Design, synthesis, molecular docking and biological screening of N-ethyl-N-methylbenzenesulfonamide derivatives as effective antimicrobial and antiproliferative agents

Article abstract of DOI:10.1016/j.molstruc.2019.04.048

Sulfonamides are the most famous agents, which have been utilized for preparation of effective antiproliferated agents. Therefore, this article describes the synthesis of new series of N-ethyl-N-methylbenzenesulfonamide derivatives having various biologically active moieties such as, thiazoles 3, 4, 11, 12, 14, 15, 21, 1,3,4-thiadiazine 6, imidazo[2,1-b]thiazole 8, 2-oxo-2H-chromene 17, and 3-oxo-3H-benzo[f]chromene 19, starting with 4-(2-bromoacetyl)-N-ethyl-N-methylbenzenesulfonamide (2), that was synthesized from the interaction of 4-acetyl-N-ethyl-N-methylbenzenesulfonamide (1) with bromine under stirring in dioxane/diethylether mixture. The newly structures were be proved via their elemental analysis and spectral data. However, they were also screened for their cytotoxic activity against two different human cell lines, alveolar adenocarcinoma carcinoma (lung) (A-549) and liver carcinoma (HepG2) and antimicrobial. Compound 8 having imidazo[2,1-b]thiazole moiety exhibited the most potent cytotoxic activity against (A-549) cell line (SI; 30.77). While, compound 11 having 2-cyanomethyl thiazole moiety showed significant cytotoxic activity against (HepG2) cell line (SI; 67.11). On the other hand, compound 9 having 4-chlorophenyl moiety exerted significant antimicrobial activity more than the reference drugs. Molecular Operating Environment (MOE) was performed for the synthesized compounds to study their mode of action as inhibitors against DHFR enzyme active sites.

Full text of DOI:10.1016/j.molstruc.2019.04.048

Accepted Manuscript
Design, synthesis, molecular docking and biological screening of N-ethyl-N-
methylbenzenesulfonamide derivatives as effective antimicrobial and antiproliferative
agents
Shimaa M. Abd El-Gilil
PII:
S0022-2860(19)30443-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.04.048
MOLSTR 26420
Reference:
To appear in: Journal of Molecular Structure
Received Date: 4 March 2019
Revised Date: 9 April 2019
Accepted Date: 10 April 2019
Please cite this article as: S.M. Abd El-Gilil, Design, synthesis, molecular docking and biological
screening of N-ethyl-N-methylbenzenesulfonamide derivatives as effective antimicrobial and
antiproliferative agents, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.04.048.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT

ACCEPTED MANUSCRIPT
Design, synthesis, molecular docking and biological screening of N-
ethyl-N-methylbenzenesulfonamide
derivatives
as
effective
antimicrobial and antiproliferative agents
Shimaa M. Abd El-Gilil
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al‐Azhar University, Nasr
City, 11754, Cairo, Egypt
ABSTRACT
Sulfonamides are the most famous agents, which have been utilized for preparation of
effective antiproliferated agents. Therefore, this article describes the synthesis of new
series of N-ethyl-N-methylbenzenesulfonamide derivatives having various biologically
active moieties such as, thiazoles 3, 4, 11, 12, 14, 15, 21, 1,3,4-thiadiazine 6,
imidazo[2,1-b]thiazole 8, 2-oxo-2H-chromene 17, and 3-oxo-3H-benzo[f]chromene 19,
starting with 4-(2-bromoacetyl)-N-ethyl-N-methylbenzenesulfonamide (2), that was
synthesized from the interaction of 4-acetyl-N-ethyl-N-methylbenzenesulfonamide (1)
with bromine under stirring in dioxane/diethylether mixture. The newly structures were
be proved via their elemental analysis and spectral data. However, they were also
screened for their cytotoxic activity against two different human cell lines, alveolar
adenocarcinoma carcinoma (lung) (A-549) and liver carcinoma (HepG2) and
antimicrobial. Compound 8 having imidazo[2,1-b]thiazole moiety exhibited the most
potent cytotoxic activity against (A-549) cell line (SI; 30.77). While, compound 11
having 2-cyanomethyl thiazole moiety showed significant cytotoxic activity against
(HepG2) cell line (SI; 67.11). On the other hand, compound 9 having 4-chlorophenyl
moiety exerted significant antimicrobial activity more than the reference drugs.
Molecular Operating Environment (MOE) was performed for the synthesized compounds
to study their mode of action as inhibitors against DHFR enzyme active sites.
1

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Keywords:
N-ethyl-N-methylbenzenesulfonamide, Thiazole, Imidazo[2,1-b]thiazole, Anticancer,
Antimicrobial, Molecular docking
1. Introduction
Thiazole, 1,3,4-thiadiazine and imidazo[2,1-b]thiazole derivatives were reported
as antimicrobial [1,2], anti-inflammatory [3] and antiviral [4-6] agents. Moreover, they
acted like antihistaminic [7], antiparasitic [8], besides as calming down agents [9].
Furthermore, thiazoles were widely used in the dye and photographic industry [10].
Additionally, sulfonamides displayed antibacterial [11-14], antifungal [15], insulin
releasing [16-18], carbonic anhydrase inhibitor [19-22], hypoglycemic [23], anesthetic
[24], anti-tumor [25,26], anti-cancer [27] and anti-inflammatory [28,29] activities. Some
active sulfonamides as anti-bacterial are also known for their immune modifying effects
[30,31]. Therefore, taking in consideration the various reports and in continuation of the
previous work [32-37], it was aimed to synthesized substituted benzenesulfonamide
conjugated with thiazole, 1,3,4-thiadiazine, imidazo[2,1-b]thiazole, 2-oxo-2H-chromene,
3-oxo-3H-benzo[f]chromene moieties that are required to medicinal chemistry utilizing
phenacyl bromide derivative 2 as a backbone.
2. Materials and methods
2.1. Chemistry
An apparatus called electrothermal melting temperature has been used to measure
melting points, which are uncorrected. IR spectra were determined as KBr discs using IR-
470 Shimadzu spectrometer, (ν, cm-1). 1H and 13C NMR spectra were monitored on
Bruker 300 MHz spectrometer in DMSO-d6. Mass spectra were run on a Shimadzu
GC/MS-QP 5000 instrument at 70 eV. Elemental analysis were done on Carlo Erba 1108
CHN Analyzer. All compounds were within ±0.4 % of the theoretical values. Besides, the
reported 4-acetyl-N-ethyl-N-methylbenzenesulfonamide (1) was prepared according to
2

