Synthesis and antimicrobial activities of some new 1,2,4-triazole derivatives

Article abstract of DOI:10.3906/kim-1004-570

The synthesis of ethyl [3-(cyanomethyl)-5-alkyl-4H-1,2,4-triazol-4-yl] carbamates (2a-d) was performed starting from ethyl 2-[ethoxy(4-(aryl)methylene] hydrazinecarboxylates (1a, 1b). The treatment of 2a with thiosemicarbazide afforded ethyl [3-[(5-amino-

Full text of DOI:10.3906/kim-1004-570

Turk J Chem
34 (2010) , 835 – 846.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1004-570
Synthesis and antimicrobial activities of some new
1,2,4-triazole derivatives
1
1
2
3
˙
˘
Hacer BAYRAK , Ahmet DEMIRBAS¸ , Hakan BEKTAS¸ , S¸engu¨l ALPAY KARAOGLU ,
1,∗
˙
Neslihan DEMIRBAS¸
1Karadeniz Technical University, Faculty of Arts and Sciences, Department of Chemistry,
61080 Trabzon- TURKEY e-mail: neslihan@ktu.edu.tr
2Giresun University, Faculty of Sciences, Department of Chemistry,
28100 Giresun- TURKEY
3Rize University, Department of Biology, 53100 Rize-TURKEY
Received 21.04.2009
The synthesis of ethyl [3-(cyanomethyl)-5-alkyl-4H -1,2,4-triazol-4-yl]carbamates (2a-d) was performed
starting from ethyl 2-[ethoxy(4-(aryl)methylene]hydrazinecarboxylates (1a, 1b). The treatment of 2a
with thiosemicarbazide afforded ethyl [3-[(5-amino-1,3,4-thiadiazol-2-yl)methyl]-5-(4-nitrophenyl)-4H -1,2,4-
triazol-4-yl]carbamates (3a), whereas compound 2b produced 5-{[4-amino-5-(4-methylphenyl)-4H -1,2,4-
triazol-3-yl]methyl}-1,3,4-thiadiazol-2-amine (3b) in the same reaction conditions. The treatment of tert-
butyl 2-[2-(4-chlorophenyl)-1-ethoxyethylidene]hydrazinecarboxylate (5) with malonohydrazide or cyanoa-
cethydrazide gave the corresponding 1,2,4-triazol-ylcarbamate derivatives (6 or 9); then the hydrolysis of
these compounds resulted in the formation of 3-{[4-amino-5-(4-chlorobenzyl)-4H -1,2,4-triazol-3-yl]methyl}-
5-(4-chlorobenzyl)-4H -1,2,4-triazol-4-amine (7) and [4-amino-5-(4-chlorobenzyl)-4H -1,2,4-triazole-3-yl]ace-
tonitrile (10), respectively. The synthesis of the Schiff base derivatives 3-(4-chlorobenzyl)-5-{[5-(4-chloroben-
zyl)- 4-[(2-hydroxyphenyl-methylene)amino]-4H -1,2,4-triazol-3-yl]methyl}-N -(2-hydroxyphenylmethylene)-
4H -1,2,4-triazol-4-amine (8), and (5-(4-chlorobenzyl)-4-{[(2,6-dichlorophenyl)methylene]amino}-4H -1,2,4-
triazol-3-yl)acetonitrile (12) was performed from the reaction of compounds 7 and 10 with salicyl alde-
hyde (for 8) or 2,6-dichlorobenzaldehyde (for 12), respectively. The treatment of compounds 5 or 10
with thiosemicarbazide gave 5-{[4-amino-5-(4-chlorobenzyl)-4H -1,2,4-triazol-3-yl]methyl}-1,3,4-thiadiazol-
2-amine (11).
All the newly synthesized compounds were screened for their antimicrobial activities and were found to
possess good or moderate antimicrobial activity.
Key Words: 1,3,4-Thiadiazole, 1,2,4-triazole, carbamate, antimicrobial activity
∗Corresponding author
835

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
Introduction
Diseases caused by pathogenic bacteria still attract significant attention from medicinal chemists and biologists
because of growing antibacterial resistance. For example, Staphylococcus aureus, which is one of the major
causes of hospital- and community-acquired infections worldwide like wound infections, bacteremia, and sepsis,
is associated with a high mortality rate.1 Another infection, tuberculosis (TB) causes the death of approximately
three million patients in the world every year. These numbers make TB one of the leading infectious causes of
death, eclipsed only by AIDS. Synthetic drugs for treating TB have been available for over half a century, but
incidences of the disease continue to rise worldwide. The causative organism, Mycobacterium tuberculosis, is a
tremendously successful colonizer of the human host and is estimated to have latently infected approximately
one-third of humanity.2−5 A growing number of immuno-compromised patients are as a result of cancer
chemotherapy, organ transplantation, and HIV infection, which are the major factors contributing to this
increase. Therefore, it is necessary to search for and synthesize new classes of antimicrobial compounds that are
effective against pathogenic microorganisms that have developed resistance to the antibiotics. In addition, the
new agents should have low side effects on the human body, because, in some cases, especially in patients
with impaired liver or kidney functions, the use of antimicrobial drugs to treat infections causes several
problems.6−9 Moreover, from the pharmacoeconomic cost-efficiency viewpoint, and seeking better patient
compliance, antimicrobial agents with high therapeutic effect, high safety, and minimum adverse effects are
considerably desirable.10,11 Therefore, there is an urgent need to develop novel antimicrobial and antitubercular
chemotherapeutic agents.
A literature survey revealed triazole derivatives belonging to an important group of heterocyclic com-
pounds that have been the subject of extensive study in the recent past. Diverse biological activities, such as
antibacterial, antifungal, anti-inflammatory, antihypertensive, and antiviral, have been associated with 1,2,4-
triazole derivatives.12−22
Keeping this observation in view and in continuation of our study on the synthesis of biologically active
nitrogen and sulfur containing heterocycles,15,18,22−29 this paper presents the synthesis of a series of some new
heterocyclic carbamates, conversion to Schiff bases, and the study of their antimicrobial activities.
Experimental
Chemistry
Melting points were determined on a Bu¨chi B-540 melting point apparatus and are uncorrected. 1 H-NMR and
13 C-NMR spectra were recorded on a Varian-Mercury 200 MHz spectrometer. The IR spectra were measured
as potassium bromide pellets using a Perkin-Elmer 1600 series FTIR spectrometer. Combustion analysis was
performed on a Costech Elemental Combustion System CHNS-O elemental analyzer. Mass spectra were obtained
for compounds 2a, 3a, 3b, 6, 7, 9, 10, and 11 on a Quattro LC-MS (70 eV) Instrument. All the chemicals
were obtained from Fluka Chemie AG Buchs (Switzerland). Compounds 1a-d, 4, and 5 were synthesized as
reported previously.30,31
836

