pyridine-2-carboxylic
acid p-tolylamide 9b
Buff crystals; (DMF); Yield 71%; m.p 273-275 oC; IR (KBr, cm−1): υ 3484, 3306 (NH2, NH),
1
3100 (Ar C), 2206 (CN), 1648 (C=O) and 1590 (C=N). H NMR (400 MHz, DMSO-d6): δ
2.30 (s, 3H, CH3), 2.61 (s, 3H, SCH3), 7.18 (d, 2H, J=8.4 Hz, Ar-H), 7.38 (t, 1H, J=6.4 Hz,
benzimidazole-H), 7.57–7.64 (m, 3H, 2Ar-H & benzimidazole-H), 7.84 (m, 3H, NH2 &
benzimidazole-H), 8.49 (d, 1H, J=8Hz, benzimidazole-H), 10.47 (br, 1H, NH) ); 13C NMR
(100 MHz, DMSO-d6): δ 21.9 (SCH3), 82.6 (Ar-C), 117.0 (CN), 119.5, 120.8, 121.9, 123.8,
124.9, 125.9, 127.6, 129.0, 130.0, 134.0, 136.4, 137.4, 138.0, 153.0, 155.8 (Ar-C), 163.9
(C=O). Anal.Calcd.For. C21H17N5OS (387.46): C% 65.10; H% 4.42; N% 18.08. Found: C%
65.35; H% 4.62; N% 18.26.
2.2.2.3. 1-Amino-4-cyano-3-methylsulfanyl-benzo[4,5]imidazo[1,2-a]pyridine-2-carboxylic
acid m-tolylamide 9c
o
Buff solid; (DMF); Yield 73%; m.p 260-262 C; IR (KBr, cm−1): υ 3408, 3323 (NH2, NH),
1
3062 (Ar C), 2222 (CN), 1642 (C=O) and 1545 (C=N). H NMR (400 MHz, DMSO-d6): δ
2.33 (s, 3H, CH3), 2.67 (s, 3H, SCH3), 6.96 (d, 1H, J=7.2 Hz, Ar-H), 7.26 (t, 1H, J=7.6 Hz,
benzimidazole-H), 7.46–7.52 (m, 2H, Ar-H & benzimidazole-H), 7.64-7.69 (m, 2H, Ar-H),
7.85 (d, 1H, J=8 Hz, benzimidazole-H), 8.27 (s, 2H, NH2), 8.60 (d, 1H, J=8.4 Hz,
benzimidazole-H), 10.71 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): δ 20.0 (CH3), 22.0
(SCH3), 83.0 (Ar-C), 116.8 (CN), 117.5, 118.0, 118.9, 120.1, 121.2, 123.4, 124.8, 125.3,
127.5, 129.7, 136.7, 137.5, 138.8, 139.6, 155.2, 157.8 (Ar-C), 164.6 (C=O). Anal.Calcd.For.
C21H17N5OS (387.46): C% 65.10; H% 4.42; N% 18.08. Found: C% 65.40; H% 4.25; N%
18.22.
2.2.3. Synthesis of 6-amino-5-(benzo[d]thiazol-2-yl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile 13
2-(Benzo[d]thiazole-2-yl)-3-(dimethylamino)acrylonitrile 11 (10 mmol) was added to a
stirred solution of cyanoacetamide 12 (10 mmol) in dry 1,4-dioxane (30 ml) containing
6

potassium hydroxide (10 mmol) and the reaction mixture was refluxed for 1 hour. The solid
precipitate was filtered off and then recrystallized from ethanol. Yellow solid; (DMF); Yield
75%; m.p
>
350 oC; IR (KBr, cm−1): υ 3451, 3202 (NH2, NH), 2285 (CN), and 1659 (C=O).
1H NMR (400 MHz, DMSO-d6): δ 7.13 (t, 1H, J=8 Hz, benzothiazole-H), 7.31 (t, 1H, J=8
Hz, benzothiazole-H), 7.61 (d, 1H, J=8 Hz, benzothiazole-H), 7.81 (d, 1H, J=8 Hz,
13
benzothiazole-H), 8.18 (s, 1H, CHpyridone); C NMR (100 MHz, DMSO-d6): δ 74.4, 75.1
(Ar-C), 119.6 (CN), 119.5, 120.0, 121.1, 122.2, 125.7, 132.5, 144.6, 154.4, 167.1 (Ar-C),
168.9 (C=O). Anal.Calcd.For. C13H8N4OS (268.29): C% 58.20; H% 3.01; N% 20.88. Found:
C% 58.51; H% 3.22; N% 20.81.
2.2.4. General procedures for the synthesis of 15a-d
2-(Benzo[d]thiazole-2-yl)-3-(dimethylamino)acrylonitrile 11 (10 mmol) was added to a
stirred solution of 2-cyano-N-(aryl)acetamide 14a-d (10 mmol) in dry 1,4-dioxane (30 ml)
containing potassium hydroxide (10 mmol) and the reaction mixture was refluxed for 1 hour.
The reaction was then cooled and poured onto iced water; the resulting precipitate was
filtered off and recrystallized from ethanol.
2.2.4.1. 6-Amino-5-(benzo[d]thiazole-2-yl)-2-oxo-1-phenyl-1,2-dihydropyridine-3-
carbonitrile 15a
o
Yellow crystals; (DMF); Yield 81%; m.p 318-320 C; IR (KBr, cm −1): υ 3374 (NH2), 2205
1
(CN), and 1665 (C=O). H NMR (400 MHz, DMSO-d6): δ 7.37-7.49 (m, 4H, 3Ar-H &
benzothiazole-H), 7.58-7.67 (m, 3H, 2Ar-H & benzothiazole-H), 7.84 (d, 1H, J=8 Hz,
benzothiazole-H), 8.06 (d, 1H, J=8 Hz, benzothiazole-H), 8.38 (s, 1H, CH pyridone); 13C
NMR (100 MHz, DMSO-d6): δ 83.7, 90.0 (Ar-C), 115.0 (CN), 119.0, 121.2, 122.0, 123.5,
125.0, 126.4, 128.8, 130.0, 133.6, 145.8, 152.4, 160.5 (Ar-C), 165.3 (C=O). Anal.Calcd.For.
C19H12N4OS (344.39): C% 66.26; H% 3.51; N% 16.27. Found: C% 66.38; H% 3.58; N%
16.45.
2.2.4.2. 6-Amino-5-(benzo[d]thiazole-2-yl)-1-(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile 15b
o
Yellow crystals; (DMF); Yield 83%; m.p 316-318 C; IR (KBr, cm−1): υ 3432 (NH2), 2209
1
(CN), and 1644 (C=O). H NMR (400 MHz, DMSO-d6): δ 7.38-7.51 (m, 4H, 2Ar-H,
2benzothiazole-H), 7.71 (d, 2H, J=8 Hz, Ar-H), 7.88 (d, 1H, J=8 Hz, benzothiazole-H), 8.08
7