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the method specified in the literature [38]. Table 8, lists the physical and analytical data
of the newly prepared compounds.
2.1.1. 4-(2-Bromoacetyl)-N-ethyl-N-methylbenzenesulfonamide (2)
To a stirred solution of acetophenone derivative 1 (2.41 g, 0.01 mol) in a 30 mL mixture
of dioxane/diethylether (1:2), drops of bromine (1.59 g, 0.01 mol) were added, leave on
side, then poured on cold water, filter the product and recrystallized from ethanol to give
2. Yield, 87 %. IR (KBr, cm-1): vmax = 3056 (CH arom.), 2909 (CH aliph.), 1701 (C=O),
1
1333, 1160 (SO2), 1179 (C-S). H NMR: δ ppm: 1.08 (t, 3H, CH3 ethyl), 2.71 (s, 3H,
CH3), 3.23 (q, 2H, CH2 ethyl), 4.63 (s, 2H, CH2), 7.87, 8.63 (dd, 4H, Ar-H, J = 8.7 Hz).
13C NMR: δ ppm: 11.7 (CH3 ethyl), 31.9 (CH2), 34.0 (CH3), 43.8 (CH2 ethyl), [129.5
(2C), 131.4 (2C), 139.3, 146.5] (6 arom. C's), 189.3 (C=O). MS m/z (%): 321 [M++2]
(25); 319 [M+] (36); 306 (100); 304 (92); 263 (26); 261 (24); 226 (41); 199 (20); 197
(21); 162 (16); 148 (16); 122 (4); 120 (8); 118 (25); 106 (20); 104 (47); 90 (29); 89 (34);
77 (6); 75 (36); 65 (4); 63 (13).
2.1.2. 4-(2-Aminothiazol-4-yl)-N-ethyl-N-methylbenzenesulfonamide (3)
2.1.2.1. Procedure A; in dioxane (40 mL), a triturated mix of I2 (2.53 g, 0.01 mole) and
thiourea (1.52 g, 0.02 mole) was added to acetophenone derivative 1 (2.41 g, 0.01 mol).
Reflux with stirring was continued for 8 h. The obtained solid was washed with aqueous
sodium thiosulfate to remove excess iodine and then with water. After that, in hot water,
dissolve the obtained solid and filter while hot. Also, NH3. H2O was added to precipitate
2-aminothiazole derivative 3, which dried and recrystallized from ethanol, Yield, 41 %.
2.1.2.2. Procedure B; a solution of phenacyl bromide derivative 2 (3.20 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mole) in ethanol was refluxed for 2 h. Then add (5 mL) of pyridine
with continuation of reflux for 5 h. Collect the gotten solid and recrystallized from
dioxane. Yield, 79 %. IR (KBr, cm-1): vmax = 3300, 3284 (NH2), 3093 (CH arom.), 2935
1
(CH aliph.), 1550 (C=N), 1364, 1181 (SO2), 1179 (C-S). H NMR: δ ppm: 1.04 (t, 3H,
CH3 ethyl), 2.69 (s, 3H, CH3), 3.27 (q, 2H, CH2 ethyl), 6.91 (s, 1H, CH-thiazole), 7.53 (s,
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2H, NH2 transferrable with D2O), 7.82, 8.67 (dd, 4H, Ar-H, J = 9.1 Hz). 13C NMR: δ
ppm: 11.9 (CH3 ethyl), 35.6 (CH3), 39.7 (CH2 ethyl), 106.9 (C5-thiazole), [127.6 (2C),
129.7 (2C), 137.3, 138.5] (6 arom. C's), 154.4 (C4-thiazole), 169.6 (C2-thiazole). MS m/z
(%): 299 [M++2] (3); 298 [M++1] (4); 297 [M+] (32); 282 (14); 239 (33); 191 (15); 176
(15); 175 (100); 133 (8); 121 (7); 105 (10); 104 (10); 89 (17); 64 (7); 52 (8); 51 (8).
2.1.3. N-Ethyl-4-(2-hydrazinylthiazol-4-yl)-N-methylbenzenesulfonamide (4)
Reflux an ethanolic solution of thiosemicarbazide (0.91 g, 0.01 mol) and 2 (3.20 g, 0.01
mol) for 1 h. Filter off the product and recrystallized from ethanol/benzene to give 4.
Yield, 55 %. IR (KBr, cm-1): vmax = 3390, 3345 (NH2), 3270 (NH), 3090 (CH arom.),
1
2981 (CH aliph.), 1590 (C=N), 1343, 1170 (SO2), 1179 (C-S). H NMR: δ ppm: 1.03 (t,
3H, CH3 ethyl), 2.61 (s, 3H, CH3), 3.32 (q, 2H, CH2 ethyl), 5.09 (br, 2H, NH2
transferrable with D2O), 7.34 (s, 1H, CH-thiazole), 7.97, 8.01 (dd, 4H, Ar-H, J = 8.7 Hz),
11.20 (s, 1H, NH transferrable with D2O). 13C NMR: δ ppm: 12.1 (CH3 ethyl), 35.2
(CH3), 37.4 (CH2 ethyl), 106.0 (C5-thiazole), [124.9 (2C), 128.6 (2C), 134.1, 137.5] (6
arom. C's), 153.2 (C4-thiazole), 176.6 (C2-thiazole). MS m/z (%): 313 [M++1] (1); 312
[M+] (2); 298 (4); 255 (5); 239 (3); 224 (6); 199 (3); 191 (4); 175 (8); 160 (16); 149 (3);
133 (4); 129 (6); 127 (7); 109 (21); 107 (21); 101 (19); 96 (14); 89 (8); 82 (100); 81 (42);
80 (98); 79 (42); 74 (14); 64 (10); 60 (17); 45 (18).
2.1.4.
N-Ethyl-N-methyl-4-(2-(phenylamino)-6H-1,3,4-thiadiazin-5-yl)benzene-
sulfonamide (6)
An equimolar mixture of 2 (3.20 g, 0.01 mol) and N-phenylhydrazinecarbothioamide
(1.67 g, 0.01 mol) was heated under reflux for 4 h in ethanol (40 mL). Collect the solid
that formed while heating and recrystallized from ethanol to give 6. Yield, 43 %. IR
(KBr, cm-1): vmax = 3215 (NH), 3065 (CH arom.), 2912 (CH aliph.), 1584 (C=N), 1356,
1
1160 (SO2), 1179 (C-S). H NMR: δ ppm: 1.04 (t, 3H, CH3 ethyl), 2.72 (s, 3H, CH3),
3.36 (q, 2H, CH2 ethyl), 4.41 (s, 2H, CH2-thiadiazine), 6.51-8.17 (m, 9H, Ar-H), 9.82 (s,
1H, NH transferrable with D2O). 13C NMR: δ ppm: 11.3 (CH3 ethyl), 27.3 (C6-
4

