Synthesis and antimicrobial evaluation of new 1,4-dihydro-4-pyrazolylpyridines and 4-pyrazolylpyridines.

BACKGROUND: Dialkyl 1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylates (1,4-DHP) have now been recognized as vital drugs. Some of these derivatives such as amlodipine, felodipine, isradipine, etc. have been commercialized. In view of wide range of biological properties associated with 1,4-DHP and owing to the biological importance of the oxidation step of 1,4-DHP, we carried out the synthesis and antimicrobial evaluation of new diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) and diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g).
RESULTS: Synthesis of a series of new diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) has been accomplished by multicomponent cyclocondensation reaction of ethyl acetoacetate, 3-aryl-1-phenyl pyrazole-4-carboxaldehyde (1a-g) and ammonium acetate. The dihydropyridines 2a-g were smoothly converted to new diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g) using HTIB ([Hydroxy (tosyloxy)iodo]benzene, Koser's reagent) as the oxidizing agent. The antimicrobial studies of the title compounds, 2a-g &3a-g, are also described.

Background

Dialkyl 1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylates (1,4-DHP; Figure 1) have now been recognized as vital drugs. Some of these derivatives, such as amlodipine, felodipine, isradipine, etc. have been commercialized, and it has been proven that their therapeutic success is related to their efficacy to bind to calcium channels and consequently to decrease the passage of the transmembrane calcium current [1-3]. Further, cerebrocrast, a dihydropyridine derivative, has been introduced as a neuroprotective agent [4]. Together with calcium channel blocker and neuroprotective activity, a number of dihydropyridine derivatives have been found as vasodilators, antihypertensive, bronchodilators, antiatherosclerotic, hepatoprotective, antitumour, antimutagenic, geroprotective, antidiabetic and antiplatelet aggregation agents [5-9]. In a recent article, 4-[5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl]-dihydropyridines have been shown to possess significant antimicrobial activity [10].

Figure 1

1,4-DHP.

In addition to above, aromatization of 1,4-DHP has also attracted considerable attention in recent years as Böcker has demonstrated that metabolism of the above drugs involves a cytochrome P-450 catalysed oxidation in the liver [11].

In view of wide range of biological properties associated with 1,4-DHP and the biological importance of the oxidation step of 1,4-DHP, we carried out the synthesis and antimicrobial evaluation of new diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) and diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g).

Results and discussion

Chemistry

The synthetic scheme used for the synthesis of diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g) is outlined in Scheme 1. Synthesis of the title compounds 2a-g was accomplished by multicomponent cyclocondensation reaction of ethyl acetoacetate, 3-aryl-1-phenyl-pyrazole-carboxaldehyde (1a-g) and ammonium acetate in ethanol. The purity of the compounds was checked by TLC and elemental analysis. Spectral data (IR, 1H NMR (see additional files 1, 2, 3, 4 and 5, mass) of the newly synthesized compounds 2a-g were in full agreement with their proposed structures. The IR spectra of compounds 2a-g exhibited characteristic peak at approximately 1697 cm-1 because of the presence of ester group (-COOEt), and peak due to -N-H stretch appeared in the region 3300-3317 cm-1. In 1H NMR of compounds 2a-g, the protons of C4-H and -NH of the dihydropyridine ring resonate between δ 5 and 6 ppm.

Scheme 1

Synthesis of 1,4-DHP (2) and aromatization of 2 to 3 using HTIB.

Hypervalent iodine (III) and iodine (V) reagents have been used as green-oxidants for a variety of substrates [12-17]. Amongst the various reagents used, HTIB has been reported to serve as a mild, fast and efficient oxidant for the aromatization of Hantzsch 1,4-dihydropyridines to pyridines [18].

Thus, diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g) were further oxidized by treating with HTIB (Koser's reagent) in dichloromethane (CH2Cl2) at room temperature to afford new diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (3a-g) in good-to-excellent yields (Scheme 1). All the compounds 3a-g were unambiguously characterized on the basis of their spectral (IR, 1H NMR (see additional files 6, 7, 8, 9, 10, 11 and 12) and mass) and elemental data.