13
(d, 1H, J=8 Hz, benzothiazole-H), 8.43 (s, 1H, CH) ); C NMR (100 MHz, DMSO-d6): δ
86.0, 89.4 (Ar-C), 116.0 (CN), 119.5, 120.3, 121.3, 122.8, 123.9, 125.0, 128.0, 129.0, 131.2,
147.5, 153.7, 159.7 (Ar-C), 160.0 (C=O). Anal.Calcd.For. C19H11ClN4OS (378.83): C%
60.24; H% 2.93; N% 14.79. Found: C% 60.56; H% 2.88; N% 14.64.
2.2.4.3. 6-Amino-5-(benzo[d]thiazole-2-yl)-2-oxo-1-(p-tolyl)-1,2-dihydropyridine-3-
carbonitrile 15c
o
Yellow crystals; (DMF); Yield 86%; m.p 256-258 C; IR (KBr, cm −1): υ 3445 (NH2), 3060
1
(Ar C), 2213 (CN), and 1669 (C=O). H NMR (400 MHz, DMSO-d6): δ 2.43 (s, 3H, CH3),
7.30 (d, 2H, J=8 Hz, Ar-H), 7.37-7.50 (m, 4H, 2Ar-H & 2benzothiazole-H), 7.86 (d, 1H, J=8
Hz, benzothiazole-H), 8.07 (d, 1H, J=8 Hz, benzothiazole-H), 8.40 (s, 1H, CHpyridone); 13C
NMR (100 MHz, DMSO-d6): δ 17.7 (CH3), 81.9, 97.0 (Ar-C), 113.0 (CN), 119.9, 120.4,
121.7, 122.5, 124.6, 125.9, 129.2, 130.6, 132.6, 144.9, 151.0, 158.3 (Ar-C), 159.9 (C=O).
Anal.Calcd.For. C20H14N4OS (358.42): C% 67.02; H% 3.94; N% 15.63. Found: C% 67.35;
H% 3.83; N% 15.90.
2.2.4.4. 6-Amino-5-(benzo[d]thiazole-2-yl)-1-(naphthalene-1-yl)-2-oxo-1,2-dihydropyridine-
3-carbonitrile 15d
o
Yellow crystals; (DMF); Yield 82%; m.p 296-298 C; IR (KBr, cm −1): υ 3419 (NH2), 2210
(CN), and 1664 (C=O). 1H NMR (400 MHz, DMSO-d6): δ 7.42-7.50 (m, 2H, naphthalene-H
& benzothiazole-H), 7.57-7.77 (m, 5H, 4naphthalene-H & benzothiazole-H), 7.85 (d, 1H, J=8
Hz, benzothiazole-H), 8.09-8.15 (m, 2H, naphthaalene), 8.20 (d, 1H, J=8 Hz, benzothiazole-
13
H), 8.56 (s, 1H, CHpyridone); C NMR (100 MHz, DMSO-d6): δ 82.0, 90.1 (Ar-C), 114.5
(CN), 119.7, 120.5, 121.9, 122.8, 123.5, 124.9, 125.9, 128.5, 129.3, 130.6, 131.8, 133.0,
147.0, 150.4, 157.8 (Ar-C), 159.0 (C=O). Anal.Calcd.For. C23H14N4OS (394.45): C% 70.03;
H% 3.58; N% 14.20. Found: C% 70.22; H% 3.28; N% 14.05.
2.2.5. General procedures for the synthesis of 21a-d
2-(Benzo[d]thiazole-2-yl)-3-(dimethylamino)acrylonitrile 11 (10 mmol) was added to a
stirred solution of 2-cyano-N-(4-alkylbenzylidine)acetohydrazide derivatives 16a-d (10
mmol) in dry 1,4-dioxane (30 ml) containing potassium hydroxide (10 mmol) and the
reaction mixture was refluxed for 2 hours. The solid precipitate was filtered off and then
recrystallized from ethanol.
8

