HETEROCYCLES

Paper | Regular issue | Vol. 78, No. 4, 2009, pp. 961-975
Received, 14th October, 2008, Accepted, 18th December, 2008, Published online, 26th December, 2008.
DOI: 10.3987/COM-08-11577

■ Synthesis, Fungicidal and Antibacterial Activity of New Pyridazine Derivatives

Henryk Foks,* Krystyna Wisterowicz, Agnieszka Miszke, Kamil Brożewicz, Katarzyna Wiśniewska, and Maria Dąbrowska-Szponar

Department of Organic Chemistry, Medical University of Gdańsk, Al. Gen. Hallera 107, 80-416 Gdańsk, Poland

Abstract

Compounds 1 - 3 were obtained in the reaction of 3,6-dichloropyridazines with phenylacetonitriles in the biphasic system - DMSO / 50% NaOH. The chlorine atom was replaced with cycloalkylamino (4 - 13) and hydrazinyl (23, 24) moiety. These last compounds were condensed with aldehydes (25 - 34). Pyridazynylphenylacetonitriles were converted into amides 14 - 18 and thioamides 19 - 22. In compounds 2, 3 the chlorine atom was replaced with thiophenyl (37, 38) and in compound 1 with thioethyl and thiophenyl (35, 36) functional groups. In the reactions of compounds 1, 2 with ammonium polysulfide thioamides with thiol group (39, 40) and chlorine atom (41, 42) were obtained. Compounds 1 – 17, 19 – 43 were screened for antibacterial and fungicidal activities.

INTRODUCTION
Organic compounds with pyridazine moiety exhibit wide pharmacological activity. Many compounds act as antiphlogistic and antirheumatic agents like naphthylpyridinylpyridazine1 and bis-4-methoxy-3,5-diphenylpyridazine2,3 derivatives. Rohet et al.4 showed that 6-oxo-3,5-diphenylpyridazines analgetic and antiphlogistic effects are stronger than for acetylsalicylic acid and indomethacine with simultaneous absence of ulcerogenesis. Pyridazine phenylpiperazinyl derivative, beyond its own activity, strengthened morphine analgesis.5 Some pyridazine derivatives possess antiepileptic activity6-8 and are useful in Alzhimer’s disease theraphy.9,10 Reported antineoplastic,11-13 antiviral,14-15 antibacterial,16-18 and fungicidal19 activities deserve attention. Due to these facts essential is the search for new pyridazine derivatives, investigation of their activity and synthetic methods.

RESULTS AND DISCUSSION
The present work is the continuation of diazine derivatives investigations20,21 and relates to pyridazynylphenylacetonitriles chemistry. Our method of obtaining 2-(6-chloropyridazin-3-yl)- 2-phenylacetonitrile (1) has relative advantages comparing to previously reported syntheses,22-24 which involve sodium amide and anhydrous environment. Compounds 1 - 3 were obtained in the reactions of 3,6-dichloropyridazine with phenylacetonitrile, 4-chloro- and 4-methoxyphenylacetonitrile in the biphasic system DMSO / 50% NaOH. Thus obtained compounds 1 - 3 reacted with amines: pyrrolidine, morpholine, 4-benzylpiperidine and 4-phenylpiperidine forming 6-aminopyridazineacetonitriles 4 - 13. Some of thus obtained pyridazineacetonitriles were treated with concentrated H2SO4 giving appropriate amides 14 - 18 and with ammonium polysulfide – thioamides (19 - 22). Next we obtained pyridazinylhydrazones 25 - 34 in the reactions of hydrazinylpyridazines 23, 24 with some aldehydes (Scheme 1).
Other pyridazine derivatives, which contain sulfur in their structure are sulfides 35 - 38, obtained in reaction of chloropyridazines 1 - 3 with sodium thiolate and for 35 – sodium ethanethiol. 2-[6-(Ethylthio)pyridazin-3-yl]-2-phenylacetonitrile in reaction with ammonium polysulfide gives

thioamide 43. The same compound can be synthesized via alkylation of thioamide 39, which contains free mercapto group, with ethyl iodide. In comparison with the previous works, which report antibacterial activity of thioamides,25,26 compounds 1 - 3 were transformed into thioamides in reaction with ammonium polysulfide solution.In this work method of gaining thioamides 39 - 42 allows control over sulfurilation degree of products (Scheme 2). Good yields of thioamides were obtained, in which chlorine atom in pyridazine ring was intact under the reaction conditions. Compound 39 was obtained by Yamada and co-workers23 in different conditions and exhibited gastric antisecretary activity.

MICROBIOLOGY
Antimicrobial activity of the chemical agents against three recommended reference strains: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and clinical strain Candida albicans were examined. The susceptibility of the microorganisms to the agents was determined by the broth microdilution assay according to the procedures outlined by the National

Committee for Clinical Laboratory Standards.27 The stock solutions of the agents were prepared by dissolving the chemicals in DMSO. The final concentration of the agents in 200 µL of Mueller – Hinton broth (or Sabouraud’s medium for C. albicans) ranged over 0.125 – 256 µg/mL. In order to prepare bacterial suspension, overnight culture of bacteria in 3 % Trypticase Soy Broth (or Sabouraud’s medium for C. albicans) was diluted in sterile saline to the final concentration of approximately 107 CFU/mL of bacteria. Aliquots (5 µL) of bacterial suspension were added to each agent solution. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the agent that completely inhibited growth of the bacteria after 24 hours incubation in 37 °C. The final results were the average values from two independent experiments. Gentamycine was used as the reference substance (Table 1).

In summary, the tested derivatives exhibited diversified activity against aerobic bacteria and C. albicans. The results indicated that only five of synthesized compounds possessed antibacterial activity against S. aureus (19, 21, 23, 39 and 40), with the most active compound 40 (MIC = 32 µg/mL) . Only compound 23 showed activity against E. coli, but in relative high concentration (MIC = 128 µg/mL). The highest activity against C. albicans showed derivatives 39, 40 (MIC = 128 µg/mL).

