Synthesis Of 4-Ayridone Lab Report

Pat 1
Synthesis of 4-aryl-6-indolylpyridine-2-carbonitriles and evaluation of their biological activity
Synthesis of 6-indolyl-4-aryl-3-cyanopyridine-2-one (1a-e)
Employing the MCRs approach and microwave irradiation, an equimolar amounts of aromatic aldehyde, 3-acetylindole, ethyl cyanoacetate were reacted with excess of ammonium acetate in one-pot, reaction, under the microwave irradiation, afforded a novel series of 4-aryl-6-indolyl-nicotinonitrile-2-one derivatives 1a-e. Ethylene glycol was used as a solvent and piperidine was employed as a catalyst during the microwave syntheses at a power of 250 W and 150 oC for a (15-20) min, scheme 1. All compounds were characterized by IR, mass, and NMR spectroscopy. The rate of reactions, and
product yield in the MWI method were compared to the traditional thermal heating method. In case of microwave method, the reaction time was shortened from several hours (14-18h) as in the thermal heating method to several minutes (15-20 min), with improvement in both the final product purity, and the overall yield (77-87%). The results for each entry are summarized in table 1.
Product R1 Time Yieldc (%) mp (C) MW, (min)
250W, 150C Th,(h)
150 C MW Th
1a 2-C4H3S 20 17 77 44 >300
1b 4-OCH3C6H4 20 18 79 45 >300
1c 4-FC6H4 15 10 87 56 >300
1d 4-ClC6H4 17 15 82 63 >300
1e 4-BrC6H4 17 14 83 61 >300
Table 1: The reaction time, the yield, and melting point for each product
The mechanism of nicotinonitrile syntheses proceeds through Claisen-Schimdt reaction. Where α,β-unsaturated ketones intermediate was formed between the α-ketomethylene group of the ketone derivative, and the aromatic aldehyde. A base catalyst such as; (10%) NaOH, triethylamine, or piperidine was used. This was followed by condensation with nitrile containing active methylene compounds e.g. ethyl cyanoacetate, cyanoacetamide, cyanothioacetamide, or malononitrile, via Michael addition reaction in the presence of 10-200 fold molar ratio of ammonium acetate, which direct the reaction towards the formation of pyridine ring system instead of pyrane ring system. That was followed by aromatization under the same reaction conditions to afford the corresponding 4-substituted-2-oxo-1H-pyridine-3-carbonitrile derivatives128,129, Scheme 2. Taking compound 1b as an example, the IR spectrum (chart 5) displayed characteristic stretching vibrational bands at: 3448 cm-1 for (NH indolyl ring) group, 3262 cm-1 for (NH pyridone) group, 2210 cm-1 for (CN) group, and 1644 cm-1 for (NH-C=O amide) group.
Its 1H-NMR (DMSO-d6) δ ppm spectrum (chart 6) revealed the presence of these signals at δ 3.8 (s, 3H, OCH3), 6.57 (s, 1H, H-5 pyridone), 7.08 (d, J = 8.4 Hz, 2H, aromatic), 7.18 (m, 2H, indolyl), 7.49 (d, J = 8.5 Hz, 1H, indolyl), 7.69 (d, J = 8.4 Hz, 2H, aromatic), 7.86 (d, J = 8.5 Hz, 1H, indolyl), 8.27 (d, 1H, indolyl), 12.06 (s, 1H, NH pyridone, D2O exchangeable), 12.35 (1H, d, NH indole, D2O exchangeable). The 13C-NMR (DMSO-d6) δ ppm (chart 7) revealed the existence of the following signals δ 108.82 (CN), 113.28, 114.81, 117.96, 120.09, 121.99, 123.33, 124.56, 129.13, 129.71, 129.88, 130.32, 137.35, 137.51, 147.59, 159.9, 161.47 (aromatic carbons), 162.43 (CO amide). The mass spectrum (chart 8) supported the structure of compound 1b, where the mass spectrum for 1a with the molecular formula C21H15N3O3 the molecular ion peak [M‏+] exactly at (m/z) = 341.00,
(100%).
