Synthesis of Some Novel Fluorinated/Nonfluorinated α-Amino Acids, Bearing 3-Thioxo-5-oxo-1,2,4-triazin-6-yl and Steroidal Moieties, and Evaluation of Their Amylolytic Effects against Some Fungi, Part-II
Some new fluorinated/nonfluorinated α-amino acids bearing 3-thioxo-5-oxo-1,2,4-triazin-6-yl and steroidal moieties have been obtained from condensation of the corresponding amino-triazinones with the steroid (Epiandrosterone). This was followed by the addition of HCN and, finally, acidic hydrolysis. The structure of the targets was established from their elemental analysis and spectral data. The amylolytic activity of the new products was evaluated against some fungi.
α-Amino acids are one of the most important bioactive chemical substances (proteins and nucleoproteins) forming the basic constituents of living cells. Nine proteinogenic amino acids are considered as essential biochemicals for humans: valine, threonine, tryptophan, phenylalanine, leucine, isoleucine, methionine, lysine, and histidine. For instance, 5-fluorocytosine is an analogue of nucleotide, used as a chemotherapeutic antifungal when combined with amphotericin B . Of equal importance, glycine is required for the biosynthesis of the heme group of haemoglobin; also, tryptophan is the precursor of a family of substances important in the biochemistry of the central nervous system (CNS), and tyrosine is the starting material for the biosynthesis of the skin pigment melanin .
The enzyme lactate dehydrogenase (LDH) illustrates isozymes very well (Figure 1).
1,2,4-Triazine rings have been reported in the literature as having antifungal properties alongside their antitumor and antiviral activities.  Recently, Abdul-Rahman et al. [4–9] reported the synthesis, chemistry, and medicinal and biological activity, especially 6-(2-aminophenyl)3-thioxo-1,2,4-triazin-5-one [10–14]. Among all approved medicinal and pharmaceutical chemicals, nearly 20% have at least one fluorine atom existing which enhances phase II-III clinical trials . Thus, the combination of fluorine with biomolecules such as fluorinated amino acids (FAAs), fluorinated steroids, and fluorinated nucleosides has increased, considerably, of the late years .
Incorporation of FAAs is one of the most utilized strategies in peptide and protein science. The effects of the combination of fluorinated α-amino acids into peptides and proteins on the primary and secondary structure have been widely reviewed by Koksch et al [17–19]. Furthermore, the fusion of unnatural/synthesized amino acids into peptides and proteins is generally closely accompanied with antimicrobial [20–22], antiviral , and metal chelating properties such as thrombin, trypsin, and factor VIIa inhibitory activity.
Based on all these observations, this present work aims to find new synthetic fluorinated/nonfluorinated α-amino acids bearing both 3-thioxo 5-oxo-1,2,4-triazin-6-yl and steroidal moieties and evaluates their enzymatic effects toward some fungi activities (amylolytic activity) with an objective to obtain new highly bioactive substances.
2. Results and Discussion
The main objective of this work is to synthesize fluorinated/nonfluorinated α-amino acids derived from 1,2,4-triazinone and steroid. Through condensation of either fluorinated or nonfluorinated 1,2,4-triazinones bearing systems, such as 6-(2′-amino-5′-fluorophenyl)-3-thioxo-1,2,4-triazin-5(2H, 4H)one (1a) and/or (1b) a solution in THF with dehydroepiandrosterone(B) (DHEA) in DMF reflux, yielded the 17-imino-derivatives 2a and 2b. Selective addition of HCN to an azomethine (imine) group, the Strecker reaction, is a common way to prepare α-amino acids by simple hydrolysis of the α-aminonitrile addition product. HCN was added to the imino group of 2a or 2b under specific conditions [25, 26] affording the α-amino-acids analogous 3a and 3b, which were hydrolysed to 4a and 4b, respectively (Scheme 1).
Synthesis pathway for the targeted compounds 2–5:
New α-amino acid derivatives 4a and 4b bearing a 6-(fluorinated)/nonfluorinated-aryl-1,2,4-triazin-one and C-(steroid) moieties were isolated by the acidic hydrolysis of 3a and 3b using a diluted HCl. The new fluorinated α-amino acids 5a and 5b were obtained by the reflux of compounds 4a and 4b with 4-fluoroaniline in EtOH (Scheme 1).