EXPERIMENTAL
All melting points were obtained with Boetius apparatus and are uncorrected. The IR spectra were taken using Thermo Mattson Satellite spectrophotometer and the 1H NMR were taken with a Varian Gemini 200 MHz apparatus. The results of elemental analyses for C, H, N and S were in agreement with the calculated values within +/- 0.3% range.
General procedure for the synthesis of compounds 1-3.
3,6-Dichloropyridazine (7.45 g, 0.05 mol) was dissolved in DMSO (20 mL) and then 2-phenylacetonitrile (5.85 g, 0.05 mol) (for 1), 2-(4-chlorophenyl)acetonitrile (7.58 g, 0.05 mol) (for 2) or 2-(4-methoxyphenyl)acetonitrile (7.36 g, 0.05 mol) (for 3) and 50 % aqueous NaOH (10 mL) were added. The mixture was stirred and heated at 60oC for 2 h. Next ice (150 g) was added and the mixture was acidified with concentrated HCl. The precipitated solid was filtered off, washed with water and crystallized from the appropriate solvent.
2-(6-Chloropyridazin-3-yl)-2-phenylacetonitrile (1)
The crude product was crystallized from MeOH to give a white solid (7.46 g, 65 %), mp 128-129°C; 1H NMR (CDCl3) δ 5.52 (1H, s), 7.35 (5H, m), 7.55 (2H, s); IR (KBr) νmax 3052, 2935, 2257, 1410, 1149, 1066, 862, 697 cm-1; Anal. Calcd for C12H8ClN3: C, 62.76; H, 3.51; N, 18.30. Found: C, 62.63; H, 3,49; N, 18.26.
2-(4-Chlorophenyl)-2-(6-chloropyridazin-3-yl)acetonitrile (2)
The crude product was crystallized from MeOH to give a white solid (9.24 g, 70 %), mp 144-146°C; 1H NMR (CDCl3) δ 5.65 (1H, s), 7.40 (4H, m), 7.56 (2H, s); IR (KBr) νmax 3057, 2935, 2250, 1570, 1490, 1412, 1152, 1097, 824 cm-1; Anal. Calcd for C12H7Cl2N3: C, 54.57; H, 2.67; N, 15.91. Found: C, 54.41; H, 2.66; N, 15.87.
2-(6-Chloropyridazin-3-yl)-2-(4-methoxyphenyl)acetonitrile (3)
The crude product was crystallized from MeOH to give a white solid (7.79 g, 60 %), mp 129-132°C; 1H NMR (CDCl3) δ 3.82 (3H, s), 5.62 (1H, s), 6.90 (1H, s), 6.95 (1H, s), 7.36 (1H, s), 7.40 (1H, s), 7.54 (2H, s); IR (KBr) νmax 3041, 2923, 2251, 1610, 1512, 1409, 1253, 1178, 1150, 1027, 821 cm-1; Anal. Calcd for C13H10ClN3O: C, 60.12; H, 3.88; N, 16.18. Found: C, 59.98; H, 3.87; N, 16.14.
General procedure for the synthesis of compounds 4-13.
6-Chloropyridazine derivative (1-3) (5 mmol) and suitable amine (13 mmol) were dissolved in 1,4-dioxane (15 mL) and refluxed for 2 h. Then the solution was evaporated in vacuo, ice (30 g) was added to the residue and the mixture was extracted with CHCl3 (3x50 mL) and dried with MgSO4. Solution was concentrated and the solid formed was crystallized from the appropriate solvent.
2-Phenyl-2-[6-(pyrrolidin-1-yl)pyridazin-3-yl]acetonitrile (4)
Reaction with pyrrolidine. Product 4 was crystallized from EtOH to give a white solid (yield 47 %), mp 176-177°C; 1H NMR (CDCl3) δ 2.10 (4H, m), 3.55 (4H, m), 5.51 (1H, s), 6.60 (1H, d, J = 9.5 Hz), 7.15 (6H, m); IR (KBr) νmax 3035, 2919, 2864, 2240, 1594, 1542, 1475, 1455, 1030, 741, 700 cm-1; Anal. Calcd for C16H16N4: C, 72.70; H, 6.10; N, 21.20. Found: C, 72.51; H, 6.08; N, 21.14.
2-(6-Morpholinopyridazin-3-yl)-2-phenylacetonitrile (5)
Reaction with morpholine. Product 5 was crystallized from EtOH to give a white solid (yield 63 %), mp 141-143°C; 1H NMR (CDCl3) δ 3.65 (4H, m), 3.85 (4H, m), 5.56 (1H, s), 6.90 (1H, d, J = 9.4 Hz), 7.25 (6H, m); IR (KBr) νmax 3057, 2968, 2857, 2246, 1601, 1544, 1439, 1269, 1242, 1116, 1029, 845, 735, 699 cm-1; Anal. Calcd for C16H16N4O: C, 68.55; H, 5.75; N, 19.99. Found: C, 68.35; H, 5.73; N, 19.93.
2-[6-(4-Benzylpiperidin-1-yl)pyridazin-3-yl]-2-phenylacetonitrile (6)
Reaction with 4-benzylpiperidine. Product 6 was crystallized from MeOH to give a white solid (yield 45 %), mp 176-177°C; 1H NMR (CDCl3) δ 1.30 (2H, m), 1.85 (3H, m), 2.58 (2H, m), 2.95 (2H, m), 4.40 (2H, d, J = 13.0 Hz), 5.52 (1H, s), 6.90 (1H, d, J = 9.6 Hz), 7.10-7.51 (11H, m); IR (KBr) νmax 3064, 2944, 2915, 2846, 2245, 1599, 1444, 1256, 756, 697 cm-1; Anal. Calcd for C24H24N4: C, 78.23; H, 6.57; N, 15.21. Found: C, 77.99; H, 6.55; N, 15.17.
2-Phenyl-2-[6-(4-phenylpiperazin-1-yl)pyridazin-3-yl]acetonitrile (7)
Reaction with phenylpiperazine. Product 7 was crystallized from MeOH to give a white solid (yield 35 %), mp 174-177°C; 1H NMR (CDCl3) δ 3.35 (4H, m), 3.90 (4H, m), 5.54 (1H, s), 7.02-7.51 (12H, m); IR (KBr) νmax 3059, 2846, 2245, 1599, 1495, 1444, 1236, 948, 756, 697 cm-1; Anal. Calcd for C22H21N5: C, 74.34; H, 5.96; N, 19.70. Found: C, 74.15; H, 5.94; N, 19.64.