2.2.5.1. 8-(Benzo[d]thiazole-2-yl)-2-(4-fluorophenyl)-5-oxo-3,5-dihydro[1,2,4]triazolo[1,5-
a]pyridine-6-carbonitrile 21a
o
Brown crystals; (DMF); Yield 73%; m.p > 350 C; IR (KBr, cm −1): υ 3404 (NH), 2204
1
(CN), and 1610 (C=O). H NMR (400 MHz, DMSO-d6): δ 7.32-7.49 (m, 4H, 2Ar-H,
2benzothiazole-H), 7.92 (d, 1H, J = 8 Hz, benzothiazole-H), 8.10 (d, 1H, J = 8 Hz,
13
benzothiazole-H), 8.34 (d, 2H, J = 8 Hz, Ar-H), 8.48 (s, 1H, CHpyridone); C NMR (100
MHz, DMSO-d6): δ 93.1, 100.0 (Ar-C), 114.6 (CN), 120.2, 121.8, 122.9, 123.4, 124.5, 125.9,
129.5, 130.2, 134.4, 149.9, 154.4, 156.2, 160.1 (Ar-C), 164.0 (C=O). Anal.Calcd.For.
C20H10FN5OS (387.39): C% 62.01; H% 2.60; N% 18.08. Found: C% 62.35; H% 2.68; N%
18.05.
2.2.5.2. 8-(Benzo[d]thiazole-2-yl)-2-(4-chlorophenyl)-5-oxo-3,5-dihydro[1,2,4]triazolo[1,5-
a]pyridine-6-carbonitrile 21b
Brown crystals; (DMF); Yield 75%; m.p > 350 oC; IR (KBr, cm−1): υ 3415 (NH), 2207 (CN),
1
and 1612 (C=O). H NMR (400 MHz, DMSO-d6): δ 7.33 (t, 1H, J=8 Hz, benzothiazole-H),
7.47 (t, 1H, J=8 Hz, benzothiazole-H), 7.65 (d, 2H, J=8 Hz, Ar-H), 7.91 (d, 1H, J=8 Hz,
benzothiazole-H), 8.10 (d, 1H, J=8 Hz, benzothiazole-H), 8.30 (d, 2H, J=8 Hz, Ar-H), 8.47
(s, 1H, CHpyridone); 13C NMR (100 MHz, DMSO-d6): δ 91.0, 102.5 (Ar-C), 115.9 (CN),
119.5, 120.9, 122.6, 123.9, 124.8, 127.5, 130.9, 131.7, 134.7, 151.0, 155.2, 157.0, 159.0 (Ar-
C), 162.2 (C=O). Anal.Calcd.For. C20H10ClN5OS (403.84): C% 59.48; H% 2.50; N% 17.34.
Found: C% 59.11; H% 2.10; N% 17.07.
2.2.5.3. 8-(Benzo[d]thiazole-2-yl)-2-(4-methoxyphenyl)-5-oxo-3,5-
dihydro[1,2,4]triazolo[1,5-a]pyridine-6-carbonitrile 21c
o
Brown crystals; (DMF); Yield 70%; m.p > 350 C; IR (KBr, cm −1): υ 3423 (NH), 2195
(CN), and 1606 (C=O). 1H NMR (400 MHz, DMSO-d6): δ 3.77 (s, 3H, OCH3), 7.33-7.39 (m,
3H, 2Ar-H, benzothiazole-H), 7.47 (t, 1H, J=8 Hz, benzothiazole-H), 7.91 (d, 1H, J=8 Hz,
benzothiazole-H), 8.11 (d, 1H, J=8 Hz, benzothiazole-H), 8.24 (d, 2H, J=8 Hz, Ar-H), 8.46
13
(s, 1H, CHpyridone); C NMR (100 MHz, DMSO-d6): δ 52.1 (OCH3), 95.2, 102.4 (Ar-C),
117.7 (CN), 119.6, 120.9, 122.8, 123.9, 125.8, 127.6, 128.8, 132.9, 137.0, 152.5, 156.9,
158.5, 159.9 (Ar-C), 161.8 (C=O). Anal.Calcd.For. C21H13N5O2S (399.43): C% 63.15; H%
3.28; N% 17.53. Found: C% 63.35; H% 3.15; N% 17.44.
9

2.2.5.4. 8-(Benzo[d]thiazole-2-yl)-5-oxo-2-(p-tolyl)-3,5-dihydro-[1,2,4]triazolo[1,5-a]
pyridine-6-carbonitrile 21d
o
Brown crystals; (DMF); Yield 72%; m.p > 350 C; IR (KBr, cm−1): υ 3374 (NH), 3121 (Ar
C), 2205 (CN), and 1665 (C=O). 1H NMR (400 MHz, DMSO-d6): δ 2.40 (s, 3H, CH3), 7.33-
7.39 (m, 3H, 2Ar-H, benzothiazole-H), 7.47 (t, 1H, J=8 Hz, benzothiazole-H), 7.92 (d, 1H,
J=8 Hz, benzothiazole-H), 8.11 (d, 1H, J=8 Hz, benzothiazole-H), 8.20 (d, 2H, J=8 Hz, Ar-
H), 8.47 (s, 1H, CHpyridone); 13C NMR (100 MHz, DMSO-d6): δ 19.5 (CH3), 97.2, 108.9
(Ar-C), 115.0 (CN), 119.0, 120.3, 122.0, 124.6, 125.3, 128.0, 129.4, 131.2, 135.4, 151.8,
155.4, 157.2, 158.5 (Ar-C), 160.7 (C=O). Anal.Calcd.For. C21H13N5OS (383.43): C% 65.78;
H% 3.42; N% 18.27. Found: C% 65.94; H% 3.68; N% 18.20.
2.3. Biological evaluation
2.3.1. In vitro antibacterial and antifungal screening
Antimicrobial tests were carried out at the Microbiology Unit in Biochemistry Central Lab,
Faculty of Science, Cairo University, Cairo, Egypt. At the honest, Agar-diffusion method was
used for the determination of the preliminary antibacterial and antifungal activities of the
synthesized compounds [36]. For the purpose of comparison against standard drugs,
gentamicin, ampicillin and nystatin were also used. All the compounds and standard drugs
were tested in vitro against staphylococcus aureus and Streptococcus mutans (Gram positive
bacteria), Escherichia coli, Pseudomonas aeruginosa and klebsiella pneumonia (Gram
negative bacteria) and Candida albicans fungal strains using nutrient agar medium. The
results were all recorded for each tested compound as the average diameter of the inhibition
zones of the microbial growth around the disks in mm ± SD, as summarized in Table 2.
DMSO was used as solvent control. The compounds were tested at a concentration of 15
mg/ml against both bacterial and fungal strains.
2.3.2. Method of testing
The sterilized media was poured onto the sterilized Petri dishes (20-25 ml, each petri dish)
and allowed to solidify at room temperature. Microbial suspension was prepared in sterilized
saline equivalent to McFarland 0.5 standard solution (1.5x 10 5 CFU mL-1) and its turbidity
was adjusted to OD = 0.13 using spectrophotometer at 625 nm. Optimally, within 15 minutes
after adjusting the turbidity of the inoculum suspension, a sterile cotton swab was dipped into
10