The IR spectrum of 4a showed new functional groups at 3500, 1710 , referring to OH and C17=O of the carboxylic acid group bearing steroids, with bonds at 3500, 3210, and 3090 for OH (steroids) and 2NH of 1,2,4-triazine and bonds at ν 1235 and 1195 attributed for C-F and C=S, while that of 5a showed a lack of the C=S group.
The 1H-NMR spectrum of 4a recorded the resonated singlet signals at δ 1.05, 1.12 for 6H (18CH3-19CH3), triplet signal for β-proton at δ 3.6 for methine H of C3, singlet signal at δ 5.36 for H of the OH group at C3, and 13.79 and 10.22 ppm for 2NH of 1,2,4-triazine, in addition to δ at 8.16 (1H, NH bonded) and 7.97–7.16 (aromatic CH). The 13C-NMR spectrum of 4a gives a signal at δ 70.99 (C-3), 142.53 (C-4), 39.11 (C-10), 42.51 (C-13), 31.99 (C-17), 143.88 (C-18), 19.51 (C-19), 173.43 (C=O), and 201.55 (C=S), in addition to signal s at δ 128–131 (aromatic carbons) and at δ 141.15 ppm (C-F). Finally, a mass spectral study of compound 4a represented that the molecular ion has the intensity below 5%; however, fragments of the (M+-1) and (M+-2) peaks with a base peak at m/z 121 attributed to stable moiety (Figure 2). The 19FNMR spectrum of compound 4 showed δ at −124 ppm, with a coupling constant at 2JF-H = 8.6 Hz and 3JF-H = 6.3 Hz.
2.2. Biological Evaluation
The four synthesized a-amino acids, 4a, 4b, 5a, and 5b, were preliminarily tested toward some fungi such as Aspergillus flavus, Aspergillus fumigains, Aspergillus Niger, Aspergillus nidulaus, and Aspergillus terreus for their amylolytic activity according to the classical methods [27–30]. Using DMF as a solvent, about 0.01 g of each compound was dissolved in the presence of phosphate buffer saline (PBS) at PH 6.5 for 30 minutes. The amylase activity was assayed at the adjusted pH and temperature 38°C, according to the standard method. [28, 29] The activity was estimated (in mg reducing sugar out of reaction mixture), and all the results obtained are reported in Table 1.
From these data, we can conclude that most of the tested compounds exhibited a good to moderate inhibition and/or acceleration activity. In specific, compound 4a, which contains both F and S atoms, showed a stronger effect towards all the tested fungi than other systems, and markably, fluorinated compounds 4 and 5 showed a complete control on the tested fungi as A. flavus, A. nedulaus, and A. niger. Finally, we report that the active new fluorinated α-amino acids are a promising candidate to be used as enzymatic catalysts in the vital biosynthesis process due to their activity.
All reagents and solvents were purchased from commercial sources and used without further purification, unless otherwise stated. Melting points were measured using a Stuart SMB3 melting point apparatus and are uncorrected. IR spectra were recorded on a PerkinElmer Lambda 550 S spectrometer (KBr/cm−1). All the chemical shifts, 1HNMR and 13CNMR, were recorded relative to TMS and recorded on a varian-700 spectrometer (DMSO, d6 ppm).
6-(2-Amino-5-fluorophenyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (1a) and 6-(2’-aminophenyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (1b): compounds 1a and 1b were prepared according to the reported method [5, 9].
17-Imino-(2-(5′-oxo-3′-thioxo-1′,2′,4′-triazin-6′-yl)-1-aryl) dehydroepiandrosterone-3β-ols (2a and 2b): equimolar mixture of 1a and/or 1b and steroid (B), THF (50 ml), and DMF (10 ml) was refluxed for 2 h, cooled, and then, evaporated. The produced solid was crystallized from dioxane to give 2a and/or 2b, respectively.