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thiadiazin), 38.1 (CH3), 39.2 (CH2 ethyl), [121.6 (2C), 123.1, 128.8 (2C), 130.1 (2C),
131.2 (2C), 138.4, 140.1, 144.1] (12 arom. C's), 154.3 (C2-thiadiazin), 168.7 (C5-
thiadiazin). MS m/z (%): 388 [M+] (4); 341 (6); 307 (10); 306 (95); 304 (89); 286 (9); 284
(19); 278 (9); 258 (10); 212 (10); 149 (6); 148 (7); 100 (100); 99 (9); 73 (29); 55 (16); 53
(22); 46 (11).
2.1.5. N-Ethyl-4-(imidazo[2,1-b]thiazol-5-yl)-N-methylbenzenesulfonamide (8)
An equimolar mixture of phenacyl bromide derivative 2 (3.20 g, 0.01 mol) and 2-
aminothiazole (1 g, 0.01 mol) was heated under reflux for 3 h, in absolute ethanol (30
ml). Solid product was collected and recrystallized from dioxane to give 8. Yield, 46 %.
IR (KBr, cm-1): vmax = 3099 (CH arom.), 2965 (CH aliph.), 1583 (C=N), 1358, 1151
1
(SO2), 1179 (C-S). H NMR: δ ppm: 1.03 (t, 3H, CH3 ethyl), 2.71 (s, 3H, CH3), 3.34 (q,
2H, CH2 ethyl), 7.56-8.17 (m, 6H, Ar-H + CH=CH of thiazole), 8.01 (s, 1H, CH-
imidazole). 13C NMR: δ ppm: 11.2 (CH3 ethyl), 35.6 (CH3), 37.4 (CH2 ethyl), 121.5,
122.4, 124.2, [125.5 (2C), 128.4 (2C), 134.5, 137.3] (6 Arom. C's), 131.1, 148.4. MS m/z
(%): 321 [M+] (2); 306 (4); 263 (6); 228 (5); 227 (3); 226 (22); 199 (13); 197 (8); 179 (5);
124 (1); 119 (4); 108 (5); 105 (4); 104 (12); 101 (7); 100 (90); 99 (20); 89 (14); 82 (96);
81 (42); 80 (100); 79 (44); 76 (18); 73 (60); 72 (16); 58 (33); 57 (8).
2.1.6. 4-(2-((4-Chlorophenyl)amino)acetyl)-N-ethyl-N-methylbenzenesulfonamide (9)
An ethanolic solution of 2 (3.20 g, 0.01 mol) and p-chloroaniline (1.52 g, 0.012 mol) was
refluxed for 4 h, the product was recrystallize from ethanol to give 9. Yield, 62 %. IR
(KBr, cm-1): vmax = 3201 (NH), 3086 (CH arom.), 2927 (CH aliph.), 1681 (C=O), 1369,
1
1141 (SO2), 1179 (C-S), 715 (C-Cl). H NMR: δ ppm: 1.04 (t, 3H, CH3 ethyl), 2.69 (s,
3H, CH3), 3.34 (q, 2H, CH2 ethyl), 4.50 (s, 2H, CH2), 6.61, 7.32 (dd, 4H, Ar-H of
chlorophenyl, J = 8.2 Hz), 7.81, 8.24 (dd, 4H, Ar-H of benzenesulfonamide, J = 8.5 Hz),
10.22 (s, 1H, NH transferrable with D2O). 13C NMR: δ ppm: 11.5 (CH3 ethyl), 32.5
(CH3), 36.5 (CH2 ethyl), 63.1 (CH2), [115.3 (2C), 128.4, 129.5 (2C), 131.2 (2C), 132.3
(2C), 139.2, 146.2, 148.4] (12 ArC's), 197.7 (C=O). MS m/z (%): 366 [M+] (6); 364 (5);
5

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359 (15); 328 (8); 313 (21); 312 (89); 307 (100); 277 (10); 226 (20); 214 (13); 208 (16);
207 (25); 199 (37); 185 (49); 171 (12); 163 (21); 152 (12); 150 (11); 142 (12); 140 (29);
138 (31); 127 (16); 119 (15); 111 (19); 105 (88); 104 (58); 91 (25); 78 (37); 77 (41).
2.1.7. 4-(2-Cyanoacetyl)-N-ethyl-N-methylbenzenesulfonamide (10)
To an ethanolic solution of 2 (3.20 g, 0.01 mol) and potassium cyanide (0.65 g, 0.01 mol)
was added and heated under reflux for 5 h, upon reflux, a yellowish white crystals were
gotten, filtered and recrystallized from ethanol to give 10. Yield, 37 %. IR (KBr, cm-1):
vmax = 3075 (CH arom.), 2978 (CH aliph.), 2202 (C≡N), 1627 (C=O), 1363, 1155 (SO2),
1
1179 (C-S). H NMR: δ ppm: 1.03 (t, 3H, CH3 ethyl), 2.69 (s, 3H, CH3), 3.35 (q, 2H,
CH2 ethyl), 3.71 (s, 2H, CH2), 7.79, 8.31 (dd, 4H, Ar-H, J = 10.4 Hz). 13C NMR: δ ppm:
12.2 (CH3 ethyl), 27.6 (CH2), 36.3 (CH3), 39.8 (CH2 ethyl), [124.4 (2C), 127.5 (2C),
139.1, 147.3] (6 arom. C's), 136.2 (C≡N), 189.6 (C=O). MS m/z (%): 267 [M++1] (10);
266 [M+] (12); 264 (10); 263 (64); 238 (31); 226 (18); 215 (13); 208 (16); 198 (100); 186
(13); 184 (46); 175 (44); 169 (11); 166 (12); 159 (15); 149 (15); 134 (12); 121 (20); 120
(13); 119 (11); 105 (16); 103 (13); 91 (18); 89 (30); 77 (20); 76 (14); 64 (73); 60 (15); 59
(20).
2.1.8. 4-(2-(Cyanomethyl)thiazol-4-yl)-N-ethyl-N-methylbenzenesulfonamide (11)
A mixture of 2 (3.20 g, 0.01 mol) and 2-cyanoethanethioamide (1.00 g, 0.01 mol) in
ethanol was heated under reflux for 3 h. The obtained solid was recrystallized from
ethanol/benzene to give 11. Yield, 81 %. IR (KBr, cm-1): vmax = 3097 (CH arom.), 2981
1
(CH aliph.), 2198 (C≡N), 1597 (C=N), 1336, 1160 (SO2), 1179 (C-S). H NMR: δ ppm:
1.04 (t, 3H, CH3 ethyl), 2.71 (s, 3H, CH3), 3.31 (q, 2H, CH2 ethyl), 4.62 (s, 2H, CH2),
13
7.79 (s, 1H, H5-thiazole), 7.13, 8.01 (dd, 4H, Ar-H, J = 8.6 Hz). C NMR: δ ppm: 11.3
(CH3 ethyl), 22.1 (CH2), 35.2 (CH3), 38.3 (CH2 ethyl), 104.3 (C5-thiazole), 116.1 (C≡N),
[122.3 (2C), 124.1 (2C), 132.4, 137.2] (6 arom. C's), 151.1 (C4-thiazole), 169.1 (C2-
thiazole). MS m/z (%): 321 [M+] (6); 305 (25); 263 (26); 215 (10); 200 (13); 198 (100);
6