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
General Method for the Synthesis of Compound [3-(cyanomethyl)-5-alkyl-4H-1,2,4-triazol-4-
yl]carbamates (2a-d).
A mixture of corresponding compound 1 and 2-cyanoacetohydrazide was refluxed at 95-100 ◦ C for 1 h. After
the residue was cooled to room temperature, n-butyl acetate-petroleum ether (1:2) was added and the mixture
was kept in the cold overnight. The resulting solid separated was collected by filtration and recrystallized from
an appropriate solvent to afford the desired compound.
Ethyl [3-(cyanomethyl)-5-(4-nitrophenyl)-4H-1,2,4-triazol-4-yl]carbamate (2a). Recrystal-
lized from ethyl acetate. Yield 77%, mp 135-136 ◦ C. IR (KBr, υ, cm−1): 3112 (NH), 2257 (CN), 1744
(C=O), 1554 and 1525 (2C=N); Anal. Calcd. (%) for C13 H12 O4 N6 : C, 49.37; H, 3.82; N, 26.57. Found: C,
49.96; H, 3.88; N, 26.72; 1 H-NMR (DMSO-d6 , δ ppm): 1.18 (3H, t, CH3 , J = 7.7 Hz), 3.55 (2H, s, CH2), 4.10
(2H, q, CH2 , J = 6.1 Hz), 7.51 (2H, d, arH, J = 8.4 Hz), 8.20 (2H, d, arH, J = 8.4 Hz), 11.07 (1H, br, NH);
13 C-NMR (DMSO-d6 , δ ppm): 17.49 (CH3), 24.41 (CH3), 38.08-40.58 (DMSO-d6 +CH2), 116.13 (CN), arC:
[127.45 (CH), 127.97 (CH), 129.31 (CH), 129.57 (CH), 141.13 (C), 164.45 (C)], 152.27 (triazole C-3), 156.74
(triazole C-5), 184.45 (C=O).
EI MS m/z (%): 115 (16), 123 (28), 138 (16), 229 (13), 259 (13), 271 ([M-OEt]+ , 24), 285 (19), 303 (25),
332 (22), 353 (75), 390 (27).
Ethyl [3-(cyanomethyl)-5-(4-methylphenyl)-4H-1,2,4-triazol-4-yl]carbamate (2b). Recrystal-
lized from acetone. Yield 30%, mp 127-128 ◦ C. IR (KBr, υ, cm−1): 3188 (NH), 2260 (CN), 1690 (C=O),
1492 and 1453 (2C=N); Anal. Calcd. (%) for C14 H15 O2 N5 : C, 58.94; H, 5.30; N, 24.55. Found: C, 59.23; H,
5.36; N, 24.41; 1 H-NMR (DMSO-d6 , δ ppm): 1.20-1.39 (3H, m, CH3), 2.45 (2H, s, CH2), 3.40 (3H, s, CH3),
3.93-4.15 (2H, q, CH2 , J = 7.1 Hz), 7.22-7.38 (2H, d, arH, J = 6.4 Hz), 7.40-7.54 (2H, t, arH, J = 7.7 Hz), 11.07
(1H, s, NH); 13 C-NMR (DMSO-d6 , δ ppm): 14.79 (CH3), 20.88 (CH3), 24.52 (CH2), 66.60 (CH2), 116.18
(CN), arC: [127.59 (CH), 127.95 (CH), 129.05 (CH), 129.20 (CH), 140.25 (C), 149.68 (C)], 152.66 (triazole C-3),
158.72 (triazole C-5), 164.27 (C=O).
Ethyl [3-(cyanomethyl)-5-phenyl-4H-1,2,4-triazol-4-yl]carbamate (2c). Recrystallized from
dimethyl sulfoxide. Yield 30%, mp 110-111 ˚C. IR (KBr, υ, cm−1): 2262 (CN), 1743 (C=O), 1622 and
1589 (2C=N); Anal. Calcd. (%) for C13 H13 O2 N5 : C, 57.56; H, 4.83; N, 25.82. Found: C, 57.51; H, 4.79;
N, 25.85; 1 H-NMR (DMSO-d6 , δ ppm): 1.25 (3H, t, CH3 , J = 6.8 Hz), 3.88-4.15 (2H, m, CH2), 4.24 (2H, s,
CH2), 7.51-7.63 (5H, m, arH), 10.97 (1H, s, NH).
Ethyl [3-(4-chlorophenyl)-5-(cyanomethyl)-4H-1,2,4-triazol-4-yl]carbamate (2d). Recrystal-
lized from dimethyl sulfoxide. Yield 25%, mp 130-131 ˚C. IR (KBr, υ, cm−1): 3193 (NH), 2260 (CN), 1689
(C=O); Anal. Calcd. (%) for C13 H12 O2 N5 Cl: C, 50.74; H, 4.59; N, 22.76. Found: C, 51.18; H, 3.96; N, 22.85;
1 H-NMR (DMSO-d6 , δ ppm): 1.26 (3H, t, CH3 , J = 7.0 Hz), 3.90-4.17 (2H, m, CH2), 4.21 (2H, s, CH2),
7.54-7.67 (4H, m, arH), 11.01 (1H, s, NH).
General Method for the Synthesis of Compounds 3a and 3b
To a solution of compound 2 (10 mmol) in trifluoroacetic acid, 1 equiv. of thiosemicarbazide was added and the
mixture was heated in an oil bath for 10 h at 60 ◦ C. Then the reaction mixture was neutralized with ammonia.
The formed solid was filtered off and recrystallized from ethyl acetate to afford the desired compound.
837