2-(4-Chlorophenyl)-2-(6-morpholinopyridazin-3-yl)acetonitrile (8)
Reaction with morpholine. Product 8 was crystallized from IPA to give a white solid (yield 51 %), mp 102-104°C; 1H NMR (CDCl3) δ 3.65 (4H, m), 3.85 (4H, m), 5.56 (1H, s), 6.93 (1H, d, J = 9.5 Hz), 7.20-7.53 (5H, m); IR (KBr) νmax 3055, 2920, 2854, 2250, 1595, 1544, 1490, 1269, 1117, 1094, 942, 848, 827 cm-1; Anal. Calcd for C16H15ClN4O: C, 61.05; H, 4.80; N, 17.80. Found: C, 60.88; H, 4.78; N, 17.75.
2-[6-(4-Benzylpiperidin-1-yl)pyridazin-3-yl]-2-(4-chlorophenyl)acetonitrile (9)
Reaction with 4-benzylpiperidine. Product 9 was crystallized from MeOH to give a white solid (yield 44 %), mp 172-175°C; 1H NMR (CDCl3) δ 1.30 (2H, m), 1.80 (3H, m), 2.55 (2H, m), 2.90 (2H, m), 4.40 (2H, d, J = 11.7 Hz), 5.47 (1H, s), 6.89 (1H, d, J = 9.5 Hz), 7.10-7.51 (10H, m); IR (KBr) νmax 3061, 2915, 2853, 2253, 1590, 1541, 1492, 1445, 1257, 1095, 753, 704 cm-1; Anal. Calcd for C24H23ClN4: C, 71.54; H, 5.75; N, 13.91. Found: C, 71.33; H, 5.73; N, 13.87.
2-(4-Chlorophenyl)-2-[6-(4-phenylpiperazin-1-yl)pyridazin-3-yl]acetonitrile (10)
Reaction with phenylpiperazine. Product 10 was crystallized from IPA to give a white solid (yield 47 %), mp 142-146°C; 1H NMR (CDCl3) δ 3.55 (4H, s), 3.90 (4H, s), 5.51 (1H, s), 6.98 (1H, d, J = 9.3 Hz), 7.26-7.46 (10H, m); IR (KBr) νmax 3062, 2846, 2247, 1600, 1494, 1444, 1241, 1157, 1096, 948, 755 cm-1; Anal. Calcd for C22H20ClN5: C, 67.77; H, 5.17; N, 17.96. Found: C, 67.63; H, 5.16; N, 17.92.
2-(4-Methoxyphenyl)-2-(6-morpholinopyridazin-3-yl)acetonitrile (11)
Reaction with morpholine. Product 11 was crystallized from MeOH to give a white solid (yield 37 %), mp 114-116°C; 1H NMR (CDCl3) δ 3.65 (4H, m), 3.85 (7H, m), 5.50 (1H, s), 6.90-7.45 (6H, m); IR (KBr) νmax 3060, 2960, 2855, 2247, 1606, 1510, 1446, 1255, 1177, 1119, 1030, 938, 830 cm-1; Anal. Calcd for C17H18N4O2: C, 65.79; H, 5.85; N, 18.05. Found: C, 65.62; H, 5.83; N, 17.99.
2-[6-(4-Benzylpiperidin-1-yl)pyridazin-3-yl]-2-(4-methoxyphenyl)acetonitrile (12)
Reaction with 4-benzylpiperidine. Product 12 was crystallized from MeOH to give a white solid (yield 40 %), mp 155-158°C; 1H NMR (CDCl3) δ 1.30 (2H, m), 1.70 (3H, m), 2.60 (2H, m), 2.90 (2H, m), 3.80 (3H, s), 4.40 (2H, d, J = 13.0 Hz), 5.48 (1H, s), 6.20-6.90 (5H, m), 7.10-7.45 (6H, m); IR (KBr) νmax 3062, 2917, 2838, 2249, 1591, 1511, 1444, 1257, 1177, 1033, 752, 704 cm-1; Anal. Calcd for C25H26N4O: C, 75.35; H, 6.58; N, 14.06. Found: C, 75.24; H, 6.56; N, 14.02.
2-(4-Methoxyphenyl)-2-[6-(4-phenylpiperazin-1-yl)pyridazin-3-yl]acetonitrile (13)
Reaction with phenylpiperazine. Product 13 was crystallized from MeOH to give a white solid (yield 43 %), mp 123-125°C; 1H NMR (CDCl3) δ 3.27 (4H, m), 3.80 (3H, s), 3.84 (4H, m), 5.48 (1H, s), 6.84-7.41 (11H, m); IR (KBr) νmax 3070, 2960, 2841, 2247, 1599, 1511, 1442, 1255, 1235, 1030, 984, 759 cm-1; Anal. Calcd for C23H23N5O: C, 71.67; H, 6.01; N, 18.17. Found: C, 71.49; H, 5.99; N, 18.13.
General procedure for the synthesis of compounds 14-18.
2-(6-Chloropyridazin-3-yl)-2-phenylacetonitrile (0.345 g, 1.5 mmol) (for 14), 2-(4-chlorophenyl)-2- (6-chloropyridazin-3-yl)acetonitrile (0.396 g, 1.5 mmol) (for 15), 2-[6-(4-benzylpiperidin-1-yl) pyridazin-3-yl]-2-(4-chlorophenyl)acetonitrile (0.604 g, 1.5 mmol) (for 16), 2-(4-chlorophenyl)- 2-[6-(4-phenylpiperazin-1-yl)pyridazin-3-yl]acetonitrile (0.584 g, 1.5 mmol) (for 17) or 2-[6-(4-benzylpiperidin-1-yl)pyridazin-3-yl]-2-(4-methoxyphenyl)acetonitrile (0.597 g, 1.5 mmol) (for 18) was dissolved in concentrated H2SO4 (5 mL) and left for 24 h at rt. Next ice (10 g) was added and the mixture was alkalized with ammonia. The precipitated solid was filtered off and crystallized.
2-(6-Chloropyridazin-3-yl)-2-phenylacetamide (14)
The crude product was crystallized from MeOH/H2O (1:1) to give a white solid (0.152 g, 41 %), mp 79-83°C; 1H NMR (CDCl3) δ 5.44 (1H, s); 6.09 (1H, s), 6.82 (1H, s), 7.20-7.50 (6H, m), 7.65 (1H, d, J = 8.9 Hz); IR (KBr) νmax 3320, 3199, 1669, 1624, 1410, 1152, 702 cm-1; Anal. Calcd for C12H10ClN3O: C, 58.19; H, 4.07; N, 16.97. Found: C, 58.06; H, 4.05; N, 16.93.
2-(4-Chlorophenyl)-2-(6-chloropyridazin-3-yl)acetamide (15)
The crude product was crystallized from MeOH to give a white solid (0.211 g, 50 %), mp 169-172°C; 1H NMR (CDCl3) δ 3.25 (2H, s), 5.40 (1H, s), 7.36-7.46 (4H, m), 7.66-7.85 (2H, m); IR (KBr) νmax 3363, 3195, 1628, 1487, 1415, 1158, 820, 648 cm-1; Anal. Calcd for C12H9Cl2N3O: C, 51.09; H, 3.22; N, 14.89. Found: C, 50.97; H, 3.21; N, 14.85.