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172 (22); 159 (41); 134 (11); 132 (38); 93 (15); 91 (4); 90 (11); 89 (98); 87 (8); 85 (4); 79
(21); 77 (8); 75 (12); 52 (24); 51 (23).
2.1.9.
N'-(4-Chlorophenyl)-4-(4-(N-ethyl-N-methylsulfamoyl)phenyl)thiazole-2-carbo-
hydrazonoyl cyanide (12)
2.1.9.1. Procedure A; to an ethanolic icy solution of 11 (3.21 g, 0.01 mol) containing
sodium acetate (6.55 g, 0.09 mol), p-chlorobenzenediazonium chloride was added drop
wise (prepared under stirring at 0-5 oC upon adding p-chloroaniline (1.26 g, 0.01 mol) to
sodium nitrite (0.68 g, 0.01 mol) in 5 mL concentrated hydrogen chloride). The product
recrystallized from dioxane to give 12. Yield, 86 %.
2.1.9.2. Procedure B; an equimolar mixture of 2 (3.20 g, 0.01 mol) and 2-amino-N'-(4-
chlorophenyl)-2-thioxoacetohydrazonoyl cyanide (2.37 g, 0.01 mol) was refluxed in 40
mL ethanol for 2 h. The product obtained was filtered to give the compound 12, Yield, 73
%. IR (KBr, cm-1): vmax = 3170 (NH), 3097 (CH arom.), 2935 (CH aliph.), 2218 (C≡N),
1
1597 (C=N), 1345, 1151 (SO2), 1179 (C-S), 741 (C-Cl). H NMR : δ ppm : 1.04 (t, 3H,
CH3 ethyl), 2.71 (s, 3H, CH3), 3.32 (q, 2H, CH2 ethyl), 7.07, 7.24 (dd, 4H, Ar-H, of
chlorophenyl, J = 8.6 Hz), 7.51 (s, 1H, H5-thiazole), 7.81, 8.01 (dd, 4H, Ar-H, of
benzenesulfonyl, J = 8.6 Hz), 11.94 (s, 1H, NH, Discharged with D2O). 13C NMR: δ
ppm: 11.9 (CH3 ethyl), 35.3 (CH3), 37.6 (CH2 ethyl), 112.2 (C5-thiazole), [115.2 (2C),
124.3, 125.4 (2C), 126.7 (2C), 128.1 (2C), 133.4, 136.2, 143.4] (12ArC's), 122.3 (C≡N),
156.1 (C4-thiazole), 157.2 (C2-thiazole), 159.1 (C=N). MS m/z (%): 460 [M++1] (1); 459
[M+] (9); 302 (3); 255 (16); 228 (7); 225 (12); 224 (23); 222 (30); 205 (14); 160 (14); 152
(25); 147 (38); 128 (21); 114 (29); 112 (100); 77 (56); 75 (17); 74 (12); 64 (53); 52 (26);
51 (33).
2.1.10.
4-(2-(1-Cyano-2-(4-fluorophenyl)vinyl)thiazol-4-yl)-N-ethyl-N-methylbenzene-
sulfonamide (14)
2.1.10.1. Procedure A; to an ethanolic solution of mixture thiazolylacetonitrile derivative
11 (3.21 g, 0.01 mol) and p-fluorobenzaldehyde (1.23 g, 0.01 mol), add few drops of
7