A plausible mechanism for the oxidation of dihydropyridines 2 to 3 is outlined in Scheme 2. The probable mechanism might involve the attack by N-H on PhI(OH)OTs, leading to the formation of intermediate 4. The intermediate 4 finally loses a molecule of iodobenzene (PhI) to give 3.

Scheme 2

Proposed mechanism for the oxidation of 2 to 3.

Pharmacology

All the synthesized compounds, 2a-g and 3a-g, were evaluated in vitro for their antibacterial activity against two gram-positive bacterial strains, Staphylococcus aureus &Bacillus subtilis and two gram-negative bacteria, namely, Escherichia coli and Pseudomonas aeruginosa and their activities were compared with a well-known commercial antibiotic, ciprofloxacin. In addition, the synthesized compounds were also evaluated for their antifungal activity against Aspergillus niger &Aspergillus flavus and their antifungal potential was compared to reference drug, fluconazole. Compounds possessed variable antibacterial activities against Gram-positive bacteria, S. aureus, B. subtilis. However, the compounds in this series were not effective against any Gram-negative bacteria, neither against E. coli nor against P. aeruginosa. Results of antibacterial evaluation are summarized in Table 1.

Table 1

Antibacterial activity of chemical compounds through agar well diffusion method

Compound	Diameter of growth of inhibition zone (mm)a
	S. aureus	Bacillus Subtilis	E. coli	P. aeruginosa
2a	15.6	16.3	-	-
2b	18.6	15.6	-	-
2c	16.3	15.6	-	-
2d	17.6	16.3	-	-
2e	16	15.3	-	-
2f	15.6	14	-	-
2g	15.3	16.6	-	-
3a	20	19.3	-	-
3b	15	15.6	-	-
3c	15.3	16.6	-	-
3d	16.6	14.6	-	-
3e	16.6	18.3	-	-
3f	15.3	16.6	-	-
3g	16.3	18.6	-	-
Ciprofloxacin	27.6	26.3	25.0	25.3

-, No activity

aValues, including diameter of the well (8 mm), are means of three replicates

Compounds 2a-g and 3a-g showed zones of inhibition ranging between 14 and 20 mm. On the basis of the zones of inhibition produced against the test bacteria, compounds 2b and 3a were found to be most effective against S. aureus, showing the maximum zones of inhibition at 18 and 20 mm, respectively, and compounds 3a, 3e and 3g were found to be most effective against B. subtilis. The remaining compounds showed fair activity against gram-positive bacterial strains (Table 1). In the whole series, the MIC (minimum inhibitoty concentration) values of various tested chemical compounds ranged between 64 and 256 μg/mL against gram-positive bacteria. Compounds 2b and 3a displayed good antibacterial activity with the lowest MIC value, 64 μg/ml against S. aureus. Three compounds, 3a, 3e and 3g possessed antibacterial activity with MIC value of 64 μg/mL against B. subtilis (Table 2).

Table 2

MIC (in μg/mL) of compounds obtained using macrodilution method

Compound	S. aureus	Bacillus Subtilis	Compound	S. aureus	BacillusSubtilis
2a	128	128	3a	64	64
2b	64	128	3b	128	128
2c	128	128	3c	128	128
2d	128	128	3d	128	256
2e	128	128	3e	128	64
2f	128	256	3f	128	128
2g	128	128	3g	128	64
Ciprofloxacin	5	5

Amongst the synthesized compounds, six compounds 2a, 2d, 2g, 3a, 3c and 3d showed more than 50% mycelial growth inhibition against whereas compounds, 2a, 2e, 2f, 3a, 3d and 3f were found to be active against A. flavus (Table 3).

Table 3

Antifungal activity of chemical compounds through poisoned food method (mycelial growth inhibition) (%)

Compound	A. niger	A. flavus	Compound	A. niger	A. flavus
2a	51.1	58.8	3a	52.5	51.1
2b	50	44.4	3b	48.8	45.5
2c	48.8	50	3c	51.1	50
2d	52.5	48.8	3d	55.5	52.5
2e	45.5	51.1	3e	45.5	44.4
2f	47.7	52.5	3f	50	51.1
2g	51.1	48.8	3g	48.8	44.4
Fluconazole	81.1	77.7

From the overall result it is evident that compound 3a could be identified as the most biologically active member within this study with good antifungal and antibacterial profile.