Structure reactivity relationship
The electronic nature of the substituent on the aromatic aldehyde affected the syntheses rate, and the product yield in both approaches, table 1. In the presence of a strong electron withdrawing group on the in para position of the aromatic aldehydes used (e.g. 4-fluorine, 4-chloro, 4-bromo), the higher pyridone yield was achieved in shorter reaction time compared to that one having 4-methoxy substituent (electron donating groups), or thiophenyl moiety under the same reaction conditions.
Chlorination of indolylpyridones 1a-e
The treatment of compounds 1a-e with the neat, anhydrous phosphoryl chloride for 18-24 h with thermal heating at 80 oC, resulted in the chlorination of the pyridone ring, as the 2-chloro-6-indolylnicotinonitrile derivatives (2a-e) were obtained in 95-98% yield, scheme 3. The structure of the new compounds was confirmed by FT-IR, mass, and NMR. For example, the 2-Chloro-6-indolylnicotinonitrile 2a, its IR spectrum (chart 21) showed an absorption band at 3305 cm-1 for (NH indolyl), and 2224 cm-1 (CN) while, the stretching absorption bands of amidic (CO) groups disappeared. Its 1H-NMR (CDCl3) δ ppm spectrum (chart 22) revealed the absence of pyridone NH signal at δ 11.62 ppm which supported the formation of compound 2. 13C-NMR (DMSO-d6) δ ppm (chart 23), also revealed the absence of the amidic carbonyl signal at δ 162.84 ppm.
Additionally, the molecular ion peak in the mass spectrum for compound 2a (chart 24) which has the molecular formula C18H10ClN3S appeared at m/z = 335.00 [M‏+, 100%] in addition to the peak at m/z = 298 (20%) which was assigned to the cleavage of HCl fragment [M+ - HCl].
Substitution of the chlorine moiety by ethylene-1,2-diamine
The new derivatives of 2-aminoethylenamino-6-indolyl nicotinonitrile (3a-e) were prepared by the reaction of ethylene-1,2-diamine, with 2-chloro-6-indolylnicotinonitriles (2a-e), through the nucleophilic substitution of the chlorine moiety, using of trimethylamine as a base catalyst. The reaction was carried out under refluxing condition for 36-48h, in anhydrous ethanol, scheme 4. FT-IR, NMR, and mass spectroscopic techniques were used to elucidate the chemical structures of the new compounds. As a representative example, The IR spectrum for compound 3b (chart 45) revealed the presence of stretching vibrational bands at 2201 cm-1 for the (CN) group; also it displayed a broad absorption band at 3366 cm-1 for the (NH & NH2) groups. Its 1H-NMR spectrum (DMSO-d6) δ ppm (chart 46), displayed the following signals at δ 2.86 (t, J = 6.1 Hz, 2H, CH2); 3.31 (broad, 1H, NH, D2O exchangeable), 3.66 (m, 2H, CH2), 3.82 (s, 3H, OCH3), 4.09 (broad, 1H, NH), 7.07 (d, J = 8.4 Hz, 2H, aromatic), 7.14 (t, J = 77 Hz, 2H, indolyl), 7.33 (d, J = 6.1 Hz, 1H, indolyl), 7.41 (d, J = 6.1 Hz,1H, indoly), 7.60 (d, J = 8.4 Hz, 2H, aromatic), 8.31 (s, 1H, H-5 pyridine), 8.47 (d, 1H, indolyl), 11.73 (d, 1H, NH indolyl, D2O exchangeable). Additionally, the 13C-NMR (DMSO-d6) δ ppm (chart 47) showed signals at δ 40.16 (CH2), 40.33 (CH2), 55.87 (OCH3), 108.11 (CN); 112.51, 114.63, 121.17, 122.39, 122.55, 125.97, 128.86, 129.39, 130.24, 137.62, 153.44, 158.18, 158.44, 160.16, 160.70 (aromatic carbons). The mass spectrum for C23H21N5O (m/z, %) (chart 48) showed the molecular ion peak [M+] at m/z 383.00 (10), in addition to the molecular ion peak at m/z = 353 (100%) corresponds to the fragment [M+-OCH3].