2-[6-(4-Benzylpiperidin-1-yl)pyridazin-3-yl]-2-(4-chlorophenyl)acetamide (16)
The crude product was crystallized from MeOH/H2O (1:1) to give a white solid (0.435 g, 69 %), mp 215-219°C; 1H NMR (CDCl3) δ 1.30 (2H, m), 1.70 (3H, m), 2.50 (2H, m), 3.20 (2H, m), 3.40 (2H, bs), 4.22 (2H, d, J = 12.9 Hz), 5.16 (1H, s), 7.09-8.00 (11H, m); IR (KBr) νmax 3412, 2922, 1684, 1640, 1599, 1216, 1174, 1121, 1034, 1010, 696, 605 cm-1; Anal. Calcd for C24H25ClN4O: C, 68.48; H, 5.99; N, 13.31. Found: C, 68.31; H, 5.97; N, 13.28.
2-(4-Chlorophenyl)-2-[6-(4-phenylpiperazin-1-yl)pyridazin-3-yl]acetamide (17)
The crude product was crystallized from MeOH to give a white solid (0.287 g, 47 %), mp 102-105°C; 1H NMR (CDCl3) δ 3.50 (4H, s), 3.80 (4H, s), 5.10 (1H, s), 5.84 (1H, bs), 6.80-7.50 (12 H, m); IR (KBr) νmax 3375, 3183, 2847, 1678, 1597, 1492, 1445, 1386, 1231, 759 cm-1; Anal. Calcd for C22H22ClN5O: C, 64.78; H, 5.44; N, 17.17. Found: C, 64.63; H, 5.43; N, 17.12.
2-[6-(4-Benzylpiperidin-1-yl)pyridazin-3-yl]-2-(4-methoxyphenyl)acetamide (18)
The crude product was crystallized from MeOH to give a white solid (62 mg, 10 %), mp 243-247°C; 1H NMR (DMSO-d6) δ 1.20 (2H, m), 1.60 (3H, m), 2.50 (2H, m), 2.90 (2H, m), 3.40 (2H, bs), 3.72 (3H, s), 4.25 (2H, d, J = 12.8 Hz), 5.07 (1H, s), 6.75-7.85 (9H, m); IR (KBr) νmax 3425, 3196, 2920, 2851, 1679, 1642, 1602, 1492, 1450, 1182, 1032, 1010, 621 cm-1; Anal. Calcd for C25H28N4O2: C, 72.09; H, 6.78; N, 13.45. Found: C, 71.89; H, 6.76; N, 13.41.
General procedure for the synthesis of compounds 19-22.
Compound (5, 7, 8, 9) (5 mmol) was dissolved in pyridine (10 mL) and yellow ammonium polysulfide solution (3 mL) was added. The mixture was stirred at rt for 12 h and next ice (10 g) was added. The precipitated solid was filtered off and crystallized from the appropriate solvent.
2-(6-Morpholinopyridazin-3-yl)-2-phenylethanethioamide (19)
The crude product was crystallized from benzene to give a yellow solid (0.990 g, 63 %), mp 128-130°C; 1H NMR (CDCl3) δ 3.60 (4H, m), 3.90 (4H, m), 5.62 (1H, s), 6.90 (1H, d, J = 9.5 Hz), 7.20-7.60 (6H, m), 7.83 (1H, bs), 9.70 (1H, bs); IR (KBr) νmax 3271, 3096, 2964, 2852, 1592, 1444, 1252, 1124, 934, 699 cm-1; Anal. Calcd for C16H18N4OS: C, 61.12; H, 5.77; N, 17.82; S, 10.20. Found: C, 60.98; H, 5.75; N, 17.77; S, 10.17.
2-Phenyl-2-[6-(4-phenylpiperazin-1-yl)pyridazin-3-yl]ethanethioamide (20)
The crude product was crystallized from EtOH to give a white solid (0.934 g, 48 %), mp 177-179°C; 1H NMR (CDCl3) δ 3.30 (4H, m), 3.80 (4H,m), 5.61 (1H, s), 6.95 (4H, m), 7.30 (6H, m), 7.55 (2H, m), 7.88 (1H, bs), 9.76 (1H, bs); IR (KBr) νmax 3262, 3060, 2849, 1591, 1543, 1485, 1448, 1258, 1229, 1030, 708, 696 cm-1; Anal. Calcd for C22H23N5S: C, 67.84; H, 5.95; N, 17.98; S, 8.23. Found: C, 67.72; H, 5.93; N, 17.93; S, 8.20.
2-(4-Chlorophenyl)-2-(6-morpholinopyridazin-3-yl)ethanethioamide (21)
The crude product was crystallized from benzene/petr. ether to give a yellow solid (1.05 g, 60 %), mp 66-67°C; 1H NMR (CDCl3) δ 3.60 (4H, m), 3.85 (4H, m), 5.56 (1H, s), 6.90 (1H, d, J = 9.4 Hz), 7.45 (1H, d, J = 9.4 Hz), 7.20-7.40 (4H, m), 8.02 (1H, bs), 9.75 (1H, bs); IR (KBr) νmax 3288, 3169, 2962, 2853, 1594, 1545, 1489, 1442, 1254, 1116, 682 cm-1; Anal. Calcd for C16H17ClN4OS: C, 55.09; H, 4.91; N, 16.06; S, 9.19. Found: C, 54.93; H, 4.89; N, 16.02; S. 9.17.
2-[6-(4-Benzylpiperidin-1-yl)pyridazin-3-yl]-2-(4-chlorophenyl)ethanethioamide (22)
The crude product was crystallized from benzene/petr. ether to give a beige solid (1.42 g, 65 %), mp 83-84°C; 1H NMR (CDCl3) δ 0.89 (1H, m), 1.60 (2H, m), 1.80 2H, m), 2.60 (2H, m), 2.90 (2H, m), 4.35 (2H, d, J = 6.7 Hz), 5.50 (1H, s), 6.90 (1H, d, J = 9.5 Hz), 7.34 (9H, m), 7.50 (1H, d, J = 9.5 Hz), 7.85 (1H, bs), 9.98 (1H, bs); IR (KBr) νmax 3280, 3163, 3025, 2924, 2847, 1594, 1543, 1488, 1445, 1251, 743, 700 cm-1; Anal. Calcd for C24H25ClN4S: C, 65.96; H, 5.77; N, 12.82; S, 7.34. Found: C, 65.79; H, 5.75; N, 12.79; S, 7.32.
(6-Chloro-pyridazin-3-yl)hydrazine (23)