2a: yellow crystals: yield 80 %, m.p. 212°C–219°C. Analytical data found C, 65.93; H, 6.31; F, 3.59; N, 10.79; and S; 5.99 %, calculated for C28H33N4FSO2 (508), C, 66.14; H, 6.49; F, 3.74; N, 11.02; and S, 6.29 %. IR (cm−1) = 3550, 3200–3180 (2NH-OH), 3080 (aromatic CH), 2980 (aliphatic CH), 1680 (C=O) 1625 (C=N), 1580 (C=N), 1490, 1440 (defer. CH, CH2, CH3), 1250 (C-F), and 1190 (C-S). 1HNMR (δ ppm): 13.81, 13.15(each s, 2H, NH, NH-1,2,4–triazine), 7.98 (1H, dd, J = 1.7, 0.5 Hz, H-24), 7.45 (1H, dd, J = 8.5, 1.7 Hz, H-26), 7.43 (dd, J = 8.5, 0.5 Hz, H-27), 5.35 (s, 1H, OH steroid), 0.94 (3H, s, CH3 of steroid), 1.21 (3H, s, CH3 of steroid), 2.44, 2.42, 2.28, 2.22, 2.17, 2.10, 2.09, 2.07, 2.05, 1.85, 1.83, 1.68, 1.64, 1.55, 1.50, 1.47, 1.29, 1.27, 1.07, 1.03, and 1.00 (CH and CH2 of steroid). 13CNMR (δ ppm): 168 (C=O), 160 (C=S), 158.68, 148.09 (C=N), 140.92 (C-F), 140.09 (C-N), 131.21, 128.66, 120.88 (aromatic carbons), 37.2(C1), 30.70 (C2), 70.99 (C3), 42.53 (C4), 141.37 (C5), 122.55 (C6), 31.43 (C7), 31.42 (C8), 51.68 (C9), 39.11 (C10), 21.81 (C11), 39.87 (C12), 42.51 (C13), 57.53 (C14), 30.70 (C15), 30.10 (C16), 32.99 (C17), 42.14 (C18), and 19.40 (C19). 19FNMR (δ ppm): −124. The coupling constant was JF-H (2Jo = 8.6 Hz, 2Jo = 8.6 Hz, and 3Jm = 6.3 Hz).
2b: deep yellow crystals, yield 70%; m.p. 150°C–152°C. Analytical data found C, 67.98; H, 7.11, N: 11.18, and S, 6.33%, calculated for C28H34N4SO2 (490); C, 68.29; H, 7.31; N, 11.38; and S, 6.50 %. IR (cm−1): 3550 (OH), 3200, 3100 (NH), 1678 (C=O), 1620 (C=N), C 1490, 1448 (deformation CH2), and 1187 (C=S).
17-Cyano-17-(heteroarylamino)-dehydroepiandrosterone-3β-ol/17-cyano-17-(2-(3-thioxo-5-oxo-1,2,4-triazino(2H,2H)-6-yl)-1-arylamino)-epiandrosterone-3-β-ols (3a and 3b): a mixture of 2a and/or 2b (0.001 mol) and NaCN (0.001 mol, in 10 ml) with acetic acid-ethanol (1 : 1, 50 ml) was refluxed for 2 h, cooled, and was then, poured onto ice. The solid was filtered and crystallized from ethanol to give 3a and 3b, respectively.
3a: deep yellow crystals yield 60% m.p. 213°C–215°C. Analytical data found C, 64.84; H, 6.29; F, 3.35; N, 12.88; and S, 5.89%, calculated for C29H34N5FSO2 (535); C, 65.04; H, 6.35; F, 3.55; N, 13.08; and S, 5.98%. IR (cm−1) = 3500–3300 (b, NH-OH), 2220 (C≡N), 1680 (C=O), 1480, 1445 (deformation. CH2 and CH3), 1250 (C-F), and 1198 (C-S). 1HNMR (δ ppm): 0.89 (3H, s, CH3 of steroid)), 1.21 (3H, s, CH3 of steroid), 10.80, 10.76(each s, NH, NH), 8.0(s, 1H, OH of 1,2,4-triazine), 5.19 (1H, dd, J = 7.7, 7.1 Hz, =C-H steroid alkene), 7.08 (1H, dd, J = 8.7, 1.6 Hz,, H-24/26/27), 7.39 (1H, dd, J = 1.6, 0.5 Hz,, H-24/26/27), 7.56 (1H, dd, J = 8.7, 0.5 Hz,, H-24/26/27), 5.34(s, 1H, OH of steroid) 3.42, 3.41, 2.58, 2.42, 2.26, 2.25, 2.09, 2.07, 1.98, 1.84, 1.80, 1.78, 1.65, 1.55, 1.48, 1.46, 1.27, 1.25, 1.06, and 1.02 (CH & CH2 steroid). 13CNMR (δ ppm): 172 (C=S), 165 (C-F), 158 (C=O), 153 (C=N), 140.09 (C-N), 115.5 (C≡N), 131.21–118.66 (aromatic carbons), 120.00, 116.50 (alkene of steroid), 37.2, 30.70, 70.99, 42.53, 31.43, 31.42, 51.68, 39.11, 21.81, 39.87, 42.51, 57.53, 30.70, 30.10, 32.99, 42.14, and 19.40 (steroids C). 19FNMR (δ ppm): −124. The coupling constant was JF-H(2Jo = 8.8 Hz, 2Jo = 8.9 Hz, and 3Jm = 6.3 Hz).