Conclusions

A series of diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) and diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g) has been synthesized with the hope of discovering new structure leads. Compounds 2b and 3a were found to be most effective against S. aureus showing the maximum zones of inhibition of 18 and 20 mm, respectively, and compounds 3a, 3e and 3g were found to be most effective against B. subtilis. Moreover, six compounds 2a, 2d, 2g, 3a, 3c and 3d showed more than 50% mycelial growth inhibition against A. niger whereas compounds, 2a, 2e, 2f, 3a, 3d and 3f were found to be active against A. flavus; however, no compound was found superior over the reference drug.

Finally, compound 3a could be identified as the most biologically active member within this study with an interesting antibacterial and antifungal profile.

Experimental

Chemical synthesis

Melting points were taken in open capillaries and are uncorrected. IR spectra were recorded on Perkin-Elmer IR spectrophotometer. The 1H NMR spectra were recorded on Brucker 300 MHz instrument. The chemical shifts are expressed in ppm units downfield from an internal TMS standard. 3-Aryl-1-phenylpyrazole-4-carboxaldehydes (1a-h), needed for the present study, were synthesized by Vilsmeier-Haack reaction according to the literature procedure [19].

Synthesis of diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g)

General procedure: A mixture of appropriate 3-aryl-1-phenylpyrazole-4-carboxaldehyde (1, 10 mmol), ethyl acetoacetate (20 mmol) and ammonium acetate (22 mmol) in ethanol was allowed to reflux on water bath for 25-30 min. After completion of the reaction, the reaction mixture was cooled to room temperature to give pure diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g).

Characterization data of diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g)

2a: M.p.: 124°C; yield: 74%; IR (νmax, cm-1, KBr): 3323 (NH stretch), 1690 (-COOEt), 1207; 1H NMR (CDCl3, δ, ppm): 1.069-1.115 (t, 6H), 2.237 (s, 6H), 3.744-4.068 (m, 4 H), 5.318 (s, 1 H), 5.544 (s, 1 H), 7.221-7.424 (m, 4 H), 7.806 (s, 1 H), 7.681-7.868 (m, 6 H); mass: m/z 472.30 (M+ + 1, 100%).

Anal. Calcd for C28H29N3O4: C 71.33, H 6.15, N 8.91; found: C 71.34, H 6.18, N 8.94; C 71.33, H 6.15, N 8.91.

2b: M.p.: 189°C; yield: 70%; IR (νmax, cm-1, KBr): 3325 (NH stretch), 1697 (-OOEt), 1643, 1211; 1HNMR (CDCl3, δ, ppm): 1.032-1.087 (t, 6 H), 2.225 (s, 6 H), 2.401 (s, 3 H), 3.730-4.095 (m, 4 H), 5.306 (s, 1 H), 5.722 (bs, 1 H), 7.205-7.282 (m, 3 H), 7.381-7.450 (m, 2 H), 7.664-7.692 (m, 2 H), 7.733 (s, 1 H), 7.742-7.769 (d, 2 H, J = 8.1 Hz); mass: m/z 486.20 (M+ + 1, 100%).

Anal. Calcd for C29H31N3O4: C 71.75, H 6.39, N 8.66; found: C 71.71, H 6.42, N 8.66.

2c: M.p.: 139°C; yield: 78%; IR (νmax, cm-1, KBr): 3317 (NH stretch), 1697 (-COOEt), 1643, 1211; 1H NMR (CDCl3, δ, ppm): 1.079-1.127 (t, 6 H), 2.250 (s, 6 H), 3.866 (s, 3 H), 3.801-4.102 (m, 4 H), 5.288 (s, 1 H), 5.561 (s, 1 H), 6.962-6.991 (d, 2 H, J = 8.7 Hz), 7.209-7.440 (m, 3 H), 7.670-7.697 (d, 2 H, J = 8.7 Hz) 7.742 (s, 1 H), 7.785-7.814 (d, 2 H, J = 8.7 Hz); mass: m/z 502.32 (M+ + 1, 100%).

Anal. Calcd for C29H31N3O5: C 69.46, H 6.19, N 8.38; found: C 69.42, H 6.24, N 8.37.