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
Ethyl [3-[(5-amino-1,3,4-thiadiazol-2-yl)methyl]-5-(4-nitrophenyl)-4H-1,2,4-triazol-4-yl]car-
bamate (3a). Yield 37%, mp 175-176 ◦ C. IR (KBr, υ, cm−1): 3118 (NH2 +NH), 1740 (C=O), 1658, 1583
and 1457 (4C=N), 1514 and 1399 (NO2), 1109 (C-O); Anal. Calcd. (%) for C14 H14 O4 N8 S: C, 43.07; H, 3.61;
N, 28.70. Found: C, 43.12; H, 3.78; N, 28.56; 1 H-NMR (DMSO-d6 , δ ppm): 1.19 (3H, t, CH3 , J = 7.1 Hz),
3.43 (2H, br, CH2), 4.24 (2H, q, CH2 , J = 7.4 Hz), 7.17 (2H, s, NH2 , exch. D2 O), 7.52 (2H, d, arH,J = 8.7
Hz), 8.21 (2H, d, arH, J = 8.9 Hz), 11.00 (1H, s, NH); 13 C-NMR (DMSO-d6 , δ ppm): 14.76 (CH3), 29.96
(CH2), 63.01 (CH2), arC: [124.20 (2CH), 130.85 (2CH), 143.82 (C), 143.86 (C)], 147.14 (triazole C-3), 151.95
(triazole C-5 ve thiadiazole C-5), 153.70 (thiadiazole C-3), 170.20 (C=O); EI MS m/z (%): 215 (25), 390 ([M]+ ,
100), 391 ([M+1]+ , 391).
5-{[4-amino-5-(4-methylphenyl)-4H-1,2,4-triazol-3-yl]methyl}-1,3,4-thiadiazol-2-amine (3b).
Recrystallized from ethanol. Yield 32%, mp 204-205 ◦ C. IR (KBr, υ, cm−1): 3282 and 3092 (NH+NH2), 1740
(C=O), 1634, 1583, 1514 and 1470 (4C=N); Anal. Calcd. (%) for C12 H13 N7 S: C, 50.16; H, 4.56; N, 34.12.
Found: C, 50.08; H, 4.51; N, 34.07; 1 H-NMR (DMSO-d6 , δ ppm): 2.37 (3H, s, CH3), 2.52 (2H, s, CH2),
7.33 (2H, d, arH, J = 8.0 Hz), 7.66 (2H, d, arH, J = 7.8 Hz), 8.38 (4H, br, 2NH2); 13 C-NMR (DMSO-d6 ,
δ ppm): 21.42 (CH3), 38.07-40.59 (DMSO-d6 + CH2), arC: [126.66 (C), 126.99 (2CH), 130.45 (2CH), 141.89
(C)], 157.03 (triazole C-5 and thiadiazole C-3), 169.24 (triazole C-3 and thiadiazole C-5). MS m/z (%): 373
(11), 117 (44), 133 (25), 150 (88), 193 (19).
Tert-butyl 3-({4-[(tert-butoxycarbonyl)amino]-5-(4-chlorobenzyl)-4H-1,2,4-triazol-3-yl}met-
hyl)-5-(4-chlorobenzyl)-4H-1,2,4-triazol-4-ylcarbamate (6). A mixture of tert-butyl 2-[2-(4-chlorophenyl)-
1-ethoxyethylidene]hydrazinecarboxylate (5) (20 mmol) and malonohydrazide (10 mmol) was heated in an oil
bath at 95 ◦ C for 2 h. After the reaction mixture was cooled to room temperature, a few drops of ethyl acetate
was added and was kept in the cold overnight. The solid formed was filtered and recrystallized from acetone
to obtain compound 6. Yield 78%, mp 174-175 ◦ C. IR (KBr, υ, cm−1): 2979 and 2936 (2NH), 1739 (2C=O);
Anal. Calcd. (%) for C29 H34 O4 N8 Cl2 : C, 55.33; H, 5.54; N, 17.80. Found: C, 55.28; H, 5.50; N, 17.77; 1 H-
NMR (DMSO-d6 , δ ppm): 1.35 (18H, s, 6CH3), 3.89 (2H, s, CH2), 4.00 (4H, s, 2CH2), 7.24 (4H, d, arH,J =
8.3 Hz), 7.38 (4H, d, arH, J = 8.3 Hz), 10.75 (2H, br, 2NH); 13 C-NMR (DMSO-d6 , δ ppm): 27.47 (6CH3),
28.75 (3CH2), 81.64 (2C), arC: [128.24 (4CH), 130.44 (4CH), 131.35 (2C), 134.30 (2C)], 149.73 (triazole 2C-3),
153.12 (triazole 2C-5), 179.80 (2C=O); EI MS m/z (%): 121(25), 146 (28), 149 (18), 155 (13), 188 (16), 210
(14), 229 (100), 230 (23), 246 (18), 273 (38), 279 (14), 313 (30), 335 (41), 357 (26), 365 (28), 367 (29), 368 (14),
399 (24), 429 (88), 431 (64), 432 (19), 451 (46), 453 (31), 473 (73), 475 (50), 529 (49), 531 (31), 532 (13), 551
(31), 553 (26), 574 (13), 629 ([M]+ , 64), 631 (41), 652 ([M+Na]+ , 19).
3-{[4-Amino-5-(4-chlorobenzyl)-4H-1,2,4-triazol-3-yl]methyl}-5-(4-chlorobenzyl)-4H-1,2,4-
triazol-4-amine (7). A mixture of compound 6 (10 mmol) and 6N HCl (5.45 mL) in tetrahydrofuran was
refluxed in a water bath for 15 min. After the solvent was evaporated under reduced pressure a white solid
appeared. The crude product was added to a potassium carbonate solution (10 mmol) slowly and the obtained
mixture was stirred at room temperature for 30 min. The obtained solid was filtered and recrystallized from
ethanol-water (1:1). Yield 81%, mp 214-215 ◦ C. IR (KBr, υ, cm−1): 3252 and 3197 (2NH2 +ArCH), 1650,
1606, 1521 and 1493 (4C=N); Anal. Calcd. (%) for C19 H18 N8 Cl2 : C, 53.16; H, 4.23; N, 26.10. Found: C,
53.08; H, 4.17; N, 26.08; 1 H-NMR (DMSO-d6 , δ ppm): 4.09 (4H, br, 2CH2), 4.19 (2H, br, CH2), 5.97 (4H,
br, 2NH), 7.21-7.40 (8H, m, arH); 13 C-NMR (DMSO-d6 , δ ppm): 21.08 (CH2), 29.56 (2CH2), arC: [129.00
838