the adjusted suspension and was flooded on the dried agar surface then allowed to dry for 15
minutes with lid in place. Wells of 6 mm diameter was made in the solidified media with the
help of sterile borer. 100 ꢀL of the solution of the tested compound was added to each well
with the help of micropipette. The plates were incubated at 37oC for 24 hrs in case of
antibacterial activity. This experiment was carried out in triplicate and zones of inhibition
were measured in mm. scale.
2.4. Antiviral Evaluation
2.4.1. Cytotoxicity test
It was done according to literature [37,38]. Briefly, all samples (50 mg) were
dissolved in 1 mL of DMSO. Decontamination of samples was done by adding 24_µL of
100× of antibiotic–antimycotic mixture to 1 mL of each sample. Then, bi-fold dilutions were
done to 100_µL of original dissolved samples and 100 _µL of each dilutions were inoculated
in Hep-2, Vero, BGM, FRHK4, and Huh 7.5 cell lines (obtained from the Holding Company
for Biological Products & Vaccines VACSERA, Egypt) previously cultured in 96 multi well
plates (Greiner-Bio one, Germany) to estimate the non-toxic dose of the tested samples.
Cytotoxicity assay was done using cell morphology evaluation by inverted light microscope
and cell viability test applying trypan blue dye exclusion method.
2.4.2. Cell morphology evaluation by inverted light microscopy
Hep-2, Vero, BGM, FRHK4, and Huh 7.5 cell cultures (2×105 cells/mL) were
prepared separately in 96-well tissue culture plates (Greiner-Bio one, Germany). After 24 h
o
incubation at 37 C in a humidified 5% (v/v) CO2 atmosphere cell monolayers were
confluent, the medium was removed from each well and replenished with 100_µL of bi-fold
dilutions of different samples tested prepared in DMEM (GIBCO BRL). For cell controls
o
100_µL of DMEM without samples was added. All cultures were incubated at 37 C in a
humidified 5% (v/v) CO2 atmosphere for 72 h. Cell morphology was observed daily for
microscopically detectable morphological alterations, such as loss of confluence, cell
rounding and shrinking, and cytoplasm granulation and vacuolization. Morphological
changes were scored [37].
2.4.3. Cell viability assay
11

It was done by trypan blue dye exclusion method [38]. Hep-2, Vero, BGM, FRHK4,
and Huh 7.5 cell cultures (2×105 cells/mL) were grown in 12-well tissue culture plates
(Greiner-Bio one, Germany). After 24 h incubation, the same assay described above for
tested samples cytotoxicity was followed by applying 100_µL of tested samples dilutions
(bifold dilutions) per well. After 72 h the medium was removed, cells were trypsinized and an
equal volume of 0.4% (w/v). Trypan blue dye aqueous solution was added to cell suspension.
Viable cells were counted under the phase contrast microscope.
2.4.4. Determination of adenovirus 7, HAV HM175, Coxsackievirus B4, and Herpes simplex
virus type 1 Titers using plaque assay
Non-toxic dilutions were mixed (100ꢀl) with 100ꢀl of different doses of Herpes
simplex virus type 1, adenovirus 7, HAV HM175 and Coxsackievirus B4 (1X105, 1X106,
1X107). The mixture was incubated for 1/2 hr in 37 °C. The inoculation of (100ꢀl) 10 fold
dilutions of treated and untreated adenovirus 7, HAV HM175, Coxsackievirus B4 and Herpes
simplex virus type 1 was carried out separately into Hep-2, FRHK4, BGM, and Vero cell
lines respectively in 12 multi well- plates. After 1 hr of incubation for adsorption at 37 °C in
a 5% CO2-water vapor atmosphere without constant rocking. The plates were rocked
intermittently to keep the cells from drying. After adsorption, 1 mL of 2X media (Dulbecco΄s
Modified Eagle Medium, Gibco-BRL (DMEM) plus 1ml 1% agarose was added to each
well, the plates were incubated at 37 °C in a 5% CO2-water vapor atmosphere. After the
appropriate incubation period, the cells were stained with 0.4% crystal violet after formaline
fixation, and the number of plaques counted. The viral titers were then calculated, and
expressed as plaque-forming units per milliliter (pfu/mL) [39].
2.4.5. Antiviral bioassay of tested materials against ED-43/SG-Feo (VYG) replicon of
Hepatitis C virus genotype 4a
:
ED-43/SG-Feo (VYG) replicon of HCV genotype 4a was treated with the non-toxic
dose of the tested compounds. HCV RNA was quantified in algal extracts treated Huh 7.5
infected cells using qRT-PCR (Taqman probe kit, Qiagen) and according to the
manufacturer’s instructions to show a dose-dependent decrease in sub genomic RNA copies
according to the literature [40].
3. Results and Discussion
12