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piperidine and refluxed for 4 h. The solid was recrystallized from ethanol/benzene to give
14. Yield, 80 %.
2.1.10.2. Procedure B; an equimolar mixture of 2 (3.20 g, 0.01 mol) and 2-cyano-3-(4-
fluorophenyl)prop-2-enethioamide (2.04 g, 0.01 mol) was refluxed in 50 mL ethanol for
3 h. The solid was filtered and recrystallized. Yield, 67 %. IR (KBr, cm-1): vmax = 3107
(CH arom.), 2974 (CH aliph.), 2218 (C≡N), 1593 (C=N), 1365, 1181 (SO2), 1179 (C-S),
1170 (C-F). 1H NMR: δ ppm: 1.03 (t, 3H, CH3 ethyl), 2.72 (s, 3H, CH3), 3.37 (q, 2H, CH2
ethyl), 7.22, 7.81 (dd, 4H, Ar-H of fluorophenyl, J = 8.6 Hz), 7.99, 8.49 (dd, 4H, Ar-H of
benzenesulfonyl, J = 8.5 Hz), 7.51 (s, 1H, H5-thiazole), 9.02 (s, 1H, CH=C). 13C NMR: δ
ppm: 11.6 (CH3 ethyl), 35.4 (CH3), 37.6 (CH2 ethyl), 110.3 (C=CH), 112.4 (C5-thiazole),
[116.2 (2C), 123.6 (2C), 125.7 (2C), 132.1 (2C), 133.3, 137.1, 138.2, 164.3] (12ArC's),
120.1 (C≡N), 153.2 (C4-thiazole), 156.3 (C2-thiazole), 159.1 (C=CH). MS m/z (%): 428
[M++1] (11); 427 [M+] (50); 413 (7); 412 (36); 306 (17); 304 (100); 208 (3); 206 (10);
175 (25); 158 (10); 152 (20); 140 (4); 139 (5); 89 (23).
2.1.11.
4-(2-(1-Cyano-2-(dimethylamino)vinyl)thiazol-4-yl)-N-ethyl-N-methylbenzene-
sulfonamide (15)
Compound 11 (3.21 g, 0.01 mol) was heated under reflux for 4 h in xylene (40 mL) and
DMF-DMA (1.19 g, 0.01 mol). The collected solid washed with ether, and recrystallized
from benzene to give 15. Yield, 53 %. IR (KBr, cm-1): vmax = 3038 (CH arom.), 2924 (CH
1
aliph.), 2211 (C≡N), 1590 (C=N), 1355, 1161 (SO2), 1179 (C-S). H NMR: δ ppm: 1.04
(t, 3H, CH3 ethyl), 2.72 (s, 3H, CH3), 2.82 (s, 6H, (CH3)2N), 3.35 (q, 2H, CH2 ethyl), 7.31
(s, 1H, CH=C), 7.81 (s, 1H, H5-thiazole), 8.18, 8.71 (dd, 4H, Ar-H, J = 8.5 Hz). 13C
NMR: δ ppm: 11.2 (CH3 ethyl), 35.1 (CH3), 37.2 (CH2 ethyl), 44.6 (2C, (CH3)2N), 84.3
(C=CH), 112.2 (C5-thiazole), 115.2 (C≡N), [128.3 (2C), 129.2 (2C), 134.1, 137.4] (6
arom. C's), 153.2 (C4-thiazole), 156.3 (C2-thiazole), 158.6 (C=CH). MS m/z (%): 378
[M++2] (10); 377 [M++1] (18); 376 [M+] (100); 254 (33); 253 (46); 239 (9); 237 (9); 220
(12); 211 (18); 198 (13); 180 (11); 127 (15); 89 (15).
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2.1.12. N-Ethyl-N-methyl-4-(2-(2-oxo-2H-chromen-3-yl)thiazol-4-yl)benzenesulfonamide
(17)
2.1.12.1. Procedure A; to an ethanolic solution containing few drops of piperidine, a
mixture of 11 (3.21 g, 0.01 mol) and o-hydroxybenzaldehyde (1.22 g, 0.01mol) was
heated under reflux for 4 h. The collected product was recrystallized from
ethanol/benzene to give 17. Yield, 77 %.
2.1.12.2. Procedure B; an ethanolic solution of equimolar mixture of 2 (3.20 g, 0.01 mol)
and 2-oxo-2H-chromene-3-carbothioamide (2.04 g, 0.01 mol) was heated under reflux for
2 h. Filter the solid and recrystallized. Yield, 57 %. IR (KBr, cm-1): vmax = 3093 (CH
arom.), 2935 (CH aliph.), 1658 (C=O), 1600 (C=N), 1364, 1180 (SO2), 1276, 1037 (C-O-
C), 1179 (C-S). 1H NMR: δ ppm: 1.03 (t, 3H, CH3 ethyl), 2.72 (s, 3H, CH3), 3.36 (q, 2H,
CH2 ethyl), 7.04 (s, 1H, H5-thiazole), 7.38-7.52 (m, 4H, Ar-H), 7.81, 8.01 (dd, 4H, Ar-H,
J = 8.5 Hz), 9.08 (s, 1H, H4-coumarin). 13C NMR: δ ppm: 11.8 (CH3 ethyl), 32.5 (CH3),
36.7 (CH2 ethyl), 113.2 (C5-thiazole), [114.2, 118.5, 123.3, 125.2 (2C), 128.6 (2C),
129.2, 130.8, 131.1, 134.5, 141.3, 144.2, 156.1] (14ArC's), 157.1 (C4-thiazole), 158.2
(C2-thiazole), 163.7 (C=O). MS m/z (%): 427 [M++1] (9); 426 [M+] (17); 425 (71); 410
(26); 408 (72); 306 (14); 304 (22); 303 (100); 288 (17); 286 (14); 259 (5); 258 (16); 151
(37); 134 (11); 89 (34).
2.1.13.
N-Ethyl-N-methyl-4-(2-(3-oxo-3H-benzo[f]chromen-2-yl)thiazol-4-yl)benzene-
sulfonamide (19)
An ethanolic solution of a mixture of thiazolylacetonitrile derivative 11 (3.21 g, 0.01
mol) and o-hydroxy-1-naphthaldehyde (1.73 g, 0.01 mol) containing drops of pipredine
was heated under reflux for 6 h. Filter the product obtained and recrystallized from acetic
acid to give 19. Yield, 69 %. IR (KBr, cm-1): vmax = 3062 (CH arom.), 2873 (CH aliph.),
1660 (C=O), 1590 (C=N), 1333, 1179 (SO2), 1208, 1042 (C-O-C), 1179 (C-S). 1H NMR:
δ ppm: 1.04 (t, 3H, CH3 ethyl), 2.72 (s, 3H, CH3), 3.35 (q, 2H, CH2 ethyl), 7.36 (s, 1H,
H5-thiazole), 7.91-8.22 (m, 10H, Ar-H), 8.86 (s, 1H, H1-benzocoumarin). 13C NMR: δ
ppm: 11.2 (CH3 ethyl), 32.6 (CH3), 37.1 (CH2 ethyl), 112.0 (C5-thiazole), [113.5, 118.2,
121.2, 122.9, 125.7, 126.9 (2C), 128.0 (2C), 129.1, 130.0, 130.9, 131.7, 132.1, 135.3,
9

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138.9, 144.8, 154.1] (18ArC's), 155.8 (C4-thiazole), 156.8 (C2-thiazole), 164.3 (C=O).
MS m/z (%): 478 [M++2] (5); 477 [M++1] (8); 476 [M+] (15); 475 (14); 459 (23); 457
(31); 354 (16); 353 (14); 336 (23); 252 (6); 240 (9); 238 (8); 198 (5); 193 (56); 191 (13);
177 (31); 176 (25); 175 (28); 165 (22); 164 (57); 162 (19); 160 (10); 150 (11); 149 (22);
139 (29); 128 (14); 133 (28); 132 (12).
2.1.14. 2-Cyano-2-(4-(4-(N-ethyl-N-methylsulfamoyl)phenyl)thiazol-2-yl)ethanedithioic
acid (21)
Carbon disulfide (0.75 g, 0.01 mol) was added slowly to an icy solution of compound 11
(3.21 g, 0.01 mol) in 25 mL N,N-dimethylformamide containing potassium hydroxide
(1.12 g, 0.02 mol). Left mix for 24 h at room temperature followed by trituration with 40
mL icy water, neutralized with 1N hydrogen chloride. Wash the collected solid with
excess water, and recrystallized from ethanol to give 21. Yield, 44 %. IR (KBr, cm-1):
vmax = 3094 (CH arom.), 2927 (CH aliph.), 2591 (SH), 2218 (C≡N), 1613 (C=N), 1366,
1
1172 (SO2), 1344 (C=S), 1179 (C-S). H NMR: δ ppm: 1.04 (t, 3H, CH3 ethyl), 1.69 (s,
1H, SH), 2.73 (s, 3H, CH3), 3.37 (q, 2H, CH2 ethyl), 5.34 (s, 1H, CH), 7.71 (s, 1H, H5-
thiazole), 7.98, 8.92 (dd, 4H, Ar-H, J = 8.5 Hz). 13C NMR: δ ppm: 11.4 (CH3 ethyl), 35.3
(CH3), 37.5 (CH2 ethyl), 59.3 (CH), 110.3 (C5-thiazole), 113.6 (C≡N), [125.8 (2C), 126.5
(2C), 134.5, 137.4] (6ArC's), 158.4 (C4-thiazole), 167.7 (C2-thiazole), 224.6 (C=S). MS
m/z (%): 397 [M+] (1); 368 (1); 348 (1); 339 (2); 334 (1); 326 (3); 324 (13); 320 (25); 310
(19); 306 (48); 282 (16); 297 (45); 263 (28); 224 (45); 200 (10); 199 (60); 159 (100); 132
(40); 89 (64); 64 (26); 63 (16); 58 (21); 52 (12).
3. In-vitro anticancer screening
3.1. Cytotoxicity Assessment
All novel compounds were evaluated for their cytotoxicity toward two human cell
lines, alveolar adenocarcinoma carcinoma (lung) (A-549) and liver carcinoma (HepG2).
Doxorubicin (DOX) was a reference control, (Tables 1-5).
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3.2. Cytotoxicity evaluation using viability assay
Cell lines were purchased from the American Type Culture collection and their
accession number as follows: A-549 (ATCC CCL-185™) lung carcinoma cell line, and
HepG2 (ATCC® HB-8065™) liver carcinoma cell line. Exponentially, place cells for 24
h in 96-well plates, and then add fresh medium which containing different concentration
of the tested sample. Sequential two-fold dilutions of the tried sample were added using a
o
multichannel pipette. Moreover, all cells were cultivated at 37 C, 5 % CO2 and 95 %
o
moisture. In addition, incubation of control cells occurred at 37 C. However, after
incubation for 24 h different concentrations of tested sample were added (50, 25, 12.5,
6.25, 3.125 & 1.56 µg/L) and continued the 48 h incubation, then, add 1 % crystal violet
solution to each well for 0.5 h to examine viable cells. Rinse the wells using water until
no stain. After that, add 30 % glacial acetic acid to all wells with shaking plates on
Microplate reader (TECAN, Inc.) to measure the absorbance, using a test wavelength of
490 nm. Besides, compare the treated samples with the control cell. The cytotoxicity was
estimated by IC50 in µM. Additionally, selectivity index (SI), the concentration ratio
which causes death of 50 % in baby hamster kidney (BHK) cell line (CC50) divided to the
concentration which causes death of 50 % in human carcinoma (IC50) [39-42] was also
determined.
4. In Vitro Antimicrobial evaluation
Bacterial and fungal strains were obtained from culture collection of the Regional
Center for Mycology and Biotechnology (RCMB), Al-Azhar University. The testing of
the efficacy of all novel derivatives was carried out by standardized disc-agar diffusion
method [43]. Using Mueller Hinton Agar. Activation of various strains of bacteria
occurred by using a loop that full of bacterial strain in the broth and incubated for 24 h at
37o C. Furthermore, 0.1 mL of the suspension of strains was poured on the agar, spread
well and left to solidify. Moreover, using a sterile cork, about 0.9 cm cut was made and
were filled completely with the tested compound solution. The wells were incubated at 37
11