3b: orange crystals yield 70%; m.p. >290°C. Analytical data found C, 66.95; H, 6.85; N, 13.25; and S, 5.90%. Calculated for C29H35N5SO2 (517); C, 67.05; H, 7.12; N, 13.48; and S, 6.16%. IR (cm−1): 3550–3350 (b, NH, OH), 2230 (C≡N), 1677 (C=O), 1480, 1440 (deformation CH2), and 1190 (C=S).
17-(6-(2–Arylamino)-3-thioxo–5–oxo-1,2,4-triazino-6-yl)-17-carboxy-17-heteroaryl amino or (2-(3-thioxo-5-oxo-1,2,4-triazin(2H, 4H)-6-yl-4-fluoro-1-amino)-dehydroepiandrosterone-3-β-ols (4a and 4b): compounds 3a and/or 3b (0.5 gm) diluted HCl (10%, 50 ml) were in reflux for 2 h, cooled, and poured onto ice. The solid, thus, obtained was filtered off and crystallized from ethanol to give 4a and/or 4b, respectively.
4a: orange-yellow crystals, yield 65 %: m.p. 225–227°C. Analytical data found C, 62.66; H, 6.05; F, 3.21; N, 10.00; and S, 5.55%, calculated for C29H35N4FSO4 (554); C, 62.81; H, 6.31; F, 3.42; N, 10.10; and S, 5.77%. IR (cm−1) ν = 3500, 3300 and 3210 (OH, NH, NH), 1710, 1680 (2C=O), 1235 (C-F), and 1195 (C-S). 1HNMR((δ ppm): 0.89 (3H, s, CH3), 1.21 (3H, s, CH3), 13.79, 10.22(each s, 2H, NH, NH), 8.16(s, 1 H, NH bounded), 1.39–1.85 (12H, 1.63 (m), steroids), 5.19 (1H, dd, J = 7.7, 7.1 Hz steroid alkene), 4.28 (dddd, J = 7.6, 3.2, 2.6, 2.3 Hz, H3), 7.07 (1H, dd, J = 8.8, 1.6 Hz, H24/26/27), 7.32 (1H, dd, J = 8.8, 0.5 Hz, H24/26/27), 7.39 (1H, dd, J = 1.6, 0.5 Hz, H24/26/27), 5.36 (s, 1H, OH of steroid), 6.5 (s, 1H, OH of acid), 1.67, 1.58, 1.46, 1.49, 1.75, 1.58, and 1.53 Hz (CH and CH2 of steroid). 13CNMR (δppm): 172 (C=S), 165 (C-F), 158 (C=O), 151.55 (C=O), 131.21–120.88 (aromatic carbons), 37.20 (C1), 30.70 (C2), 70.99 (C3), 42.53 (C4), 141.37 (C5), 122.55 (C6), 31.43 (C7), 31.42 (C8), 51.68 (C9), 39.11 (C10), 21.81 (C11), 39.87 (C12), 42.51 (C13), 57.53 (C14), 30.70 (C15), 30.70 (C16), 32.99 (C17), 42.14 (C18), 19.42 (C19), M/Z (Int.%) 554 (0.0), 275 (5), 235 (5), 197 (100), 121 (100), 189 (25), 99 (10), 78 (40), and 56 (14). 19FNMR (δ ppm): −124. The coupling constant was JF-H(2Jo = 8.8 Hz, 2Jo = 8.9 Hz, and 3Jm = 6.3 Hz).
t4b: orange crystals, yield 78%, m.p. 270–272°C. Analysis data found C, 64.53; H, 6.88; N, 10.25; S, 5.7 %, calculated for C29H36N4SO4 (536); C, 64.68; H, 7.06; N, 10.40; and S, 5.94%. IR (cm−1): ν = 3500 (OH), 3320, 3180 (NH), 1690, 1670 (C=O), 1480, 1440 (deformation CH2), and 1190 (C=S).
17-(6-(2’-Arylamino)-3(4’-fluoro-phenylamino)-1,2,4-triazin-5(4H)ones)-17-carboxy-dehydroeniandrosterone-3-β-ols(5a and 5b): equimolar of 4a and/or 4b and 4-fluoroailine with ethanol(100 ml) was refluxed for 4 h, cooled, and then, poured onto ice. The solid produced filtered off and crystallized from ethanol to give 5a and 5b, respectively.