Initially, compound N-aryl-2-cyano-3,3-bis(methylthio)acrylamide 3a-f was
synthesized using the procedure reported in the literature [41]. The synthesized compounds
3a-f were reacted with benzothiazoleylacetonitrile 4 in the presence of potassium hydroxide
in 1,4-dioxane at room temperature for one hour to afford the 4-cyano-1-imino-3-
(methylthio)-N-aryl-1-H-pyrido[1,2-a]benzothiazole-2-carboxamide 7a-f, Scheme 1. The
structures of these derivatives were established on the basis of elemental analysis and spectral
1
data (IR), H NMR and 13C NMR. For instance, the IR spectra of compound 7b showed
characteristic bands at 3439 and 3260 cm-1 for NH groups, a band at 2201 cm-1 associated
with the cyano group, and a peak at 1644 cm-1 for the carbonyl group. The 1H NMR spectrum
of 7b revealed a singlet peak at δ 2.54 ppm for the protons of SCH3 group, and two broad
13
peaks at δ 8.27 and 10.79 ppm for two protons of two NH groups. The C NMR showed a
signal at δ 12.7 ppm for the SCH3 carbon and a signal at δ 118.9 ppm for the CN carbon.
Moreover, signals appeared at δ 158.1 ppm were attributed to the carbonyl group. The
corresponding data of these compounds confirmed the fused structures of compounds 7a-f.
The reaction is assumed to proceed via Micheal addition of the active methylene group of
compound 4 to the double bond of compound 3 followed by the elimination of CH3SH to
form the intermediate acyclic adduct 5. Tautomerism occurred through proton transfer in an
intramolecular fashion from the methylene group to the nitrogen atom of the benzothiazole
ring to produce intermediate 6. The later cyclized by the addition of NH proton to the cyano
group to yield the novel pyrido[1,2-a]benzothiazole derivatives 7a-f.
13

O
O
CS2
NC
Ar
CH3I
NC
Ar
N
N
H
H
KS SK
KOH / DMF
RT
RT/24h
1
2
N
O
Ar
NH
O
S
CN
NC
Ar
4
N C
N
N
SCH3
CN
H
KOH / Dioxane
RT/1h
H3CS
SCH3
S
H
5
3
Ar
NH
O
O
Ar
NH
HN
N C
NH
SCH3
N
SCH3
CN
S
S
CN
6
7
7
Ar
7
Ar
C6H5
4-Cl-C6H4
3-Cl-C6H4
4-CH3-C6H4
3-CH3-C6H4
2-CH3-C6H4
a
b
c
d
e
f
Scheme 1. Synthesis of 4-cyano-1-imino-3-methylsulfanyl-benzo[4,5]thiazo[1,2-a]pyridine-
-2-carboxylic acid arylamides 7a-f
The course of the reaction between N-aryl-2-cyano-3,3-bis(methylthio)acrylamide
derivatives 3 and benzothiazole-2-yl-acetonitrile 4 prompted us to investigate similar reaction
between compound 3 and 2-benzoimidazole-2-yl-acetonitrile 8 under similar conditions. The
products obtained were shown to form via the same mechanism to afford the
methylthiobenzo[4,5]imidazo[1,2-a]pyridine-2-carboxamide 9a-c, Scheme 2. The structure of
the synthesized fused pyridine derivatives were also confirmed by elemental analysis and
spectral data.
14

3 b,d,e
N
KOH / Dioxane
RT/1h
N
H
CN
8
Ar
O
NH
H2N
SCH3
CN
N
N
9
9
a
Ar
4-Cl-C6H4
b 4-CH3-C6H4
3-CH3-C6H4
c
Scheme 2. Synthesis of 1-amino-4-cyano-3-methylsulfanyl-benzo[4,5]imidazo[1,2-
a]pyridine-2-carboxylic acid arylamides 9a-c
Furthermore, reaction of benzothiazole-2-yl-acetonitrile
4
with N,N-
dimethylformamide dimethyl acetal (DMF-DMA) 10 in ethyl alcohol at room temperature for
10 minutes afforded the 2-(benzo[d]thiazol-2-yl)-3-(dimethylamino)acrylonitrile 11 in high
yield [42]. The latter was further reacted with cyanoacetamide 12 in presence of potassium
hydroxide in 1,4-dioxane under reflux conditions to afford substituted 2-pyridylbenzothiazole
13, Scheme 3. The reaction proceeded via Micheal addition followed by the elimination of
NH(CH3)2 and intermolecular cyclization through the addition of NH2 proton to the cyano
group as to produce the substituted 2-pyridylbenzothiazole 13. According to IR spectra of
compound 13, the bands of NH2 and NH groups were observed at 3451 and 3202 cm-1,
respectively, while the bands of CN and carbonyl group observed at 2285 and 1659 cm-1,
respectively.
15

H3C O CH3
N
H3C O CH3
4
+
10
EtOH
RT/10min
CN
N
S
CH3
N
CH3
11
O
KOH/Dioxane
/2h
NC
NH2
12
N
S
CN
O
H2N
N
H
13
Scheme 3. Synthesis of 6-amino-5-(benzo[d]thiazol-2-yl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile 13
In order to further investigate the scope of this reaction and to establish whether the
reaction of 2-(benzo[d]thiazol-2-yl)-3-(dimethylamino)acrylonitrile 11 with aryl
cyanoacetamides could be extended to provide a general approach for the synthesis of N-
substituted 2-pyridylbenzothiazole, the reaction of compound 11 with other functionalized
cyanoacetamides was investigated. The reaction of compound 11 with N-aryl-2-
cyanoacetamides 14a-d and potassium hydroxide in 1,4-dioxane under reflux afforded N-
substituted 2-pyridylbenzothiazole 15a-d, Scheme 4.
16

11
O
NC
Ar
KOH/Dioxane
/2h
N
H
14
N
S
CN
O
H2N
N
Ar
15
15
Ar
C6H5
4-Cl-C6H4
4-CH3-C6H4
a
b
c
d
Scheme 4. Synthesis of 6-amino-1-aryl-5-(benzo[d]thiazole-2-yl)-2-oxo-1,2-
dihydropyridines 15a-d
The structures of the newly synthesized compounds 15a-d were also established on
1
the basis of their elemental analysis and spectral data. For example, H NMR spectrum of
compound 15c showed a singlet signal at δ 2.43 ppm for the protons of the CH3 group,
doublet at δ 7.30 ppm for the two protons of 4-tolyl group, two doublets at δ 7.86 and 8.07
ppm for two protons of benzothiazole ring and singlet peak at δ 8.40 ppm for the CH proton.
In order to establish the chemical structure of these derivatives, X-ray crystallographic
studies of compound 15c was performed. X-ray analysis confirmed the exclusive presence of
15c in the solid state, Fig. 1.
17