3,6-Dichloropyridazine (4.50 g, 30 mmol) was dissolved in dioxane (20 mL) and then triethylamine (4.2 mL, 30 mmol) and hydrazine (1.6 mL, 33 mmol) were added. The mixture was refluxed for 2 h. The mixture was concentrated in vacuo and ice (20 g) was added to the residue. The precipitated solid was filtered off and crystallized from IPA to give a white solid (3.18 g, 73 %), mp 123-126°C; 1H NMR (DMSO-d6) δ 4.40 (2H, bs), 7.10 (1H, d, J = 9.4 Hz), 7.40 (1H, d, J = 9.4 Hz), 8.20 (1H, bs); IR (KBr) νmax 3255, 3030, 2925, 1600, 1452, 1154, 833, 641 cm-1; Anal. Calcd for C4H5ClN4: C, 33.23; H, 3.49; N, 38.76. Found: C, 33.15; H, 3.48; N, 38.66.
(6-Hydrazino-pyridazin-3-yl)-(4-methoxyphenyl)-acetonitrile (24)
Compound 3 (1.3 g, 5 mmol) was dissolved in EtOH (20 mL) and hydrazine monohydrate (3 mL, 61 mmol) was added. The mixture was refluxed for 3 h. Next the mixture was concentrated in vacuo and MeOH (3 mL) was added. After cooling down the precipitated solid was filtered off and crystallized from MeOH to give a white solid (0.64 g, 50 %), mp 153-157°C; 1H NMR (DMSO-d6) δ 3.73 (3H, s), 4.30 (2H, s), 5.79 (1H, s), 6.85-7.40 (6H, m), 8.10 (1H, s); IR (KBr) νmax 3307, 3236, 3017, 2931, 2250, 1608, 1513, 1430, 1262, 1178, 1101, 1033, 825, 641 cm-1; Anal. Calcd for C13H13N5O: C, 61.17; H, 5.13; N, 27.43. Found: C, 61.01; H, 5.11; N, 27.38.
General procedure for the synthesis of compounds 25-34.
Compound 23 or 24 (2 mmol) was dissolved in EtOH (30 mL) and then suitable aldehyde (2.5 mmol) was added. The mixture was refluxed for 1 h. After cooling down water (20 mL) was added, and precipitated
solid was filtered off and crystallized from the appropriate solvent.
3-(2-Benzylidenehydrazinyl)-6-chloropyridazine (25)
Reaction with benzaldehyde. Product 25 was crystallized from dioxane to give a white solid (yield 53 %), mp 254-256°C; 1H NMR (DMSO-d6) δ 7.37-7.72 (7H, m), 8.13 (1H, s); 11.72 (1H, s); IR (KBr) νmax 3024, 2921, 2847, 1614, 1593, 1530, 1413, 1135, 1064, 751,687 cm-1; Anal. Calcd for C11H9ClN4: C, 56.78; H, 3.90; N, 24.08. Found: C, 56.63; H, 3.88; N, 24.01.
3-[2-(4-Bromobenzylidene)hydrazinyl]-6-chloropyridazine (26)
Reaction with 4-bromobenzaldehyde. Product 26 was crystallized from dioxane to give a white solid (yield 62 %), mp 278-279°C; 1H NMR (DMSO-d6) δ 7.54-7.70 (6H, m), 8.09 (1H, s), 11.80 (1H, s); IR (KBr) νmax 3204, 2921, 1616, 1600, 1530, 1485, 1416, 1397, 1130, 1070, 831, 812 cm-1; Anal. Calcd for C11H8BrClN4: C, 42.40; H, 2.59; N, 17.98. Found: C, 42.30; H, 2.58; N, 17.93.
3-Chloro-6-[2-(4-nitrobenzylidene)hydrazinyl]pyridazine (27)
Reaction with 4-nitrobenzaldehyde. Product 27 was crystallized from dioxane to give a lateritious solid (yield 52 %), mp 277-279°C; 1H NMR (DMSO-d6) δ 7.60-8.25 (7H, m), 12.09 (1H, s); IR (KBr) νmax 2925, 2845, 1621, 1527, 1429, 1395, 1345, 1139, 1105, 1074, 851, 839, 746, 685 cm-1; Anal. Calcd for C11H8ClN5O2: C, 47.58; H, 2.90; N, 25.22. Found: C, 47.48; H, 2.89; N, 25.16.
3-Chloro-6-{2-[(5-nitrofuran-2-yl)methylene]hydrazinyl}pyridazine (28)
Reaction with 5-nitrofuran-2-carbaldehyde. Product 28 was crystallized from MeOH to give a yellow solid (yield 90 %), mp 276-278°C; 1H NMR (DMSO-d6) δ 7.20 (1H, d, J = 4.2 Hz), 7.60-8.10 (3H, m), 8.06 (1H, s), 12.28 (1H, s); IR (KBr) νmax 3135, 2922, 2850, 1594, 1471, 1417, 1387, 1353, 1257, 1138, 1028, 809 cm-1; Anal. Calcd for C9H6ClN5O3: C, 40.39; H, 2.26; N, 26.17. Found: C, 40.29; H, 2.25; N, 26.11.
3-Chloro-6-{2-[(5-nitrothiophen-2-yl)methylene]hydrazinyl}pyridazine (29)
Reaction with 5-nitrothiophene-2-carbaldehyde. Product 29 was crystallized from DMF/H2O (1:1) to give an orange solid (yield 69 %), mp 303-305°C; 1H NMR (DMSO-d6) δ 7.46 (1H, d, J = 3.0 Hz), 8.10 (1H, d, J = 3.0 Hz), 7.63 (1H, d, J = 9.0 Hz), 7.73 (1H, d, J = 9.0 Hz), 8.28 (1H, s), 12.27 (1H, s); IR (KBr) νmax 3100, 2917, 2819, 1610, 1573, 1436, 1419, 1330, 1286, 1221, 1140, 812, 732 cm-1; Anal. Calcd for C9H6ClN5O2S: C, 38.10; H, 2.13; N, 24.69; S, 11.30. Found: C, 38.02; H, 2.12; N, 24.62; S, 11.27.
2-[6-(2-Benzylidenehydrazinyl)pyridazin-3-yl]-2-(4-methoxyphenyl)acetonitrile (30)
Reaction with benzaldehyde. Product 30 was crystallized from dioxane to give a white solid (yield 54 %), mp 263-265°C; 1H NMR (DMSO-d6) δ 3.74 (3H s), 5.91 (1H, s), 6.90-7.80 (11H, m), 8.13 (1H, s), 11.65 (1H, s); IR (KBr) νmax 3189, 2919, 2849, 2248, 1615, 1540, 1513, 1428, 1257, 1137, 1031, 824, 693 cm-1; Anal. Calcd for C20H17N5O: C, 69.96; H, 4.99; N, 20.40. Found: C, 69.99; H, 4.97; N, 20.35.
2-{6-[2-(4-Bromobenzylidene)hydrazinyl]pyridazin-3-yl}-2-(4-methoxyphenyl)acetonitrile (31)