5a: orange-yellow crystals, yield 65%; m.p. 220–221°C. Analytical data found C, 66.3; H, 5.89; F, 5.89; and N, 10.98%, calculated for C35H41N5F2O4(633); C, 66.56; H, 6.18; F, 6.02; N, and 11.09%. IR(cm−1): ν = 3480, 3300, 3210, 3190 (3NH, OH), 2980, 2880(aliphatic CH), 1610(C=N), 1250(C-F), and 650(C-F). 1HNMR (δppm): 13.73, 12.96, 10.23 (each s, 3NH), 7.96, 7.95, 7.35, 7.17, 7.16 6.87 (2H, ddd, J = 8.5, 1.6, 0.5 Hz), 6.94–7.04 (3H, 6.97 (dd, J = 8.8, 1.6 Hz), 7.01 (ddd, J = 8.5, 1.9, 0.5 Hz), 7.27–7.34 (2H, 7.33 (dd, J = 1.6, 0.5 Hz), 7.30 (dd, J = 8.8, 0.5 Hz) (aromatic CH), 5.36(s, 1H, OH of steroid), 6.20 (s, 1H, OH of acid), 4.19 (1H, dd, J = 7.7, 7.1 Hz, alkene steroid), 0.89 (3H, s, CH3 steroid), 1.21 (3H, s, CH3 steroid), 1.39–1.85 (12H, 1.63 (dddd, J = 13.1, 7.6, 2.9, 2.0 Hz), 3.49, 2.56–2.68, 2.59, 2.30, 2.11, 2.09, 2.08, and 1.94–1.88(CH & CH2 of steroid). 13CNMR (δ ppm): 170.43 (C-F), 165.5 (C-F), 162.1 (C=O), 160.0 (C=O), 141.53 (C=N), 140.15 (C-N), 130–123(aromatic carbons), 119, 116 (C5-C6, -1,2,4-triazine), 37.11 (C1), 30.70 (C2), 71.01 (C3), 42.6 (C4), 141.3 (C5), 122.6 (C6), 32.0 (C7), 31.5 (C8), 51.7 (C9), 39.0 (C10), 21.9 (C11), 21.9 (C11), 39.9 (C12), 45.6 (C13), 57.63 (C14), 30.70 (C15), 30.72 (C16), 32.98 (C17), 42.11 (C18), and 19.40 (C19). 19FNMR((δ ppm): −124. The coupling constants are JF-H(2Jo = 8.8 Hz, 2Jo = 8.9 Hz, and 3Jm = 6.3 Hz) and JF-H(2Jo = 8.9 Hz and 3Jm = 5.6 Hz).
5b: orange crystals, yield 70%; m.p. 245°C–247°C. Analytical data found C, 67.99; H, 6.66; F, 2.91; and N, 11.19%, calculated for C35H42FN5O4 (615); C, 68.29; H, 6.82; F, 3.08; and N, 11.38%. IR (cm−1): 3500 (OH), 3300, 3200, 3180 (NH), 2980, 2880 (aliphatic CH), 1610 (C=N), 1230 (C-F), and 680 (C-F).
In a search for new α-amino acids, the present work reports a simple route to synthesize fluorinated and nonfluorinated α-amino acids derived from the corresponding 1,2,4-triazinone bearing an amino-group and a steroidal component. The new fluorinated synthetic skeletons exhibit an amylolytic activity greater than nonfluorinated systems against some fungi.
The data used to support the study can be made available upon request to the corresponding author.
Conflicts of Interest
The authors have no conflicts of interest to declare.
Figure I: a graphical abstract of all newly synthesized compounds 4a, 4b, 5a, and 5b. (Supplementary Materials)