Fig. 1 Molecular structure of compound 15c
Moreover, compound 11 was allowed to react with 2-cyano-N'-(4-substituted
benzylidene)acetohydrazide 16a-d to afford 2-aryl-8-(benzo[d]thiazole-2-yl)-5-oxo-3,5-
dihydro[1,2,4]triazolo[1,5-a]pyridine-6-carbonitrile 21a-d, Scheme 5. The most probable
reaction pathway involved the addition of the active methylene group of the 2-cyano-N'-(4-
substituted benzylidene)acetohydrazide 16a-d to the double bond of compound 11 to form
intermediate Michael adducts with the subsequent elimination of the HN(CH3)2 group to
yield the intermediate 17a-d. Intramolecular cyclization through the addition of the NH group
to the cyano group forming the intermediate 18a-d followed by additional intramolecular
cyclization involving the bonding of the amino group to the ylidene carbon, which is
followed by the hydrolysis and the removal of H2 molecule to finally yield the novel 2-aryl-8-
(benzo[d]thiazole-2-yl)-5-oxo-3,5-dihydro[1,2,4]triazolo[1,5-a]pyridine-6-carbonitrile 21a-d.
Elemental analysis and spectral data of compounds 21a-d were consistent with its proposed
structure. For example, IR spectra of compound 21d showed bands for stretching vibrations
of NH group at 3374 cm-1, CN group at 2205 cm-1, and carbonyl group at 1665 cm-1. The 1H
NMR spectrum of compound 21d showed a singlet signal at δ 2.40 ppm, which indicated the
presence of CH3 protons, a multiplet at δ 7.33-7.39 ppm assigned for the aromatic protons
and a singlet peak at δ 8.47 ppm of the CH proton.
18

N
KOH/Dioxane
/2h
H
O
S
CN
O
NC
N
Ar
+
11
N
N
HN
N
H
16
Ar
17
N
N
H
CN
S
S
HN
CN
O
H2N
N
N
O
N
N
Ar
Ar
19
18
N
N
- H2
CN
O
CN
S
S
N
NH
N
N
NH
O
HN
Ar
Ar
20
21
Ar
21
4-F-C6H4
4-Cl-C6H4
a
b
c 4-CH3O-C6H4
4-CH3-C6H4
d
Scheme 5. Synthesis of 2-aryl-8-(benzo[d]thiazole-2-yl)-5-oxo-3,5-
dihydro[1,2,4]triazolo[1,5-a]pyridines 21a-d
Interestingly, some of the synthesized compounds showed fluorescence properties
which prompted us to study its photophysical properties. The photophysical properties of 7b,
7d-f, 9b, 13, 15a-d are summarized in Fig. 2-4 and Table 1. Fig. 2 shows the absorption
19

spectra of the examined compounds 7b, 7d-f, 9b, 13, 15a-d in ethanol. It can be clearly
observed that the absorption spectrum of 15c exhibited strong absorption band at 379 nm
with other two weak absorption bands at 326 and 480 nm. It is most likely that the absorption
bands at 326 and 379 nm arise from π−π transition, while the absorption band at the longer
wavelength of 480 nm arise from the n- π transition.
Similar observations were recorded for the other derivatives 15a-d. The absorption
maxima were listed in Table 1. Upon excitation, the samples with 370 nm absorbance, the
fluorescence spectra of the examined compounds in ethanol showed strong emission bands in
the range of 418-425 nm as shown in Fig. 3 and Table 1. From the listed values, it is clearly
seen that the fluorescence maxima are slightly change (1-5 nm) with changing the
substituents of 15a-d suggesting minor effect of the substituents in the fluorescence behavior.
Fig. 2 UV-vis absorption spectra of 7b, 7d-f, 9b, 13, 15a-d in ethanol
Fig. 3. Fluorescence spectra of 7b, 7d-f, 9b, 13, 15a-d in ethanol; λex = 370 nm
20

Fig. 4. Fluorescent images of 7b, 7d-f, 9b, 13, 15a-d in ethanol when excited with 370 nm light
To check the ability of the examined compounds to be utilized as fluorescent
materials in laser devices and optoelectronic applications, the fluorescence quantum yields
were determined. In general, the quantum yield of photo-excited fluorescence (FF) is defined
as the ratio of the number of photons emitted and the number of photons absorbed. The
fluorescence quantum yields of the examined compounds in ethanol were calculated by the
steady-state comparative method using Coumarin 460 as a standard (Φst = 0.73, ethanol) [22]
according to the following equation:
Where ΦF is the emission quantum yield of the samples, Φst is the emission quantum yield of
the standard, Ast and Ax represent the absorbance of the standard and sample at the excitation
wavelength, respectively, while Sst and Sx are the integrated emission band areas of the
standard and sample, respectively, and nst and nx the solvent refractive index of the standard
and sample, x and st refer to the unknown and standard, respectively. This method relies on
the fact that the unknowns of equation (1) should be quantitatively the same for different
fluorescent solutions as compared under identical conditions of excitation i.e. at the same
excitation wavelength with the same aperture settings (slit widths). By comparing the
intensity of fluorescence from a sample with that from a material whose QY is known, it is
straightforward to estimate QF.
Table 1: Fluorescent properties of synthesized compounds 7b, 7d-f, 9b, 13, 15a-d
abs
flu
Compd. No.
λmax
λmax
Φf
395
421
422, 492, 531
423
7b
7d
7e
0.10
0.02
0.03
399, 484
315, 400, 468
21