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
(4CH), 131.38 (4CH), 131.87 (2C), 136.49 (2C)], 151.54 (triazole 2C-3), 154.48 (triazole 2C-5); EI MS m/z
(%):116.65 (71), 134.69 (42), 148.65 (46), 163.49 (42), 179.90 (36), 180.72 (27), 196.63 (23), 208.76 (46), 229.07
(100), 244.66 (50), 245.10 (29), 273.27 (20), 287.92 (14), 313.07 (17), 357.09 (54), 367.21 (39), 367.34 (19),
371.05 (15), 399.21 (13), 412.92 (39), 415.31 (21), 429.21 ([M]+ , 88), 431.09 (71), 451.15 ([M-1+Na]+ , 71),
453.04 ([M+1+Na]+ , 41).
3-(4-Chlorobenzyl)-5-{[5-(4-chlorobenzyl)-4-[(2-hydroxyphenylmethylene)amino]-4H-1,2,4-
triazol-3-yl]methyl}-N-(2-hydroxyphenylmethylene)-4H-1,2,4-triazol-4-amine (8). A mixture of
compound 7 (10 mmol) and salicyl aldehyde (20 mmol) was stirred in glacial acetic acid for 15 min at room
temperature. Then the mixture was refluxed for 4 h. After the solvent was evaporated under reduced pressure a
white solid appeared and was recrystallized from ethanol to give the desired product 8. Yield 87 %, mp 207-208
◦ C. IR (KBr, υ, cm−1): 3413 (2OH), 3043 (Ar CH), 1621, 1596, 1491 and 1452 (4C=N); Anal. Calcd. (%) for
C
33 H26 N8 O2 Cl2 : C, 62.17; H, 4.11; N, 17.58. Found: C, 62.15; H, 4.08; N, 17.55; 1 H-NMR (DMSO-d6 , δ
ppm): 4.13 (4H, s, 2CH2), 4.44 (2H, s, CH2), 6.83-6.96 (4H, m, arH), 7.16 (4H, d, arH, J = 8.2 Hz), 7.38 (4H,
d, arH, J = 8.2 Hz), 7.42 (2H, t, arH, J = 7.4 Hz), 7.62 (2H, d, arH, J = 8.2 Hz), 8.78 (2H, s, 2CH), 10.37
(2H, s, 2OH); 13 C-NMR (DMSO-d6 , δ ppm): 29.57 (2CH2), 38.45-40.17 (DMSO-d6 +CH2), 116.47 (2CH),
arC:[117.70 (2C), 119.33 (2CH), 127.90 (2CH), 128.24 (4CH), 130.27 (4CH), 131.22 (2C), 134.47 (2CH), 134.79
(2C), 146.44 (2C)], 149.25 (triazole 2C-3), 158.42 (triazole 2C-5), 163.31 (2CH). EI MS m/z (%): 104.98 (41),
134.92 (54), 152.99 (13), 169.00 (13), 209.95 (18), 349.18 (34), 351.18 (14), 399.07 (14), 518.17 (29), 540.17 (23),
542.17 (14), 637.21 ([M]+ , 59), 659.15 ([M+1+Na]+ , 81), 675.15 (100), 677.16 ([M+1+K]+ , 77).
Tert -butyl [3-(4-chlorobenzyl)-5-(cyanomethyl)-4H-1,2,4-triazol-4-yl]carbamate (9). A mix-
ture of compound 5 (10 mmol) and cyanoacethydrazide (10 mmol) was heated in an oil bath at 100 ◦ C for
1.5 h. After the residue was cooled to room temperature, 2-3 mL of ethyl acetate-petroleum ether (1:2) was
added and the mixture was kept overnight in the cold. The resulting solid separated was collected by filtration
and recrystallized from ethyl acetate-petroleum ether (1:2) to yield compound 9. Yield 72%, mp 105-106 ◦ C.
IR (KBr, υ, cm−1): 3141 (NH), 2261 (CN), 1728 (C=O); Anal. Calcd. (%) for C16 H18 N5 O2 Cl: C, 55.25;
H, 5.22; N, 20.14. Found: C, 55.23; H, 5.22; N, 20.11; 1 H-NMR (DMSO-d6 , δ ppm): 1.38 (9H, s, 3CH3),
4.01 (2H, s, CH2), 4.19 (2H, s, CH2), 7.20 (2H, d, arH, J =8.3 Hz), 7.39 (2H, d, arH, J = 8.3 Hz), 10.79
(1H, br, NH); 13 C-NMR (DMSO-d6 , δ ppm): 29.51 (3CH2), 36.34 (CH2), 38.55-41.07 (DMSO-d6 + CH2),
62.98 (CN), 74.98 (C), arC:[129.05 (2CH), 131.29 (2CH), 132.27 (C), 134.78 (C)], 151.30 (triazole C-3), 154.42
(triazole C-5), 170.43 (C); EI MS m/z (%): 153 (16), 229 (22), 248 (25), 294 (31), 314 (36), 316 (11), 348 ([M]+ ,
19), 370 ([M+Na]+).
[4-Amino-5-(4-chlorobenzyl)-4H-1,2,4-triazole-3-yl]acetonitrile (10). To a solution of com-
pound 9 (10 mmol) in tetrahydrofuran was added 6N HCl (21.49 mL) and the mixture was refluxed in a
water bath for 15 min. After the solvent was evaporated under reduced pressure a white solid appeared. The
solid was added to a potassium carbonate solution (10 mmol) slowly and was stirred at room temperature for
30 min. The mixture was kept overnight in the cold, and the resulting solid separated was collected by filtration
and recrystallized from ethanol-water (1:1). Yield 80%, mp 130-131 ◦ C. IR (KBr, υ, cm−1): 3235 and 3116
(NH2), 2261 (CN); Anal. Calcd. (%) for C11 H10 N5 Cl: C, 53.34; H, 4.07; N, 28.26. Found: C, 53.29; H, 4.03;
N, 28.24; 1 H-NMR (DMSO-d6 , δ ppm): 4.09 (2H, s, CH2), 4.18 (2H, s, CH2), 5.94 (2H, s, NH2), 7.28-7.41
(4H, q, arH, J = 8.4 Hz); 13 C-NMR (DMSO-d6 , δ ppm): 14.56 (CH2), 29.12 (CH2), 116.19 (CN), arC: [128.77
839