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oC for 24 h. Each assay was done in triplicate. ZOI was determined using the mean value.
Tested compound giving high ZOI value reflecting its significant antibacterial activity.
The tested strains included three strains of gram-positive bacteria, namely;
Staphylococcus aureus (RCMB 010027), Streptococcus pneumoniae (RCMB 010010) and
Bacillus subtilis (RCMB 010067) compared to Ampicillin as a reference drug. In addition
to four strains of gram-negative bacteria, namely; Pseudomonas aeruginosa (RCMB
010043), Mycobacterium tuberculosis (RCMB 010120), Klebsiella pneumoniae (RCMB
0010093) and Escherichia coli (RCMB 010052) ATCC 25955 compared to Gentamicin as
a reference drug. Besides two different fungal strains namely; Aspergillus fumigatus
(RCMB 02568) and Candida albicans (RCMB 05036) compared to Amphotericin B as a
control drug, (Table 6).
5. Docking assay
5.1. Materials
The study was done in the Department of Pharmaceutical Organic Chemistry,
Faculty of Pharmacy (Girls), Al‐Azhar University, on an Intel(R) Core(TM) i7-3632QM
2.20 GHz processor, 8.00 GB memory with windows 7 Ultimate operating system using
Molecular Operating Environment (MOE 2015.10; Chemical Computing Group,
Montreal, Canada) as the computational software. All the minimizations were performed
with MOE until a RMSD gradient of 0.05 Kcal mol-1 Å-1 with MMFF94X force field.
5.2. General Methodology
It depends on downloading from Protein Data Bank (PDB ID: 1BID), the
bounding of 3D structure of doxorubicin (DOX) to DHFR enzyme (PDB ID: 4DFR).
Hydrogen atoms were further added to the structure of the enzyme. In addition,
molecules of water and bound ligands were deleted manually and their energy was be
minimized. MOE-Alpha site finder has been used to generate the active site. However,
the alpha spheres created Dummy atoms. Besides, docking of ligands via the active sites
12

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of DHFR was obtained using MOE-Dock and MMFF94X molecular mechanics force
field for 8000 connections. Furthermore, the conformation having the lowest energy was
chosen and forced to minimization of energy using MMFF94X force field. Docking
results were mentioned as Score. E_conf; The energy of the conformer. E_place; Score
from the placement stage E_score1; Score from the 1strescoring stage. E_score2; Score
from the 2nd rescoring stage. E_refine; Score from the refinement stage.
6. Results and discussion
6.1. Chemistry
4-(2-bromoacetyl)-N-ethyl-N-methylbenzenesulfonamide (2) was synthesized by
reaction of 4-acetyl-N-ethyl-N-methylbenzenesulfonamide (1) with bromine, which was
employed as a building unit for synthesis of novel thiazole (3) (Scheme 1). The active
function that attached to N-ethyl-N-methylbenzenesulfonamide derivatives determined
the degree of its pharmacological activity. 1H NMR spectrum of 2 displayed a singlet at δ
= 4.63 ppm due to active CH2 of bromoacetyl group. Besides, 13C NMR spectrum
exhibited a vital signal at δ = 31.9 ppm corresponding to active CH2 group. However, MS
spectrum showed molecular ion peaks at m/z = 319 and 321 corresponding bromine
isotopes. On the other hand, 2-Aminothiazole derivative 3, was obtained by two various
pathways. One of them included refluxing of compound 1 with thiourea and iodine.
Besides that, the solid product has low yield, iodine has pollution problems. Therefore,
the effort extended to other pathway at which in ethanolic solution compound 2 was
refluxed with thiourea, this method gave yield about 79 %. IR spectrum of 3 exhibited
1
forked bands at ν = 3300, 3284 cm-1 mentioned to NH2 function. H NMR spectrum
shown a singlet of one proton at δ = 6.91 ppm, attributed to CH-thiazole and deuterium
13
oxide transferrable signal at δ = 7.53 ppm, referred to NH2 protons. However, C NMR
spectrum of 3 displayed signals at δ = 106.9 and 169.6 ppm due to C5-thiazole and C2-
thiazole, respectively.
13