2d: M.p.: 175°C; yield: 72%; IR (νmax, cm-1, KBr): 3333 (NH stretch), 1697 (-COOEt), 1643, 1211; 1H NMR (CDCl3, δ, ppm): 0.940-0.975 (t, 6 H), 2.521 (s, 6 H), 4.102-4.132 (m, 4 H), 5.175 (s, 1 H), 5.562 (s, 1 H), 6.962-6.991 (d, 2 H, J = 8.7 Hz), 7.281-7.513 (m, 5 H), 7.734 (d, 2 H, J = 7.5 Hz), 7.922 (s, 1 H); mass: m/z 490.26 (M+ + 1, 100%)

Anal. Calcd for C28H28N3O4F: C 68.71, H 5.73, N 8.58; found: C 68.72, H 5.75, N 8.56.

2e: M.p.: 185°C; Yield: 76%; IR (νmax, cm-1, KBr): 3317 (NH stretch), 1697 (-COOEt), 1636, 1211; 1H NMR (CDCl3, δ, ppm): 1.072-1.119 (t, 6 H), 2.280 (s, 6 H), 3.790-4.080 (m, 4 H), 5.285 (s, 1 H), 5.551 (s, 1 H), 7.235-7.454 (m, 5 H), 7.668-7.694 (d, 2 H) 7.814 (s, 1 H), 7.863-7.891 (d, 2 H, J = 8.4 Hz); mass: m/z 506.26, 508.24.

Anal. Calcd for C28H28N3O4Cl: C 66.47, H 5.54, N 8.31; found: C 66.47, H 5.55, N 8.31.

2f: M.p.: 174°C; yield: 72%; IR (νmax, cm-1, KBr): 3564 (NH stretch), 1728 (-COOEt), 1242; 1H NMR (CDCl3, δ, ppm): 1.072-1.119 (t, 6 H), 2.275 (s, 6 H), 3.764-4.104 (m, 4 H), 5.284 (s, 1 H), 5.581 (s, 1 H), 7.234-7.452 (m, 3 H), 7.561-7.588 (d, 2 H, J = 7.8 Hz), 7.665-7.691 (d, 2 H, J = 7.8 Hz) 7.753 (s, 1 H), 7.806-7.834 (d, 2 H, J = 8.4 Hz); mass: m/z 550.31, 552.31.

Anal. Calcd for C28H28N3O4Br: C 61.20, H 5.10, N 7.65; found: C 61.09, H 5.14, N 7.64.

2g: M.p.: 198°C; yield: 70%; IR (νmax, cm-1, KBr): 3302 (NH stretch), 1697 (-COOEt), 1636, 1211; 1H NMR (CDCl3, δ, ppm): 1.026-1.071 (t, 6 H), 2.325 (s, 6 H), 3.775-4.047 (m, 4 H), 5.335 (s, 1 H), 5.766 (s, 1 H), 7.282-7.473 (m, 4 H), 7.684-7.709 (d, 2 H, J = 7.5 Hz), 7.801 (s, 1 H), 8.254-8.344 (m, 3 H); mass: m/z 517.29 (M+ + 1, 100%).

Anal. Calcd for C28H28N4O6: C 65.11, H 5.42, N 10.85; found: C 65.13, H 5.47, N 10.83.

Synthesis of diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g)

General procedure: To a solution of appropriate 1,4-DHP (2, 10 mmol) in dichloromethane, was added HTIB (12 mmol) and the mixture was stirred at room temperature. The progress of the reaction was monitored by TLC. Reaction was completed in 4-5 min. After the completion of reaction, the reaction mixture was washed with aqueous NaHCO3 solution. Organic phase was then separated, dried and concentrated on water bath. Crude product, thus obtained, was purified by silica gel column chromatography using Pet ether/EtOAc (20:1) as eluent to afford pure diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g).

Characterization data of dimethyl 2,6-dimethyl-4-pyrazolylpyridine-3,5-dicarb oxylates (3a-g)

3a: M.p.: 111°C; yield: 68%; IR (νmax, cm-1, KBr): 1736, 1233; 1H NMR (CDCl3, δ, ppm): 0.911-0.997 (t, 6 H), 2.613 (s, 6 H), 3.910-4.07 (m, 4 H), 7.110-7.313 (m, 4 H), 7.817 (s, 1 H), 7.581-7.690 (m, 6 H); mass: m/z 470.20 (M+ + 1, 100%).