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
(2CH), 131.11 (2CH), 131.80 (C), 135.46 (C)], 147.27 (triazole C-3), 155.18 (triazole C-5); EI MS m/z (%): 153
(44), 214 (41), 259 (17), 280 (34).
5-{[4-Amino-5-(4-chlorobenzyl)-4H-1,2,4-triazol-3-yl]methyl}-1,3,4-thiadiazol-2-amine (11).
Method 1: The mixture of compound 10 (10 mmol) and thiosemicarbazide (10 mmol) in tetrahydrofuran
was refluxed in an oil bath at 80 ◦ C for 9 h. After cooling to room temperature, the reaction mixture was
neutralized with ammonia, and kept in cold overnight. The resulting solid separated was collected by filtration
and recrystallized from acetone to afford the desired product.
Method 2: The mixture of compound 9 (10 mmol) and thiosemicarbazide (10 mmol) in tetrahydrofuran
was refluxed in an oil bath at 80 ◦ C for 9 h. After cooling to room temperature, the reaction mixture was
neutralized with ammonia, and kept in the cold overnight. The solid formed was filtered and recrystallized
from acetone to afford the desired product. Yield 79%, mp 247-248 ◦ C. IR (KBr, υ, cm−1): 3337 and 3261
(NH2), 1631, 1607, 1518 and 1491 (4C=N); Anal. Calcd. (%) for C12 H12 N7 SCl: C, 44.79; H, 3.76; N, 30.47.
Found: C, 44.75; H, 3.76; N, 30.46; 1 H-NMR (DMSO-d6 , δ ppm): 4.08 (2H, s, CH2), 4.31 (2H, s, CH2), 5.90
(2H, s, NH2), 7.14 (2H, s, NH2), 7.27-7.40 (4H, q, arH, J = 8.4 Hz); 13 C-NMR (DMSO-d6 , δ ppm): 25.34
(CH2), 28.77 (CH2), arC: [128.17 (2CH), 130.59 (2CH), 131.14 (C), 135.52 (C)], 151.24 (triazole C-3), 152.48
(thiadiazole C-2), 153.63 (triazole C-5), 169.21 (thiadiazole C-5); EI MS m/z (%): 306 (17), 322 ([M]+ , 100),
324 (41), 344 (31), 346 (11).
5-(4-Chlorobenzyl)-4-{[(2,6-dichlorophenyl)methylene]amino}-4H-1,2,4-triazol-3-yl)aceto-
nitrile (12). To the solution of compound 10 (10 mmol) in glacial acetic acid was added 2,6-dichloro
benzaldehyde (10 mmol) and the mixture was allowed to reflux for 3 h. The reaction solvent was evaporated
under reduced pressure. Then 2-3 mL of ethanol was added to the obtained oily product and kept overnight
in the cold. The resulting solid that separated was collected by filtration and recrystallized from acetone to
yield the title compounds. Yield 83%, mp 209-210 ◦ C. IR (KBr, υ, cm−1): 2235 (CN); Anal. Calcd. (%)
for C18 H12 N5 Cl3 : C, 53.42; H, 2.99; N, 17.31. Found: C, 53.41; H, 2.96; N, 17.34; 1 H-NMR (DMSO-d6 , δ
ppm): 4.19 (2H, s, CH2), 6.32 (2H, s, CH2), 7.37 (4H, d, arH, J = 8.2 Hz), 7.41-7.59 (1H, t, arH, J = 7.9 Hz),
7.70 (2H, d, arH, J = 8.0 Hz), 8.56 (1H, s, CH); 12 C-NMR (DMSO-d6 , δ ppm): 29.23 (CH2), 37.23-40.14
(DMSO-d6 +CH2), 108.28 (CN), 113.91 (CH), arC:[126.84 (CH), 128.53 (2CH), 128.61 (2CH), 129.00 (2CH),
131.27 (CH), 132.033 (C), 133.16 (C), 135.46 (C), 143.16 (C)], 146.81 (triazol C-3), 157.22 (triazole C-5).
Antimicrobial activity
Antimicrobial activity assessment
All bacterial and yeast strains were obtained from the Re?k Saydam Hıfzıssıhha Institute (Ankara, Turkey) and
were as follows: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Yersinia pseudotuberculosis
ATCC 911, Enterobacter aeruginosa ATCC 13048, Pseudomonas aeruginosa ATCC 27853, Staphylococcus
aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus cereus 702 Roma, Mycobacterium smegmatis
ATCC607, Candida albicans ATCC 60193, and Candida tropicalis ATCC 13803. All the newly synthesized
compounds were dissolved in dimethyl sulfoxide (DMSO) and ethanol to prepare chemicals of stock solution of
10 mg/1 mL.
840