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Scheme 1. Synthesis of starting material 2 and 2-aminothiazole derivative 3.
Moreover, alteration of compound 2 into different heterocyclic compound when
reacted with thiosemicarbazide were unsuccessful, as the reaction afforded also thiazole
derivative 4, instead of the expected 2-aminothiadiazine derivative 5, which confirmed by
1
IR spectrum (as it showed band at ν = 3269 cm-1 conforming to NH function) and H
NMR spectrum (as it indicated a singlet at δ = 11.20 ppm) (Scheme 2).
Scheme 2. Synthesis of 2-hydrazinylthiazole derivative 4.
However, thiadiazin derivative 6 was obtained via the reaction of compound 2
with N-phenylhydrazinecarbothioamide. While, the other expected structure 4-(3-amino-
2-(phenylimino)-2,3-dihydrothiazol-4-yl)-N-ethyl-N-methylbenzene-sulfonamide (7) was
ruled out depending on spectral data. IR spectrum of 6 showed a specific band at ν =
3215 cm-1 attributed to NH moiety. Moreover, 13C NMR spectrum revealed carbons of
thiadiazine moiety at δ = 27.3 ppm. Besides, the MS spectrum confirmed structure 6 as it
showed m/z = 388. The goal was raised to focus on different fused heterocyclic rings
activity. Therefore, refluxing of compound 2 with 2-aminothiazole in ethanolic solution
afforded imidazothiazole derivative 8. Also, reaction of 2 with 4-chloroaniline and
potassium cyanide afforded compounds 9 and 10, respectively. Thus, IR spectrum of 10
14

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showed an absorption band at ν = 2220 cm-1 corresponding to cyano function.
1
Additionally, H NMR spectrum showed a singlet at δ = 3.71 ppm corresponding to
active methylene group. (Scheme 3).
Scheme 3. Synthesis of compounds 6, 8-10 from starting material 2.
Our aim was extended to synthesis other thiazole derivatives via refluxing of 2
with 2-cyanoethanethioamide in ethanolic solution to afford 4-(2-(cyanomethyl)thiazol-4-
yl)-N-ethyl-N-methylbenzenesulfonamide 11. IR, 1H NMR and 13C NMR spectra
confirmed the product. As methylene group is established to be very reactive. Therefore,
coupling of equimolar amount of 11 with 4-chlorobenzenediazonium chloride in cold
o
ethanolic solution (0-5 C) containing sodium acetate, yellow crystalline solid of one of
two structures 12 or 13 be possible. Spectral data provided a definite support for only
structure 12 as IR spectrum exhibited band at ν = 3170 cm-1 due to NH function. Besides,
1
thiazole proton was appeared as a singlet at δ = 7.51 ppm in H NMR spectrum.
Additionally, other pathway could be used for preparation of compound 12 through
refluxing of phenacyl bromide derivative 2 with 2-amino-N'-(4-chlorophenyl)-2-
thioxoacetohydrazonoyl cyanide. Moreover, condensation of compound 11 with 4-
fluorobenzaldehyde afforded thiazolylacrylonitrile derivative 14. However, refluxing of
compound 2 with 2-cyano-3-(4-fluorophenyl)prop-2-enethioamide gave also compound
14. (Scheme 4).
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Scheme 4. Strategy synthesis of novel sulfonamide thiazole derivatives 11, 12 and 14
utilizing 2 as a starting material.
Furthermore, refluxing of 11 with DMF-DMA in xylene afforded compound 15,
1
which confirmed by H NMR and 13C NMR spectra. Our goal was also discovered the
condensation of 11 with o-phenolic aldehydes [44], such as o-hydroxybenzaldehyde and
o-hydroxy-1-naphthaldehyde. Therefore, refluxing of 11 with o-hydroxybenzaldehyde in
ethanolic solution containing few drops of piperidine afforded compound 17. In addition,
it can be prepared via Knovenagel condensed intermediate 16, at which intramolecular
cyclization occurred via Michael-type addition reaction [45] followed by formation of
carbonyl group by hydrolysis of the imino group. Similar hydrolysis reactions have been
reported [46,47]. Another pathway at which phenacyl bromide derivative 2 was refluxed
with 2-oxo-2H-chromene-3-carbothioamide in ethanol can also synthesize it. In the same
manner, upon reaction of 11 with 2-hydroxy-1-naphthaldehyde, it gave the intermediate
benzo[f]chromen-3-imine derivative 18 which by hydrolysis afforded benzo[f]chromen-
3-one derivative 19. (Scheme 5).
16

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Scheme 5. Synthesis of newly compounds 15, 17 and 19.
At room temperature, in dry DMF and in presence of KOH, reaction of 11 with
carbon disulfide can be proceeded and gave potassium dithiolate salt 20 which upon
treatment with 1N hydrogen chloride, it transformed into ethanedithioic acid derivative
21, which confirmed by spectral data. (Scheme 6).
Scheme 6. Strategy synthesis of newly sulfonamide ethanedithioic acid derivative 21
from 2-cyanomethylthiazole derivative 11.
6.2. In vitro anticancer activity
Activity of the prepared compounds toward (A-549) and (HepG2) cell lines was
depends on their IC50 values which tabulated in (Tables 1 and 2); respectively as well as
(Charts 1 and 2); respectively. While, CC50 values against baby hamster kidney (BHK)
cell line represented in (Table 3). Furthermore, CC50, IC50 and SI=CC50/IC50 of the tested
compounds toward lung (A-549), (HepG2) cell lines compared to human normal baby
hamster kidney (BHK) cell lines are tabulated in (Tables 4 and 5); respectively as well
as (Charts 3 and 4); respectively. From the obtained results in the previous (Tables 1-5)
and adopting that increasing selectivity index SI values give a selective toxicity against
cancer cells, it can be noted that, compound 3 bearing 2-amino thiazole moiety exerted
significant increase in cytotoxicity against (HepG2) cell line having SI value 64.52, while
it showed comparable activity against lung cancer cell line (A-549), SI value 4.00,
17