D. S. Page, Principles of Biological Chemistry, Willard Grant Press, Boston, MA, USA, 2nd edition, 1981.
R. M. Abdel-Rahman, M. S. T. Makki, and A. N. Al-Romaizan, “Synthesis of novel fluorine substituted isolated and fused heterobicyclic nitrogen systems bearing 6-(2'-phosphorylanilido)-1,2,4-triazin-5-one moiety as potential inhibitor towards HIV-1 activity,” International Journal of Organic Chemistry, vol. 4, no. 4, pp. 247–268, 2014.View at: Publisher Site | Google Scholar
A. N. Al-Romaizan, M. S. T. Makki, and R. M. Abdel-Rahman, “Synthesis of new fluorine/phosphorus substituted 6-(2'-amino phenyl)-3-thioxo-1,2,4-triazin-5(2H, 4H)one and their related alkylated systems as molluscicidal agent as against the snails responsible for bilharziasis diseases,” International Journal of Organic Chemistry, vol. 4, no. 2, pp. 154–168, 2014.View at: Publisher Site | Google Scholar
R. M. Abdel-Rahman, “Role of uncondensed 1,2,4-triazine compounds and related heterobicyclic systems as therapeutic agents,” Die Pharmazie, vol. 56, no. 1, pp. 18–22, 2001.View at: Google Scholar
R. Abdel-Rahman, “Synthesis of some new heterobicyclic nitrogen systems bearing the 1,2,4-triazine moiety as anti-HIV and anti-cancer drugs, part II,” Die Pharmazie, vol. 54, no. 9, pp. 791–863, 1999.View at: Google Scholar
R. M. Abdel-Rahman, “Synthesis of some new fluorine bearing tri-substituted 3-thioxo-1,2,4-triazine-5- one as potential anti cencer agent,” Farmaco (Societa Chimica Italiana: 1989), vol. 47, no. 3, pp. 319–326, 1992.View at: Google Scholar
R. M. Abdel-Rahman, “Synthesis and anti human immune virus activity of some new fluorine containing substituted -3-thioxo-1,2,4-triazin-5-ones,” Farmaco (Societa Chimica Italiana: 1989), vol. 46, no. 2, pp. 379–389, 1991.View at: Google Scholar
M. Spinella, R. De Marco, E. L. Belsito, A. Leggio, and A. Liguori, “The dimethylsulfoxonium methylide as unique reagent for the simultaneous deprotection of amino and carboxyl function of N-Fmoc-α-amino acid and N-Fmoc-peptide esters,” Tetrahedron, vol. 69, no. 8, pp. 2010–2016, 2013.View at: Publisher Site | Google Scholar
A. Srivastava, S. Singh, and S. Samanta, “(±)-CSA catalyzed Friedel-Crafts alkylation of indoles with 3-ethoxycarbonyl-3-hydoxyisoindolin-1-one: an easy access of 3-ethoxycarbonyl-3-indolylisoindolin-1-ones bearing a quaternary α-amino acid moiety,” Tetrahedron Letters, vol. 54, no. 11, pp. 1444–1448, 2013.View at: Publisher Site | Google Scholar
J. Taira, Y. Kida, H. Yamaguchi, K. Kuwano, Y. Higashimoto, and H. Kodama, “Modifications on amphiphilicity and cationicity of unnatural amino acid containing peptides for the improvement of antimicrobial activity against pathogenic bacteria,” Journal of Peptide Science, vol. 16, no. 11, pp. 607–612, 2010.View at: Publisher Site | Google Scholar
W. Horne, S. Gellman, and L. Johnson, “Methods of biologically active α-β peptides,” 2010, US Patent 20090578993.View at: Google Scholar
N. Zhou, H.-J. Fu, D. Rong, M.-S. Cheng, and K.-L. Liu, “Design, synthesis of unnatural amino acids with chelating functional groups and their application in bio-active peptide,” Chemical Research in Chinese Universities, vol. 28, pp. 668–671, 2007.View at: Google Scholar
M. S. Sigman and E. N. Jacobsen, “Enantioselective addition of hydrogen cyanide to imines catalyzed by a chiral (salen) Al(III) complex,” Journal of the American Chemical Society, vol. 120, no. 21, pp. 5315-5316, 1998.View at: Google Scholar
R. M. Abdel-Rahman and M. S. Abdel-Malik, “Synthesis of some new 3,6-diheteroarryl-1,2,4-triazine-5-one and their effect on amylolytic activity of some fungi,” Pakistan Journal of Scientific and Industrial Research, vol. 33, pp. 142–147, 1990.View at: Google Scholar
J. C. Gould and J. M. Bowie, “The determination of bacterial sensitivity to antibiotics,” Edinburgh Medical Journal, vol. 59, no. 4, pp. 178–199, 1952.View at: Google Scholar
A. A. Singh, R. Dhakarey, and G. Saxena, “Magnetic and spectral behaviour of semicarbazone derivatives of manganese (II), copper (II), iron (III) and chromium (III) and their antimicrobial screening,” Journal of the Brazilian Chemical Society, vol. 73, pp. 339–342, 1996.View at: Google Scholar