314, 403, 464
402, 488
422, 490, 534
470, 530
418, 438
421
7f
0.01
0.01
0.29
0.28
0.24
0.29
0.26
9b
326, 399
13
326, 378, 480
327, 378, 481
326, 379, 480
328, 381, 482
15a
15b
15c
15d
419
421
425
Fluorescence quantum yields of 13 and 15a-d were determined to be 0.29, 0.28, 0.24,
0.29 and 0.26, respectively. The considerably smaller value of 15b compared to other
samples (15a,c,d) may rationalized by the presence of the electron withdrawing chlorine
atom. In general, the determined fluorescence quantum yields values are in an excellent
agreement with the fluorescent images obtained by irradiation at 360 nm, Fig. 4.
Accordingly, it is clear that the newly synthesized N-substituted 2-pyridylbenzothiazole
compounds 13 and 15a-d showed remarkably higher quantum yield values than those of
pyrido[2,1-b]benzothiazole 7b, 7d-f and pyrido[2,1-b]benzoimidazole compounds 9b. Such
relatively high fluorescence quantum yields of N-substituted 2-pyridylbenzothiazoles suggest
the usefulness of these compounds as fluorescent probes in different optoelectronic
applications.
The antimicrobial activities of the newly synthesized compounds were evaluated in-
vitro against different bacterial strains such as Escherichia coli, Klebsiella pneumonia,
Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans as well as
against Candida albicans fungal strain. The synthesized compounds of benzothiazole-
substituted pyridines such as 7a, 7f, 9a, 9b, 13, 15a, 15d, 21a, 21b, and 21d showed some
activities against tested bacterial and fungal strains while other compounds such as 7b, 7d,
7e, 15b and 15c showed no reactivity toward all tested bacteria, Table 2. Interestingly,
compound 21a was found to be the most potent among all synthesized compounds towards
Klebsiella pneumonia strain (Inhibition Zone 24.3±0.6 mm) with comparable performance to
that of gentamicin, Table 2. It also showed moderate activities towards Staphylococcus
aureus (Inhibition Zone 20.3±0.6 mm) and Streptococcus mutans (Inhibition Zone 20.3±0.5
mm) as compared to ampicillin. Alternatively, compound 9b was the most potent among all
synthesized compounds toward Streptococcus mutans (Inhibition Zone 21.3±1.5 mm). It was
found that other compounds such as 9a, 13, 15a, and 21d have exhibited relatively low
activities toward Staphylococcus aureus when compared to ampicillin. In addition, some of
22

synthesized compounds such as 9a, 9b, 15a, and 21d showed activity against Candida
albicans as compared to Nystatin.
The structure-activity relationship (SAR) evaluated for the synthesized compounds
revealed that substituted benzene ring with methyl group at the ortho position 7f had some
activities toward Escherichia coli, Klebsiellla pneumonia and Streptococcus mutans while
derivatives with methyl group at the para and meta positions such as compounds 7d and 7e,
respectively, showed no activity toward all tested bacterial and fungi strains. Surprisingly,
substituted benzene ring with electron withdrawing group such as chlorine atom at the para
and meta positions, compound 7b and 7c, respectively, exhibited no activity toward all tested
bacterial and fungi strains. Moreover, replacing the benzothiazole moiety with a
benzoimidazole one in the presence of the methyl group at the para position of the benzene
ring, compound 9b, led to increasing the potency against Escherichia coli, Klebsiellla
pneumonia, Streptococcus mutans and Candida albicans strains. Interestingly, the presence
of the chlorine atom at the para position of the benzene ring such as compound 9a showed an
activity toward Staphylococcus aureus and Candida albicans strains only. Compound 13,
which contains the N-unsubstituted 2-pyridylbenzothiazole ring was shown to be more potent
than N-substituted 2-pyridylbenzothiazole derivatives 15a-d against Pseudomonas
aeruginosa and Staphylococcus aureus strains. Only compound 15a with a phenyl group at
the N-position of 2-pyridylbenzothiazole showed some activity toward fungi strain, Candida
albicans, while compound 15d with naphthyl group at the N-position of 2-
pyridylbenzothiazole showed some activity toward Klebsiellla pneumonia stain as compared
with other N-substituted 2-pyridylbenzothiazole derivatives 15a-c. Triazolo-pyridone
derivative with fluorine atom at the para position of the benzene ring, compound 21a, was
more potent than other derivatives containing - in place of the fluorine atom - a chlorine atom
or a methyl group, compounds 21b and 21d, respectively. Interestingly, the presence of the
methyl group as an electrondonating group at the para position of the benzene ring,
compound 21d, rendered the compound more potent than similar derivatives with a chlorine
atom (an electron withdrawing group) at the same position, compound 21b, against
Klebsiellla pneumonia, Staphylococcus aureus and Candida albicans.
Table 2: Antibacterial inhibition zone in mm ± standard deviation of synthesized compounds
Diameter of the inhibition zone (mm)
Compd No.
Gram (-ve) bacteria
Gram (+ve) bacteria
Fungi
23