Reaction with 4-bromobenzaldehyde. Product 31 was crystallized from dioxane to give a white solid (yield 61 %), mp 256-259°C; 1H NMR (DMSO-d6) δ 3.75 (3H s), 5.93 (1H, s) 7.00-7.63 (10H m), 8.10 (1H, s), 11.75 (1H, s); IR (KBr) νmax 3186, 2934, 2837, 2249, 1603, 1540, 1510, 1487, 1428, 1255, 1139, 1033, 827 cm-1; Anal. Calcd for C20H16BrN5O: C, 56.89; H, 3.82; N, 16.58. Found: C, 56.74; H, 3.80; N, 16.54.
2-(4-Methoxyphenyl)-2-{6-[2-(4-nitrobenzylidene)hydrazinyl]pyridazin-3-yl}acetonitrile (32)
Reaction with 4-nitrobenzaldehyde. Product 32 was crystallized from dioxane to give a lateritious solid (yield 40 %), mp 273-275°C; 1H NMR (DMSO-d6) δ 3.74 (3H, s), 5.94 (1H, s), 6.85-8.30 (11H, m), 12.05 (1H, s); IR (KBr) νmax 3195, 2841, 2249, 1611, 1573, 1541, 1524, 1429, 1341, 1256, 1143, 1032, 857, 827 cm-1; Anal. Calcd for C20H16N6O3: C, 61.85; H, 4.15; N, 21.64. Found: C, 61.72; H, 4.13; N, 21.59.
2-(4-Methoxyphenyl)-2-{6-{2-[(5-nitrofuran-2-yl)methylene]hydrazinyl}pyridazin-3-yl}acetonitrile (33)
Reaction with 5-nitrofuran-2-carbaldehyde. Product 33 was crystallized from MeOH to give a lateritious solid (yield 56 %), mp 224-227°C; 1H NMR (DMSO-d6) δ 3.74 (3H, s), 5.96 (1H, s), 6.90-7.80 (8H, m), 8.10 (1H, s), 12.25 (1H, s); IR (KBr) νmax 3123, 2936, 2838, 2247, 1583, 1512, 1477, 1431, 1351, 1307, 1252, 1020, 810 cm-1; Anal. Calcd for C18H14N6O4: C, 57.14; H, 3.73; N, 22.21. Found: C, 57.02; H, 3.72; N, 22.16.
2-(4-Methoxyphenyl)-2-{6-{2-[(5-nitrothiophen-2-yl)methylene]hydrazinyl}pyridazin-3-yl}aceto- nitrile (34)
Reaction with 5-nitrothiophene-2-carbaldehyde. Product 34 was crystallized from dioxane to give a lateritious solid (yield 62 %), mp 206-208°C; 1H NMR (DMSO-d6) δ 3.33 (3H, s), 5.97 (1H, s), 6.95-8.10 (8H, m), 8.29 (1H, s), 12.25 (1H, s); IR (KBr) νmax 3106, 2933, 2838, 2248, 1607, 1510, 1434, 1333, 1254, 1033, 816, 732 cm-1; Anal. Calcd for C18H14N6O3S: C, 54.81; H, 3.58; N, 21.31; S, 8.13. Found: C, 54.72; H, 3.57; N, 21.26; S, 8.10.
2-[6-(Ethylthio)pyridazin-3-yl]-2-phenylacetonitrile (35)
To a round bottomed flask (100 mL) containing solution of 0.888 mL (0.745 g, 12 mmol) EtSH in 20 mL of anhydrous dioxane 0.276 g (12 mmol) of sodium was added and gently heated until all of sodium was reacted (ca. 1.5h). Clear solution was then cooled to rt and the solution of 1.378 g (6 mmol) of 2-(6-chloropyridazin-3-yl)-2-phenylacetonitrile in 20 mL of anhydrous dioxane was dropped into within 5 min. After heating 3.5 h in reflux the mixture was concentrated in vacuo and 30 mL of ice-cold water was added. Precipitated solid was collected and crystallized from MeOH to give a beige solid (0.991 g, 65 %), mp 157-158°C; 1H NMR (CDCl3) δ 1.48 (3H, t, J = 7.3 Hz), 3.35 (2H, q, J = 7.3 Hz), 5.57 (1H, s), 7.25-7.55 (7H, m); IR (KBr) νmax 3063, 3039, 2978, 2934, 2248, 1576, 1415, 1153, 1052, 854, 697 cm-1; Anal. Calcd for C14H13N3S: C, 65.85; H, 5.13; N, 16.46; S, 12.56. Found: C, 65.71; H, 5.11; N, 16.41; S, 12.53.
General procedure for the synthesis of compounds 36-38.
To a round bottomed flask containing 20 mL of anhydrous dioxane and 12 mmol of appropriate thiol,12 mmol of sodium was added and then heated for 15 min. in reflux. After cooling, solution of 4-chloropyridazinephenylacetonitrile (6 mmol) in 20 mL of anhydrous 1,4-dioxane was dropped into. After heating in reflux for 3.5 h the mixture was concentrated in vacuo and 30 mL of water was added. The crude product was collected and crystallized from MeOH.
2-Phenyl-2-[6-(phenylthio)pyridazin-3-yl]acetonitrile (36)
Reaction with 2-(6-chloropyridazin-3-yl)-2-phenylacetonitrile. A white solid (0.45 g, 25 %), mp 111-113°C; 1H NMR (CDCl3) δ 5.60 (1H, s), 7.00 (1H, d, J = 9.1 Hz), 7.20 - 7.70 (11H, m); IR (KBr) νmax 3032, 2931, 2250, 1406, 1149, 1067, 748, 699 cm-1; Anal. Calcd for C18H13N3S: C, 71.26; H, 4.32; N, 13,85; S, 10.57. Found: C, 71.11; H, 4.30; N, 13.81; S, 10.54.
2-(4-Chlorophenyl)-2-[6-(phenylthio)pyridazin-3-yl]acetonitrile (37)
Reaction with 2-(4-chlorophenyl)-2-(6-chloropyridazin-3-yl)acetonitrile. A white solid (1.05 g, 52 %), mp 163-165°C; 1H NMR (CDCl3) δ 5.58 (1H, s), 7.00 (1H, d, J = 9.1 Hz), 7.23 - 7.64 (10H, m); IR (KBr) νmax 3063, 2879, 2247, 1570, 1493, 1408, 1149, 1094, 819, 746, 687 cm-1; Anal. Calcd for C18H12ClN3S: C, 64.00; H, 3.58; N, 12.44; S, 9.49. Found: C, 63.87; H, 3.57; N, 12.41; S, 9.46.
2-(4-Methoxyphenyl)-2-[6-(phenylthio)pyridazin-3-yl]acetonitrile (38)