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
Minimal inhibition concentration method
The antimicrobial effects of compounds 3a, 3b, and 9-11 were tested quantitatively in respective broth media
by using double dilution and the minimal inhibition concentration (MIC) values (μg/mL) were determined.32
The antibacterial and antifungal assays were performed in Mueller-Hinton broth (MH) (Difco, Detroit, MI,
USA) at pH 7.3 and buffered Yeast Nitrogen Base (Difco, Detroit, MI, USA) at pH 7.0, respectively. The
MIC was defined as the lowest concentration that showed no growth. Ampicillin (10 μg) was used as standard
antibacterial and antifungal drug, respectively. Dimethyl sulfoxide with dilution of 1:10 was used as solvent
control. The results are shown in the Table.
Table. Antimicrobial activity of the newly synthesized compounds (μg/mL).
Microorganisms and Minimal Inhibition Concentration Values
Comp. No.
Ec
Ea
Yp
Kp
Pa
Sa
Ef
Bc
Ms
Ca
Ct
2a
2b
2c
> 500 > 500 > 500
125
> 500 > 500 > 500 > 500 > 500 > 500 > 500
> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500
> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500
3a
< 0.12
> 500
250
1.95
250
0.49
250
0.24
> 500 > 500
250
1.95
15.6
250
125
31.3
250
> 500 > 500 > 500
250 > 500 > 500
6
> 500
7
> 500 > 500
> 500 > 500 > 500 > 500 > 500 > 500 > 500
8
> 500 > 500 > 500 > 500
250
< 0.12 < 0.12 < 0.12
> 500
0.24
1.95
0.24
250
250
0.24
1.95
0.49
> 500
10
250
0.24
0.49
0.49
250
15
> 500 > 500 > 500
> 500 > 500 > 500
> 500 > 500 > 500
> 500 > 500 > 500
9
< 0.12
< 0.12
< 0.12
0.24
0.49
0.98
10
0.49
0.24
0.98
0.24
250
11
< 0.12 < 0.12
12
> 500 > 500 > 500 > 500
10 10 > 18 > 18
250
> 500 > 500
Amp.
Strep.
Flu.
> 18
35
35
25
25
Ec: Escherichia coli ATCC 25922, Yp: Yersinia pseudotuberculosis ATCC 911, Ea: Enterobacter aeruginosa ATCC
13048, Kp: Klebsiella pneumoniae ATCC 13883, Pa: Pseudomonas aeruginosa ATCC 43288, Sa: Staphylococcus aureus
ATCC 25923, Ef: Enterococcus faecalis ATCC 29212, Bc: Bacillus cereus 702 Roma, Ms: Mycobacterium smegmatis
ATCC607, Ca: Candida albicans ATCC 60193, Candida tropicalis ATCC 13803, Amp.: Ampicillin, Flu.: Fluconazole,
Strep.: Streptomycin.
Results and discussion
The synthetic routes to obtain the designed compounds and their intermediates are described in Schemes 1 and
2. Compounds 1a-d and 5 underwent nucleophilic attack by 2-cyanoacetohydrazide to give 2a-d and 9. The IR
spectra of 2a-d, 9, 10, and 12 exhibited a sharp peak between 2235 and 2262 cm−1 due to cyano absorption.
In the 13 C-NMR spectra of these compounds, the cyano group was observed between 62.98 and 116.19 ppm.
841

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
Due to slight solubility in any NMR solvent of compounds 2c and 2d, satisfactory 13 C-NMR spectra of these
compounds could not be obtained.
O
N
N
1,2
R
N
N
N
N
NNHC
O
CN
a:
b:
c:
d:
-NO2
-CH3
-H
-Cl
R
O
NH2
N
N
S
N
NH2NHCOCH2CN
H2NCSNHNH2
NH
O
R
R'
H
R
O
3a: R= NO ,
3b: R= CH3, R'=-H
R'= -CO2Et
1a-d
2
2a-d
Scheme 1. Synthetic pathway for the preparation of compounds 2a-d and 3a, 3b.
Cl
Cl
O
NNHC
NNH.HCl
O
C
C
H2NNHCOOC(CH3)3
O
O
5
4
H2NNHCOCH2CN
H2NNHCOCH2CONHNH2
Cl
Cl
CI
N
N
N
N
N
N
CN
N
NH
N
NH
N
NH
O
O
O
O
O
O
9
6
7
HCI
2
1)
2)
1) THF/HCl
2) K2CO3
H2NNHCSNH2
K CO
3
CI
Cl
CI
N
N
N
N
N
N
Cl
N
N
N
N
CN
N
NH2
N
NH2
NH2
N
NH2
H2NNHCSNH2
N
NH2
11
S
10
OH
Cl
CHO
CHO
Cl
CI
Cl
CI
N
N
N
N
N
N
CN
Cl
N
N
N
N
N
N
HO
OH
Cl
12
8
Scheme 2. Synthetic pathway for the preparation of compounds 5-12.
842

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
The cyclocondensation of compounds 2a, 2b, and 9 with thiosemicarbazide was carried out by an analog
way reported earlier.33 This method containing the heating of the reaction mixture in trifluoroacetic acid at 60
◦ C for 10 h yielded compounds 3a, 3b, and 11, whereas the reactions of compounds 2c and 2d in the same
conditions resulted in decomposition. Another way for the synthesis of compound 11 involved the reaction
of compound 10 with thiosemicarbazide. In the 1 H-NMR spectra of compounds 3a and 3b, the signal was
observed at 7.17 ppm (for 3a) and 8.38 ppm (for 3b), attributed to the amino group on position 5 of the 1,3,4-
thiadiazole ring. The 1 H-NMR spectrum of compound 11, 2 signals were recorded at 5.90 ppm and 7.14 ppm,
which were checked by changing with D2 O. The one that appeared at the downfield region represents the –NH2
group on the 1,3,4-thiadiazole ring, while the upfield one points to the amino group on the 1,2,4-triazole ring.
In addition, the IR spectra of compounds 3a, 3b, and 11 displayed no signal originating from the cyano group.
Moreover, in the 13 C-NMR spectra of these compounds the signal derived from the cyano group disappeared,
while additional signals belonging to thiadiazole C-2 and C-5 were recorded at 158.3 and 147.0-147.7 ppm,
respectively. Furthermore, compounds 3a, 3b, and 11 gave molecular ion peak and elemental analysis data
consistent with the assigned structures.
Our efforts towards the synthesis of tert-butyl 3-({4-[(tert-butoxycarbonyl)amino]-5-(4-chlorobenzyl)-
4H -1,2,4-triazol-3-yl}methyl)-5-(4-chlorobenzyl)-4H-1,2,4-triazol-4-ylcarbamate (6) involved the reaction of
tert-butyl 2-[2-(4-chlorophenyl)-1-ethoxyethylidene]hydrazinecarboxylate (5) with malonohydrazide according
to the method developed by us earlier.34 In the 1 H-NMR spectrum of compound 6, the signals derived from
the tert-butoxy group were recorded at 1.35 ppm. This group resonated at 27.47 ppm in the 13 C-NMR spectrum.
Moreover, the 1 H-NMR spectrum of compound 6 displayed a singlet at 10.75 ppm integrating for 2 protons,
which points to the presence of 2 exocyclic -NH- groups (controlled with changing by D2 O) in the structure.
Furthermore, the mass spectrum of 6, as a typical example, contained a peak at m/z = 629.54 that agreed
with the molecular weight of the molecular formula C29 H34 O4 N8 Cl2 of the assigned structure. Moreover,
compound 6 gave satisfactory elemental analysis data.
Compound 7 was prepared starting from compound 6 in 2 steps. In the first step, the hydrolysis of
2 tert-butoxy groups was achieved by the treatment of compound 6 with 6N HCl. However, the reaction
produced the desired compound (7) as its hydrochloride, which was not isolated in the present study. In the
second step, the treatment of the intermediate forming in the first step, with aqueous K2 CO3 , yielded the
target amino compound, 3-{[4-amino-5-(4-chlorobenzyl)-4H-1,2,4-triazol-3-yl]methyl}-5-(4-chlorobenzyl)-4H-
1,2,4-triazol-4-amine (7). The IR spectrum of 7 contained 2 absorption bands originated from 2 –NH2 groups
at 3252 and 3197 cm−1 . In the 1 H-NMR spectrum, a singlet signal belonging to the amino groups appeared
at 5.97 ppm integrating for 4 protons (controlled with changing by D2 O), while no signal representing tert-
butoxy- and exocyclic –NH- groups was present. In addition, mass spectrum containing a peak at m/z= 429
and elemental analysis data of compound 7 corresponded to the molecular weight of the molecular formula
C19 H18 N8 Cl2 of the assigned structure.
Compound 7 was treated with salicyl aldehyde in glacial acetic acid at room temperature to yield
the corresponding Schiff base 3-(4-chlorobenzyl)-5-{[5-(4-chlorobenzyl)-4-[(2-hydroxyphenylmethylene)amino]-
4H -1,2,4-triazol-3-yl]methyl}-N-(2-hydroxyphenylmethylene)-4H -1,2,4-triazol-4-amine (8). The structure of
8 was proven by IR, 1 H- and 13 C-NMR spectroscopic techniques and elemental analysis. The IR spectrum of
compound 8 contained no peak corresponding to amino groups. Similarly, no signal was present in the 1 H-NMR
843