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compared to the reference drug doxorubicin having SI values 22.90 and 5.51,
respectively. Moreover, Imidazo[2,1-b]thiazole derivative 8 (IC50 = 1.55 µg/mL)
exhibited strong anticancer activity against lung cancer cell line (A-549) and revealed SI
value 30.77, however, it showed moderate activity against (HepG2) cell line (IC50 = 10.6
µg/mL) having SI value 4.50 compared to doxorubicin (IC50 = 20.23 and 4.87 µg/mL; SI
value 5.51 and 22.90), respectively. Furthermore, compound 9 bearing 4-chlorophenyl
moiety resulted in improvement in activity against lung cancer cell line (A-549), SI value
5.94, while SI value of doxorubicin 5.51. However, it revealed nearly activity to
doxorubicin (SI value 22.90) toward (HepG2) cell line (SI value 17.77). Additionally, the
presence of 2-cyanomethyl thiazole moiety as in compound 11 showing increase in
cytotoxic activity against (HepG2) cell line (SI value 67.11), while it showed comparable
activity against lung cancer cell line (A-549), SI value 4, compared to the reference drug
doxorubicin having SI values 22.90 and 5.51, respectively. Moreover, 4-fluorophenyl
derivative 14 showed marked increase in anticancer activity against both lung cancer (A-
549) and (HepG2) cell lines exerting SI values 6.82 and 25.98, respectively. Beside,
compound 17 having 2-oxo-2H-chromen moiety exhibited slightly better anticancer
activity against both lung cancer (A-549) and (HepG2) cell lines exerting SI values 5.93
and 31.17, respectively. On the other hand, the presence of 2-cyano ethanedithioic acid
moiety as in compound 21 indicated significant increase in anticancer activity against
(HepG2) cell line (SI value 35.78), however, it exists a promising activity as doxorubicin
toward lung cancer cell line (A-549), SI value 4. It can be concluded that according to SI
values of the synthesized compounds, the cytotoxicity order toward lung cancer (A-549)
cell line is: 8 > 14 > 9 > 17 > DOX > 10 > 6 > 2, 11, 19, 15, 21, 12, 3 > 4. While, the
order against (HepG2) cell line is: 11 > 3 > 21 > 17 > 14 > DOX > 10 > 9 > 12 > 2 > 8 >
19, 15 > 6 > 4.
6.3. Antimicrobial screening
Novel tested compounds were screened for the preliminary antimicrobial efficacy
toward various microorganisms representing Gram +ve bacteria (Staphylococcus aureus,
Streptococcus pneumonia and Bacillis subtilis), Gram -ve bacteria (Pseudomonas
18

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aeruginosa, Mycobacterium tuberculosis, Klebsiella pneumoniae and Escherichia coli),
fungi (Aspergillus fumigatus and Candida albicans). The obtained results in (Table 6) as
well as (Chart 5) showed that Compound 9 exhibited dual activities as antibacterial and
antifungal agents on all tested microbial strains. Regarding antibacterial activities, it can
be clearly observed that, compounds 2, 3, 4, 6 and 8 showed increase activities against
two strains of Gram-negative bacteria; Mycobacterium tuberculosis and Klebsiella
Pneumoniae. All results were compared to the reference drugs.
6.4. Docking and molecular modeling
A well-founded procedure used to expect the compound interaction with the most
suitable binding site. Various types of interactions between ligand and amino acid were
possibly determined. The main enzymes that were concerned in studying antimicrobial
and anticancer activities are thymidylate synthase and dihydrofolate reductase [48,49].
All dock runs were done by Molecular Operating Environment (MOE) module to observe
the cytotoxicity of the tested compounds. This work revealed DHFR interaction with its
substrate DOX and with the following representative active anticancer compounds 2-4, 6,
8-12, 14, 15, 17, 19 and 21 using MOE program [50] on DHFR active sites. The energy
scores were tabulated in (Table 7).
6.4.1. Docking of DOX into DHFR; illustrated that (OH) function of tetracene acted as a
H-bond donor to backbone Asp 21 with 2.65 Å having a strength of 1.1 %. However,
(OH) function of acetyl pyran acted as a H-bond acceptor with Ser 59 (3.02 Å) having a
strength of 16.2 %. Moreover, (NH2) function acted as a H-bond donor with Glu 30 (3.31
Å) with strength of 5 %. Furthermore, there is arene-arene contact among phenyl ring of
tetracene and Phe 31. In addition to, hydrophobic interactions involving: Val 8, Gly 20,
Asp 21, Leu 22, Phe 31, Phe 34, Thr 56, Ile 60, Pro 61, Asn 64, Leu 67, Val 115 and Tyr
121, Fig. 1.
19

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Fig. 1. Docking of DOX into DHFR
6.4.2. Docking of compounds 2-4, 6, 8-12, 14, 15, 17 and 21 into DHFR; (Figures 2-13
and 15), respectively (supplementary data).
6.4.3. Docking of compound 19 into DHFR; In a similar manner, it proved that H-bond
acceptor interactions were established between either (O) atom of (C=O) function and Ser
59 (2.82 Å) having a strength of 10.3 % or one (O) atom of (SO2) function and Arg 70
(2.52 Å) with a strength of 70.2 %. Besides, hydrophobic interactions among: Val 8, Leu
22, Phe 31, Phe 34,Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Leu 67, Lys 68, Arg
70, Val 115 and Tyr 121, Fig. 14.
Fig. 14. Docking of Compound 19 into DHFR
20

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6.5. Conclusion of docking assay:
Docking study exerted various interactions between the target and ligand, which
are expected to have better anticancer activity. The compounds under investigation are 2-
4, 6, 8-12, 14, 15, 17, 19 and 21. Particularly noteworthy, only compounds 19, 17, 12, 14,
15 and 6, were suggested to exhibit their efficacy via inhibition of the active sites of
DHFR enzyme (Table 7). It was clarified that, score energy of the tested compounds
follows the order 19 > 17 > 12 > 14 > 15 > 6 > 9 > 21 > 4 > 11 > 3 > 8 > 10 > 2 (Chart
6).
6.6. Structure Activity Relationship (SAR)
For the well understanding of the antitumor and antimicrobial activities, SAR of
the tested compounds has to be indicated it proved that; thiazole derivatives as in
compounds 3, 11, 14 and 21 existed marked increase in anticancer activity against human
liver cancer (HepG2) cell line, while introduction of imidazo moiety to thiazole
derivatives (imidazo[2,1-b]thiazole as in compound 8) lead to significant increase in the
anticancer activity against human lung cancer (A-549) cell line, and marked decrease in
the activity against (HepG2) cell line. Additionally, introduction of bulky group as 2-oxo-
2H-chromene in compound 17 showed anticancer activity against both cell lines more
than DOX as a reference drug. Moreover, the presence of 1,3,4-thiadiazine as in
compound 6 exerted significant decrease in anticancer activity but showed promising
antibacterial activity especially against gram negative bacteria. On the other hand, the
presence of 4-chlorophenyl moiety, compound 9 showed marked increase in both
anticancer and antimicrobial activities.
7. Conclusion
This article proved that compounds having 4-chlorophenyl moiety 9 exhibited
dual activities, antimicrobial and anticancer especially against human lung cancer (A-
549) cell line in comparable to the positive reference doxorubicin. In addition, compound
21

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2 exhibited antibacterial action toward Gram-positive bacteria. However, compound 3
showed excellent antibacterial and cytotoxic activities against (HepG2) cell line.
Compound 8 showed promising cytotoxic activity toward human lung cancer (A-549)
cell line, while, compound 11 exhibited its activity toward human liver cancer (HepG2)
cell line. The compounds exerted their cytotoxic action via inhibition of the active sites of
DHFR.
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