Reaction with 2-(6-chloropyridazin-3-yl)-2-(4-methoxyphenyl)acetonitrile. A white solid (1.00 g, 50 %), mp 135-137°C; 1H NMR (CDCl3) δ 3.80 (3H, s), 5.55 (1H, s), 6.68 - 7.66 (11H, m); IR (KBr) νmax 3067, 3028, 2933, 2830, 2248, 1511, 1407, 1252, 1172, 1010, 819, 745, 688 cm-1; Anal. Calcd for C19H15N3OS: C, 68.45; H, 4.53; N, 12.60; S, 9.62. Found: C, 68.32; H, 4.51; N, 12.57; S, 9.59.
General procedure for the synthesis of compounds 39, 40.
Compound 1 (0.92 g, 4 mmol) or compound 2 (1.05 g, 4 mmol) was dissolved in pyridine (5 mL, 61 mmol), MeOH (2 mL) and yellow solution of ammonium polysulfide (3 mL) were added. The mixture was stirred at rt for 72 h. Next ice (50 g ) was added and the precipitated solid was filtered off. The filtrate was acidified with concentrated acetic acid. The precipitated solid was filtered off and crystallized from appropriate solvent.
2-(6-Mercaptopyridazin-3-yl)-2-phenylethanethioamide (39)
The crude product was crystallized from benzene to give a yellow solid (0.68 g, 65 %), mp 156-157°C; 1H NMR (DMSO-d6) δ 5.39 (1H, s), 7.10 (1H, d, J = 9.4 Hz), 7.30‐7.60 (6H, m), 9.63 (1H, bs), 9.83 (1H, bs), 14.10 (1H, bs); IR (KBr) νmax 3412, 3275, 3181, 3146, 3074, 2995, 2922, 2884, 1620, 1572, 1416, 1402, 1232, 1089, 1028, 698, 634, 551 cm-1; Anal. Calcd for C12H11N3S2: C, 55.14; H, 4.24; N, 16.08; S, 24.54. Found: C, 55.03; H, 4.23; N, 16.03; S, 24.49.
2-(4-Chlorophenyl)-2-(6-mercaptopyridazin-3-yl)ethanethioamide (40)
The crude product was crystallized from MeOH to give a yellow solid (0.99 g, 85 %), mp 179-181°C; 1H NMR (CDCl3) δ 5.41 (1H, s), 7.15 (1H, d, J = 9.2 Hz), 7.45‐7.55 (5H, m), 9.65 (1H, bs), 9.98 (1H, bs), 14.58 (1H, bs); IR (KBr) νmax 3309, 3136, 3054, 2993, 2922, 2854, 1624, 1563, 1488, 1407, 1086, 1027, 810, 679 cm-1; Anal. Calcd for C12H10ClN3S2: C, 48.72; H, 3.41; N, 14.21; S, 21.68. Found: C, 48.62; H, 3.39; N, 14.17; S, 21.62.
General procedure for the synthesis of compounds 41, 42.
Compound 1 (0.92 g, 4 mmol) or compound 2 (1.05 g, 4 mmol) was dissolved in pyridine (5 mL, 61 mmol), MeOH (2 mL) and yellow solution of ammonium polysulfide (2.5 mL) were added. The mixture was stirred at rt for 2 h. Next ice (50 g) was added, the precipitated solid after 12 h was filtered off and crystallized.
2-(6-Chloropyridazin-3-yl)-2-phenylethanethioamide (41)
The crude product was crystallized from MeOH to give a yellow solid (0.89 g, 85 %), mp 192-195°C; 1H NMR (CDCl3) δ 5.83 (1H,s), 7.30‐7.55 (7H, m), 7.92 (1H, bs), 8.82 (1H, bs); IR (KBr) νmax 3302, 3153, 3046, 1621, 1418, 1148, 855, 719, 697 cm-1; Anal. Calcd for C12H10ClN3S: C, 54.65; H, 3.82; N, 15.93; S, 12.16. Found: C, 54.53; H, 3.80; N, 15.89; S, 12.13.
2-(4-Chlorophenyl)-2-(6-chloropyridazin-3-yl)ethanethioamide (42)
The crude product was crystallized from cyclohexane to give a yellow solid (0.53 g, 45 %), mp 112-113°C; 1H NMR (CDCl3) δ 5.91 (1H, s), 7.33‐7.58 (6H, m), 7.94 (1H, bs), 9.07 (1H, bs); IR (KBr) νmax 3303, 3160, 2925, 2851, 1617, 1491, 1415, 1149, 1092, 916, 810 cm-1; Anal. Calcd for C12H9Cl2N3S: C, 48.33; H, 3.04; N, 14.09; S, 10.75. Found: C, 48.21; H, 3.03; N, 14.05; S, 10.72.
2-[6-(Ethylthio)pyridazin-3-yl]-2-phenylethanethioamide (43)
Method A
Compound 35 (0.255 g, 1 mmol) was dissolved in pyridine (3 mL, 37 mmol) and dioxane (5 mL). Next yellow solution of ammonium polysulfide was added and the mixture was stirred at rt for 12 h. After this time the mixture was concentrated in vacuo and ice (10 g) was added to the residue. Water was decanted and MeOH (3 mL) was added to the oily residue. Water was added and the solid formed was filtered off and crystallized from MeOH to give a yellow solid (0.202 g, 70 %), mp 121-122°C; 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.3 Hz), 3.36 (2H, q, J = 7.3 Hz), 5.69 (1H, s), 7.20‐7.60 (7H, m), 7.95 (1H, bs), 9.38 (1H, bs); IR (KBr) νmax 3305, 3156, 3062, 3029, 2971, 2932, 1618, 1429, 1412, 1219, 1160, 717, 700 cm-1; Anal. Calcd for C14H15N3S2: C, 58.10; H, 5.22; N, 14.52; S, 22.16. Found: C, 57.99; H, 5.20; N, 14.49; S, 22.10.
Method B
Compound 39 (0.65 g, 2.5 mmol) was dissolved in a methanolic KOH solution (0.14 g, 2.5 mmol)/10 mL and then MeI (0.28 mL, 4.5 mmol) was added. The mixture was stirred at rt for 12 h. Next solvent was evaporated and ice (10 g) was added. The precipitate was filtered off and crystallized from MeOH to give a yellow solid (0.41 g, 57 %).