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
spectrum of compound 8 derived from the –NH2 group. Instead, new signals due to 2-hydroxyphenylmethylene
group were recorded at the related chemical shift values.
The treatment of compound 9 or 10, prepared by an analogue way with compound 7, with thiosemi-
carbazide produced compound 11. Similar to compounds 3a and 3b, the 1 H-NMR spectrum of compound
11 exhibited 2 NH2 signals at 5.90 ppm and 7.14 ppm (controlled by changing with D2 O) belonging to 1,2,4-
triazole-NH2 and 1,3,4-thiadiazole-NH2 , respectively. Furthermore, compound 11 displayed a mass spectrum
containing a molecular ion peak and elemental analysis data consistent with the assigned structure.
The synthesis of compound 12 was performed by refluxing of 10 with 2,6-dichlorobenzaldehyde in ethanol.
Compound 12 exhibited IR, 1 H-NMR, 13 C-NMR spectral data, and elemental analysis consistent with the
proposed structure.
It was reported that the compounds having an arylidene-amino structure may exist as E/Z geometrical
isomers about the –N=CH- double bond.26,27,35,36 The literature survey revealed that compounds containing
an imine bond may be present in higher percentages in dimethyl-d6 sulfoxide solution in the form of geometrical
E isomer about the –N=CH- double bond.34 The Z isomer can be stabilized in less polar solvents by an
intramolecular hydrogen bond. However, the 1 H- and 13 C-NMR spectra of compounds 8 and 12 showed
the existence of only one isomer. It can be concluded that compounds 8 and 12 exist as their E geometrical
isomers considering bulky arylidene substituent, which is in agreement with the literature. 37−43
Antimicrobial activities of the newly synthesized compounds were determined by the minimal inhibition
concentration method. Among ethyl [3-(cyanomethyl)-4H -1,2,4-triazol-4-yl]carbamates (2a-d), compound 2a
containing a 4-nitrophenyl group at the position 5 of the 1,2,4-triazole ring exhibited slight activity against
K. pneumonia with the MIC value 125 μg/mL. The conversion of the cyano group into a 5-amino-1,3,4-
thiadiazole ring (for compounds 3a, 3b) greatly increased the antibacterial activity, while no antimicobacterial
and antifungal activity was observed for these compounds.
Compound 6, which is a carbamate derivative containing two 1,2,4-triazole rings linked to each other via
a methylene linkage, was found to possess activity on E. aeroginosa, Y. pseudotuberculosis, S. aureus, B. cereus,
and M. smegmatis. On the other hand, the replacement of the carbamate group by an amino group caused
activity against only E. coli and K. pneumonia. In contrast to other newly synthesized compounds, compounds
6 and 12 were found to be active towards M. smegmatis .Nonpigmented rapidly growing mycobacteria (M.
smegmatis is one of them) constitute a group of species of the genus Mycobacterium that share some charac-
teristics. Due to differences between strains, these organisms require individualized treatment, which needs to
be selected on the basis of results obtained from in vitro susceptibility tests. Compounds 9-11 exhibited good
antibacterial activities, but no antimycobacterial and antifungal activity was observed for these compounds. In
compound 12, the conversion of amino function into 2,6-dichlorophenylmethylenamino moiety greatly decreased
the antimicrobial effect; on the other hand, this conversion caused slight activity for compound 12 towards M.
smegmatis.
Acknowledgements
¨
˙
This project was supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Project
no: 107T333) and Karadeniz Technical University, BAP, Turkey (Ref. No. 2007.111.002.5), and is gratefully
acknowledged.
844