Anal. Calcd for C28H27N3O4: C 71.64, H 5.76, N 8.95; found: C 71.63, H 5.79, N 8.93.

3b: M.p.: 105°C; yield: 69%; IR (νmax, cm-1, KBr): 1720, 1234; 1H NMR (CDCl3, δ, ppm): 0.913-0.960 (t, 6 H), 2.611 (s, 6 H), 2.468 (s, 3H), 3.923-4.072 (q, 4 H), 6.839-6.868 (d, 2H, J=8.7 Hz), 7.280-7.501 (m, 5 H), 7.732-7.759 (d, 2 H, J = 8.7 Hz), 7.905 (s, 1 H); mass: m/z 484.40 (M+ + 1, 100%).

Anal. Calcd for C29H29N3O4: C 72.05, H 6.00, N 8.70; found: C 72.06, H 6.05, N 8.70.

3c: M.p.: 136°C; Yield- 72%; IR (νmax, cm-1, KBr): 1740, 1034; 1H NMR (CDCl3, δ, ppm): 0.913-0.998 (t, 6 H), 2.612 (s, 6 H), 3.808 (s, 3 H), 3.924-4.08 (q, 4 H), 6.835-6.864 (d, 2 H, J = 8.7 Hz), 7.311-7.501 (m, 5 H), 7.732-7.759 (d, 2 H, J = 8.7 Hz), 7.905 (s, 1 H); mass: m/z 500.29 (M+ + 1, 100%).

Anal. Calcd for C29H29N3O5: C 69.73, H 5.81, N 8.41; found: C 69.71, H 5.83, N 8.40.

3d: M.p.: 121°C; yield: 70%; IR (νmax, cm-1, KBr): 1728, 1236, 1037; 1H NMR (CDCl3, δ, ppm): 0.924-0.971 (t, 6 H), 2.615 (s, 6 H), 3.905-4.105 (q, 4 H), 6.987-7.044 (m, 2 H), 7.280-7.365 (m, 1 H), 7.469-7.622 (m, 4 H), 7.733-7.759 (d, 2 H, J = 7.8 Hz), 7.923 (s, 1 H); mass: m/z 488.36 (M+ + 1, 100%).

Anal. Calcd for C28H26N3O4F: C 68.99, H 5.38, N 8.62; found: C 68.95, H 5.37, N 8.63.

3e: M.p.: 101-102°C, lit [20] M.p.: 101-102°C; Yield: 65%.

3f: M.p.: 115°C; yield: 70%; IR (νmax, cm-1, KBr): 1734, 1030; 1H NMR (CDCl3, δ, ppm): 0.940-0.962 (t, 6 H), 2.617 (s, 6 H), 3.957-4.039 (q, 4 H), 7.200-7.495 (m, 7 H), 7.732-7.756 (d, 2 H, J = 7.2 Hz), 7.921 (s, 1 H); mass: m/z 548.20, 550.20.

Anal. Calcd for C28H26N3O4Br: C 61.42, H 4.75, N 7.68; found: C 61.31, H 4.79, N 7.69.

3g: M.p.: 172°C; yield: 68%; IR (νmax, cm-1, KBr): 1728, 1234, 1034; 1H NMR (CDCl3, δ, ppm): 0.895-0.941 (t, 6 H), 2.632 (s, 6 H), 3.923-4.039 (m, 4 H), 7.279-7.410 (m, 3 H), 7.499-7.769 (m, 4 H), 7.960 (s, 1 H), 8.178-8.207 (d, 2 H, J = 7.5 Hz); mass: m/z 515.26 (M+ + 1, 100%).

Anal. Calcd for C28H26N4O6: C 64.37, H 4.98, N 10.73; found: C 65.34, H 5.08, N 10.87.