Evaluation of the anti-proliferative activity The prepared novel indolylincotinonitrile derivatives, were evaluated for their anti-proliferative activities against three different cancer cell lines namely; human ovarian adenocarcinoma (SK-OV-3), breast adenocarcinoma (MCF-7), and cervix adenocarcinoma (HeLa) cells at concentration (50 μM), after 72 h incubation. DMSO (3% in H2O) was used as a negative control, and doxorubicin (Dox, 10 μM) was used as a positive control for the assay. The results suggested that, compounds (1a, 1c, 1d, 1e, 1a, 1c, and 2d) did not exhibit a significant anti-proliferative activity against the three cancer cell lines, figure 1. Meanwhile, compounds (1b), (2b), and (3a-e) exhibited moderate to strong anti-proliferative potency. However, compounds (3b, 3d, and 3e) showed comparable potency with that of Dox in HeLa cells and significantly higher activity in SK-OV-3 and MCF-7 cells than Dox. For example, compounds (3b, 3d, and 3e) inhibited the growth of HeLa, SK-OV, and MCF-7 cancer cell lines by 62-67%, 85-88%, and 84-87% respectively. Moreover, compounds (3b, 3d, and 3e) were more effective on SK-OV-3 and MCF-7 cells compared to HeLa cells, which means that the activity of those three derivatives is cell-specific.

Structure activity relationship
The results of the anti-proliferative evaluation assay revealed that, the ethylenediamine moiety plays a significant role in enhancing the cytotoxic activity of the compounds against the three cancer cell lines. However, compound (3c) which has a p-fluorophenyl substituent at C4 did not show similar potency in comparison to the other indolylnicotinonitriles in this series, suggesting that the presence of a small sized, strong electron withdrawing group such as fluorine moiety is not productive. On the other hand, the presence of a thiophenyl ring (3a), or methoxy group as an electron donating group (3b) at C4 of the nicotinonitrile ring enhanced the cytotoxic activity. Thus, the electronic effect of the substituent on the indolylnicotinonitrile ring seems to have a direct effect on their cytotoxic activity. Figure 1. Anti-proliferative activity of compounds (1a-e), (2a-e), and (3a-e) against cancer cell lines (Hela, SK-OV-3, and MCF-7).
Based on the results from the anti-proliferation screening, compounds (3a, 3b, 3d, and 3e) were selected to determine their IC50 "the concentration that causes 50% inhibition of cancer cell growth" figure 2, and table 2. Figure 2. IC50 determination for compounds 3a, 3b, 3d, and 3e
Entry HeLaa SK-OV-3 MCF-7
3a 13.4 4.7 4.1
3b 7.2 6.5 8.1
3d 6.8 5.9 7.1
3e 8.8 5.8 6.8
Dox 0.15130a 3.2 7.5130b
Table 2.
IC50 values of four selected compounds
Antimicrobial activity of compounds (3b, d and e)
Compounds (3b, d, and e), were investigated for their antimicrobial activity at concentration (2 mg/ mL) using DMSO (50% in water) as negative control by well diffusion method, against Bacillus subtilis, Staphylococcus aureus (gram positive), Escherichia coli (gram negative) and Candida (yeast). Each well in the agar plate was seeded by (100 µL) from each tested compound or (50% DMSO). The plates were incubated at 37°C for 24 hours. the inhibition zone around each well in mm were measured, table 3. strain 3b 3d 3e
Bacillus subtilis 2 4.3 3.3
Staphylococcus aureus - 3.67 3.67
Escherichia coli - - -
Candida 1.33 3.67 2.6
Table 3. The antimicrobial activity for compounds 3b, d, and e
As we can see compound 3b exhibited minor activity against the Bacillus subtilis and Staphylococcus (g. positive) aureus and Candida, meanwhile the activity of compound 3d and 3e showed mild activity. None of the three tested compounds showed any activity against the gram negative bacteria E. coli. Meaning that, their reactivity are selective to gram positive bacteria, and
yeast.
Part 2