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
References
1. Roy, B.; Chakraborty, A.; Ghosh S. K.; Basak A. Bioorg. Med. Chem. Lett., 2009, 19, 7007-7010.
2. Dye, C.; Williams, B. G. Sci. Transl. Med., 2009, 1, 3-8.
3. Dye, C. Lancet, 2006, 367, 938-940.
¨
4. Koca, M.; Servi, S.; Kirilmis, C.; Ahmedzade, M.; Kazaz, C.; Ozbek, B.; Otu¨k, G. Eur. J. Med. Chem., 2005, 40,
¨
1351-58.
5. Zalavadiya, P.; Tala, S.; Akbari, J.; Joshi, H. Arch. Pharm. Chem. Life Sci., 2009, 342, 469-475.
6. Bekhit, A. A.; El-Sayed, O. A.; Aboulmagd, E.; Park, J. Y. Eur. J. Med. Chem., 2004, 39, 249-255.
7. Farghaly, J. Y.; Bekhit, A. A.; Park, J. Y. Arch. Pharm. Pharm. Med. Chem., 2000, 333, 53-57.
8. Dixit, P. P.; Patil, V. J.; Nair, P. S.; Jain, S.; Sinha, N.; Arora, S. K. Eur. J. Med. Chem., 2006, 41, 423-428.
9. Hassan, E.; Al-Ashmawi, M. I.; Abdel-Fattah, B. Pharmazie 1983, 38, 833-835.
10. Goswami, B. N.; Kataky, J. C. S.; Baruah, J. N. J. Heterocycl. Chem., 1986, 23, 1439-1442.
11. Eweiss, N. F.; Bahajaj, A. A.; Elsherbini, E. A. J. Heterocycl. Chem., 1986, 23, 1451-1458.
12. Tsotinis, A.; Varvaresou, A.; Calageropoulou, T.; Papastaikoudi, T. S.; Tiligada, A. Arzneim. Forsch./Drug Res.,
1997, 47, 307-310.
13. Garoufalias, S. S. P.; Todoulou, O. G.; Filippatos, E. C.; Valiraki, A. E. P.; Chytirogiou-Lada, A. Arzneim.
Forsch./Drug Res., 1998, 48, 1019-1023.
14. Sui, Z.; Guan, J.; Hlasta, D. J.; Macielag, M. J.; Foleno, B. D.; Goldschmidt, R. M.; Loeloff, M. J.; Webb, G. C.;
Barrett, J. F. Bioorg. Med. Chem. Lett., 1998, 8, 1929-1934.
15. Demirba¸s, N.; U˘gurluog˘lu, R. Turk. J. Chem., 2004, 28, 679-690.
16. Holla, B. S.; Gonsalves, R.; Shenoy, S. Il Farmaco, 1998, 53, 574-578.
17. Holla, B. S.; Poorjary, N. K.; Rao, S. B.; Shivananda, M. K. Eur. J. Med. Chem., 2002, 37, 511-517.
18. Yu¨ksek, H.; Demirbas, A.; Ikizler, A.; Johansson, C. B.; C¸ elik, C.; Ikizler, A.A. Arzneim. Forsh. Drug Res., 1997,
47, 405-409.
19. Ghorab, M. M.; El-Sharief, A. M. S.; Ammar, Y. A.; Mohamed, S. I. Il Farmaco, 2000, 55, 354-361.
20. Ku¨cu¨kgu¨zel, S. G.; Rollas, S.; Erdeniz, H.; Kiraz, M. Eur. J. Med. Chem., 1999, 34, 153-159.
21. Tozkoparan, B.; Go¨khan, N.; Aktay, G.; Ye¸silada, E.; Ertan, M. Eur. J. Med. Chem., 2000, 35, 743-750.
22. Ikizler, A. A.; Uzunali, E.; Demirba¸s, A. Indian J. Pharm. Sci., 2000, 5, 289-292.
23. Demirbas, N., Alpay-Karaoglu, S.; Demirbas, A.; Sancak, K. Eur. J. Med. Chem., 2004, 39, 793-804.
24. Bayrak, H.; Demirba¸s, A.; Alpay-Karaoglu, S.; Demirba¸s, N. Eur. J. Med. Chem., 2009, 44, 1057-1066.
25. Bayrak, H.; Demirbas, A.; Demirbas, N.; Alpay-Karaoglu, S. Eur. J. Med. Chem., 2009, 44, 4362-4366.
26. Demirbas, A.; Sahin, D.; Demirbas, N.; Alpay-Karaoglu, S. Eur. J. Med. Chem., 2009, 44, 2896-2903.
27. Demirbas, N.; Ugurluoglu, R.; Demirbas, A. Bioorg. Med. Chem., 2002, 10, 3717-3723.
28. Demirba¸s, N.; U˘gurluog˘lu, R.; Turk. J. Chem., 2004, 28, 559-571.
29. Demirbas, N.; Demirbas, A.; Alpay-Karaoglu, S. Russian J. Bioorg. Chem., 2005, 31, 430-440.
845

Synthesis and antimicrobial activities of..., H. BAYRAK, et al.,
30. Pesson, M.; Dupin, S.; Antoine, M. Bull. Soc. Chim., France, 1962, 1364-1371.
˙
31. Demirba¸s, A.; Demirba¸s, N.; Ikizler, A. A. Indian J. Heterocycl. Chem., 1999, 9, 87-94.
32. National Committee for Clinical Laboratory Standards, NCCLS Document M7-A3, 1993, 13 (25), Villanova, PA.,
USA.
33. Foroumadi, A.; Emami, S.; Pournourmohammadi, S.; Kharazmi, A.; Shafiee, A. Eur. J. Med. Chem., 2005, 40,
1346-1350.
˙
˙
34. Ikizler, A.; Demirbas, N.; Ikizler, A. A. J. Heterocycl. Chem., 1996, 33, 1765-1769.
35. Galic, N.; Peric, B.; Kojic-Prodic, B.; Cimerman, Z. J. Mol. Struct. 2001, 559, 187-194.
36. Wyrzykiewicz, E.; Prukah, D. J. Heterocycl. Chem. 1998, 35, 381-387.
37. McKelvy, L. M.; Britt, R. T.; Davis, B. L.; Gillie, J. K.; Graves, B. F.; Lentz, L. A. Anal. Chem., 1998, 70,
119R-117R.
38. Salgın-G¨oksen, U.; Gokhan-Kelekci, N.; Goktas, O.; K¨oysal, Y.; Kılıc, E.; Isık, S.; Aktay, G.; Ozalp, M. Bioorganic
& Medicinal Chemistry, 2007, 15, 5738-5751.
39. Gudasi, K. B.; Patil, M. S.; Vadavi, R. S.; Shenoy, R. V.; Patil, S. A. Transition Metal Chemistry, 2006, 31,
986-991.
40. Dugave, C.; Demange, L. Chem. Rev., 2003, 103, 2475-2532.
41. Rostom, S. A. F.; Ashour, H. M. A.; Abd El Razik, H. A.; Abd El Fattah, H.; El-Din, N. N. Bioorganic & Medicinal
Chemistry, 2009, 17, 2410-2422.
42. Bekta¸s, B.; Karaali, N.; S¸ahin, D.; Demirba¸s, A.; Karaoglu, S. A.; Demirba¸s, N. Molecules, 2010, 15, 2427-2438.
43. Demirba¸s, A. Turk. J. Chem., 2004, 28, 311-323.
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