Pharmacology

Test microorganisms

Total six microbial strains were selected on the basis of their clinical importance in causing diseases in humans. Two Gram-positive bacteria (S. aureus MTCC 96 and B. subtilis MTCC 121); two Gram-negative bacteria (E. coli MTCC 1652 and P. aeruginosa MTCC 741) and two fungi (A. niger and A. flavus) the ear pathogens isolated from the patients of Kurukshetra [21], were used in the present study for the evaluation of antimicrobial activities of the chemical compounds. All the cultures were procured from Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh. The bacteria and fungi were subcultured on Nutrient agar and Sabouraud's dextrose agar (SDA), respectively, and incubated aerobically at 37°C.

In vitro antibacterial activity

The antibacterial activities of compounds, 2a-g and 3a-g, were evaluated by the agar well diffusion method. All the cultures were adjusted to 0.5 McFarland standard, which is visually comparable to a microbial suspension of approximately 1.5 × 108 cfu/mL. 20 mL of Mueller Hinton agar medium was poured into each Petri plate, and the agar plates were swabbed with 100 μL inocula of each test bacterium and kept for 15 min for adsorption. Using sterile cork borer of 8-mm diameter, wells were bored into the seeded agar plates, and these were then loaded with a 100 μL volume with concentration of 2.0 mg/mL of each compound reconstituted in the dimethylsulphoxide (DMSO). All the plates were incubated at 37°C for 24 h. Antibacterial activity of each compound was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (Hi Antibiotic zone scale). DMSO was used as a negative control whereas ciprofloxacin was used as a positive control. This procedure was performed in three replicate plates for each organism [22,23].

Determination of minimum inhibitory concentration

Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial compound that will inhibit the visible growth of a microorganism after overnight incubation. MIC of the compounds against bacterial strains was tested through a macrodilution tube method as recommended by NCCLS [24]. In this method, various test concentrations of chemically synthesized compounds were made from 256 to 1 μg/mL in sterile tubes, 1-10. 100 μL sterile Mueller Hinton Broth was poured in each sterile tube, and followed by addition of 200 μL test compound in tube 1. Twofold serial dilutions were carried out from tubes 1 to 10, and excess broth (100 μL) was discarded from the tube 10. To each tube, 100 μL of standard inoculum (1.5 × 108 cfu/mL) was added. Ciprofloxacin was used as control. Turbidity was observed after incubating the inoculated tubes at 37°C for 24 h.

In vitro antifungal activity

The antifungal activity of the synthesized chemical compounds was evaluated by poison food technique. The moulds were grown on SDA at 25°C for 7 days and used as inocula. 15 mL of molten SDA (45°C) was poisoned by the addition of 100 μL volume of each compound having concentration of 4.0 mg/mL, reconstituted in the DMSO, poured into a sterile Petri plate and allowed to solidify at room temperature. The solidified poisoned agar plates were inoculated at the centre with fungal plugs (8-mm diameter), obtained from the actively growing colony and incubated at 25°C for 7 days. DMSO was used as the negative control whereas fluconazole was used as the positive control. The experiments were performed in triplicates. Diameter of the fungal colonies was measured and expressed as percent mycelial inhibition determined by applying the following formula [25]:

where dc is the average diameter of fungal colony in negative control plates, and dt the average diameter of fungal colony in experimental plates.

Abbreviations

1,4-DHP: dialkyl 1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylates; DMSO: dimethylsulphoxide; HTIB: hydroxy (tosyloxy)iodobenzene; MIC: minimum inhibitory concentration; MTCC: microbial type culture collection; SDA: Sabouraud dextrose agar.

Competing interests

The authors declare that they have no competing interests.

Additional file 1

1HNMR spectrum of compound 2b.

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Additional file 2

1HNMR spectrum of compound 2c.

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Additional file 3

1HNMR spectrum of compound 2e.

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Additional file 4

1HNMR spectrum of compound 2f.

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Additional file 5

1HNMR spectrum of compound 2g.

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Additional file 6

1HNMR spectrum of compound 3a.

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Additional file 7

1HNMR spectrum of compound 3b.

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Additional file 8

1HNMR spectrum of compound 3c.

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Additional file 9

1HNMR spectrum of compound 3d.

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Additional file 10

1HNMR spectrum of compound 3e.

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Additional file 11

1HNMR spectrum of compound 3f.

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Additional file 12

1HNMR spectrum of compound 3g.

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