Escherichia
coli
Klebsiella
pneumonia
Pseudomonas
aeruginosa
Staphylococcus
aureus
Streptococcus
mutans
Candida
albicans
(ATCC:3008)
(ATCC:4415)
(ATCC:27853)
(ATCC:6538)
(ATCC:25175)
(ATCC:10231)
NAa
NA
NA
10.7±1.1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
7a
7b
7d
NA
NA
NA
NA
NA
NA
NA
7e
13.0±0.0
NA
14.7±0.6
NA
NA
NA
NA
11.7±0.5
NA
14.3±0.6
NA
NA
12.7±0.5
12.7±2.0
NA
7f
9a
12.7±0.5
NA
14.7±0.5
NA
NA
21.3±1.5
NA
9b
11.3±0.5
NA
13.3±0.5
10.7±0.5
NA
13
NA
NA
NA
11.7±0.6
NA
15a
NA
NA
NA
NA
15b
NA
NA
NA
NA
NA
NA
15c
NA
16.0±1.1
24.3±0.6
15.3±0.6
17.3±1.5
35.0±0.5
-
NA
NA
NA
NA
15d
16.7±1.0
NA
NA
20.3±0.6
NA
20.3±0.5
13.0±1.0
13.7±1.2
-
NA
21a
NA
NA
21b
NA
NA
14.7±0.6
-
11.7±0.5
-
21d
35.0±0.5
-
30.0±0.5
-
Gentamicn
Ampicillin
Nystatin
30.0±1.0
-
35.0±0.5
-
-
-
-
-
20.0±0.5
a NA = No activity
The antiviral activities of the newly synthesized benzothiazole-substituted pyridines
derivatives were evaluated in-vitro against Herpes simplex virus type 1, Coxsackievirus B4,
Hepatitis A virus HM 175, ED-43/SG-Feo (VYG) replicon of Hepatitis C virus genotype 4a
and Adenovirus type 7. In order to determine the appropriate doses for the antiviral studies,
the newly synthesized compounds were subjected to a cytotoxicity evaluation using cell line
FRHK-4, Hep2, BGM, Vero and Huh 7.5 as the specific hosts to the various studied viruses.
The non-toxic doses of the synthesized compounds showed no significant differences, which
ranged between 70 and 120 µg/ml, Table 3. Even though the non-toxic doses reached 120
µg/ml, the synthesized compounds showed low level of activity against studied viruses with
reduction percent less than 50%, Table 4.
Table 3. Non-toxic doses of synthesized compounds on FRHK-4, Hep2, BGM, Vero, and
Huh 7.5 cell lines.
Nontoxic dose on
FRHK-4 cell line
(µg/mL)
Nontoxic dose on Nontoxic dose on
Nontoxic dose on
Vero cell line
(µg/mL)
Nontoxic dose on
Huh 7.5 cell line
(µg/mL)
Compd
No.
Hep2 cell line
BGM cell line
(µg/mL)
(µg/mL)
24

100
90
100
90
100
90
100
90
100
90
7a
7b
100
90
100
90
100
90
100
90
100
90
7d
7e
100
90
100
90
100
90
100
90
100
90
7f
9a
70
70
70
70
70
9b
80
80
80
90
80
9c
90
90
90
90
90
13
90
90
90
90
90
15a
15b
15c
15d
21a
21b
21c
21d
120
100
100
80
120
100
100
80
110
100
90
80
90
120
100
100
80
120
100
90
80
90
90
90
90
90
110
90
110
90
110
90
110
90
110
Table 4. Antiviral activity of non-toxic doses of synthesized compounds against Herpes
Simplex Virus, Coxsackievirus B4, Hepatitis A virus HM 175, HCVcc genotype 4 &
Adenovirus type 7.
Mean % of Reduction
Compd
No.
Herpes Simplex Coxsackievirus
Hepatitis A
HCVcc
Adenovirus
type 7
Virus
10
10
20
10
20
10
10
10
10
10
10
10
10
10
10
10
10
B4
virus HM 175 genotype 4
10
10
20
10
10
16.7
10
13.3
10
10
10
10
13.3
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
7a
7b
7d
7e
7f
20
20
10
10
13.3
10
9a
9b
10
10
9c
13
10
10
10
10
16.7
10
10
10
10
10
10
10
15a
15b
15c
15d
21a
21b
21c
21d
10
10
10
10
4. Conclusion:
25

In conclusion, eighteen novel compounds of pyrido[2,1-b]benzothiazole and N-
substituted 2-pyridylbenzothiazole derivatives were synthesized. Pyrido[2,1-b]benzothiazole
derivatives
were
synthesized
via
the
reaction
of
N-aryl-2-cyano-3,3-
bis(methylthio)acrylamide and benzothiazoleylacetonitrile while N-substituted 2-
pyridylbenzothiazole derivatives were synthesized by reacting 2-(benzo[d]thiazol-2-yl)-3-
(dimethylamino)acrylonitrile with cyanoacetamide, aryl cyanoacetamides or 2-cyano-N'-(4-
substituted benzylidene)acetohydrazide. The structures of all the compounds were confirmed
1
13
by H NMR, C NMR, IR and elemental analyses. The newly synthesized N-substituted 2-
pyridylbenzothiazole derivatives exhibited remarkable fluorescence properties. The strong
absorption bands at 326 and 379 nm most probably arise from the π−π transition, while the
absorption band at the longer wavelength of 480 nm arise from the n- π transition. Upon
excitation, samples with 370 nm absorbance showed strong fluorescence emission bands in
the range of 418-425 nm. It can be readily concluded that the fluorescence maxima may
slightly change in response to changing the aryl groups of the compounds suggesting a minor
effect of the substituents on the fluorescence behavior. Fluorescence quantum yields of these
compounds were also evaluated and determined to be in the range of 0.25-0.29.
In addition, the antimicrobial activities against Escherichia coli, Klebsiella
pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans and
Candida albicans strains have been also tested and some of examined compounds showing
some activities against the tested bacterial and fungal strains. The newly synthesized
compounds were also evaluated for their antiviral activity against HSV-1, COB4, HAV HM
175, ED-43/SG-Feo (VYG) replicon of HCV genotype 4a and HAdV7 and showed low level
of activity against examined viruses with viral reduction percent of less than 50%.
Acknowledgement
The authors thanks Prof. Peter G. Jones at the Institute for Inorganic and Analytical
Chemistry, Technical University Braunschweig, Germany for measuring the X-ray
crystallographic data and to Prof. M. El-Khouly at the Egypt Japan University for Science
and Technology (EJUST), Borg-Alarab, Alexandria, Egypt for measuring the fluorescence
data.
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