References

1. N. Tamayo, L. Liao, M. Goldberg, D. Powers, Y. Tudor, V. Yu, L. M. Wong, B. Henkle, S. Middleton, R. Syed, T. Harvey, G. Yang, R. Hungate, and C. Domnguez, Bioorg. Med. Chem. Lett., 2005, 15, 2409. CrossRef
2. T. Matsuda, T. Aoki, T. Ohgiya, T. Koshi, M. Ohkuchi, and H. Shigyo, Bioorg. Med. Chem. Lett., 2001, 11, 2369. CrossRef
3. T. Matsuda, T. Aoki, T. Koshi, M. Ohkuchi, and H. Shigyo, Bioorg. Med. Chem. Lett., 2001, 11, 2373. CrossRef
4. D. S. Doğruer, M. F. Şahin, E. Kűpeli, and E. Yeşilda, Turk. J. Chem., 2003, 27, 727.
5. F. Rohet, C. Rubat, P. Cyoudert, and J. Conquelet, Bioorg. Med. Chem., 1997, 5, 655. CrossRef
6. T. Hu, B. A. Stearns, B. T. Campbell, J. M. Arruda, Ch. Chen, J. Aiyar, R. E. Bezverkov, A. Santini, H. Schaffhauser, W. Liu, S. Venkatraman, and B. Munoz, Bioorg. Med. Chem. Lett., 2004, 14, 2031. CrossRef
7. S. Moreau, P. Coudert, C. Rubat, D. Vallee-Goyete, D. Gardette, J. C. Gramain, and J. Conquelet, Bioorg. Med. Chem., 1998, 7, 983. CrossRef
8. P. W. Harrison, G. B. Barlin, L. P. Davies, S. J. Ireland, P. Mátyus, and M. G. Wong, Eur. J. Med. Chem., 1996, 31, 651. CrossRef
9. A. Stehl, G. Seitz, and K. Schulz, Tetrahedron, 2002, 58, 1343. CrossRef
10. J. M. Contreras, I. Parrot, W. Sippl, Y. M. Rival, and C. G. Wermuth, J. Med. Chem., 2001, 44, 2707. CrossRef
11. K. F. Byth, N. Cooper, J. D. Culshaw, D. W. Heaton, S. E. Oakes, C. A. Minshull, R. A. Norman, R. A. Pauptit, J. A. Tucker, J. Breed, A. Pannifer, S. Rowsell, J. J. Stanway, A. L. Valentine, and A. P. Thomas, Bioorg. Med. Chem. Lett., 2004, 14, 2249. CrossRef
12. J. C. Bussolari, J. D. Stoeckler, and R. P. Panzica, Bioorg. Med. Chem., 1996, 4, 1725. CrossRef
13. A. Fontana, E. J. Benito, M. J. Martini, N. Sanchez, R. Alajarin, J. J. Vaquero, J. Alvarez-Builla, S. Lambel-Giraudet, S. Leonce, A. Pierre, and D. Caignard, Bioorg. Med. Chem. Lett., 2002, 12, 2611. CrossRef
14. E. A. Meade, L. L. Wotring, J. C. Drach, and L. B. Townsend, J. Med. Chem., 1997, 40, 794. CrossRef
15. M. Font, A. Monge, A. Cuartero, A. Elorriaga, J. J. Martinez-Irujo, E. Alberdi, E. Santiago, I. Prieto, J. J. Lasarte, P. Sarobe, and F. Borras, Eur. J. Med. Chem., 1995, 30, 963. CrossRef
16. A. A. Aly and A. A. F. Wasfy, Phosphorus, Sulfur and Silicon, 2003, 178, 1911. CrossRef
17. V. S. Velezheva, P. J. Brennan, V. Y. Marshakov, D. V. Gusev, I. N. Lisichkina, A. S. Peregudov, L. N. Tchernousova, T. G. Smirnova, S. N. Andreevskaya, and A. E. Medvedev, J. Med. Chem., 2004, 47, 3455. CrossRef
18. G. Heinisch, B. Matuszczak, and K. Planitzer, Arch. Pharm. Med. Chem., 2000, 333, 231. CrossRef
19. E. Akbas and I. Barber, Eur. J. Med. Chem., 2005, 40, 401. CrossRef
20. K. Wisterowicz and H. Foks, Khim. Geterotsikl. Soed., 2006, 10, 1346.
21. K. Wisterowicz and H. Foks, Khim. Geterotsikl. Soed., 2006, 10, 1534.
22. A. Abbotto, V. Alanzo, S. Bradamante, and A. G. Pagani, J. Chem. Soc., Perkin Trans. 1, 1991, 481.
23. T. Hamada, Y. Nobuhara, H. Shimamura, K. Yoshihara, A. Yamaguchi, and M. Ohki, Chem. Pharm. Bull., 1981, 29, 3433.
24. G. Hainisch and T. Langer, Synth. Commun., 1994, 24, 773. CrossRef
25. D. Libermann, M. Moyeux, N. Rist, and F. Grumbach, Compt. Rend., 1956, 234, 2409, (Chem. Abstr., 1957, 51, 404).
26. T. Taguchi and K. Yoshihira, Chem. Pharm. Bull., 1963, 11, 430.
27. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. Vol. 13, No. 25. 1993.