Synthesis and antimicrobial activity of some new pyrimidinone and oxazinone derivatives fused with thiophene rings using 2-chloro-6-ethoxy-4-acetylpyridine as starting material.

A series of pyridines, pyrimidinones, oxazinones and their derivatives were synthesized as antimicrobial agents using citrazinic acid (2,6-dihydroxyisonicotinic acid) as a starting material. α,β-Unsaturated ketones 3a-c were condensed with cyanothio-acetamide in the presence of ammonium acetate to give 2-cyanopyridinethiones 4a-c, which were reacted with ethyl chloroacetate to yield the corresponding cyano esters 5a-c. The esters 5a-c were cyclized by action of sodium methoxide to aminoesters 6a-c, which were aminolyzed with ammonia to corresponding aminoamide derivatives 7a-c. Also, the esters 6a-c were hydrolyzed with NaOH to the corresponding sodium salt 8a-c, which were treated with acetic anhydride to afford 2-methyloxazinones 9a-c. The latter compounds were treated with ammonium acetate to afford 2-methylpyrimidinones 10a-c, followed by methylation with methyl iodide to yield 2,3-dimethyl-pyrimidinones 11a-c. The antimicrobial screening showed that many of these compounds have good antibacterial and antifungal activities comparable to streptomycin and fusidic acid used as reference drugs.

1. Introduction

In previous work, we have found that certain substituted <span class="Chemical">pyridinesn> and their derivatives showed antimicrobial, analgesic, anticonvulsant, antiparkinsonian [1,2,3,4] and antitumor activities [5,6,7]. In addition, the biological and analgesic activities of many heterocyclic compounds containing a <span class="Chemical">sulfur atom have been reviewed [8,9,10]. On the other hand, <span class="Chemical">thienopyrimidine and thioxopyrimidine derivatives have promising biological [11,12] and anticancer activity [13]. Recently, some new oxazinones, thienopyrimidinones and their derivatives have been synthesized as anti-inflammatory, antimicrobial and anti-HIV agents [14,15,16,17,18]. In view of these observations and in continuation of our previous work in pyridine chemistry, we have now synthesized some novel heterocyclic compounds containing the thieno[2,3-b]pyridine moiety fused with a pyridine, oxazinone, or pyrimidinone, nucleus and tested their antimicrobial activities.

2. Results and Discussion

2.1. Synthesis

The starting materials 3a–c (Table 1) were prepared from <span class="Chemical">2,6-dihydroxyisonicotinic acidn> (1) via the corresponding <span class="Chemical">2-chloro-6-ethoxy-4-acetylpyridine 2 according to literature methods [1,19]. Acryloyl derivatives 3a–c were condensed with <span class="Chemical">2-cyanothioacetamide in the presence of ammonium acetate to give the corresponding cyanopyridine thione derivatives 4a–c (Table 1). Treatment of 4a–c with ethyl chloroacetate in the presence of anhydrous K2CO3 gave the corresponding ethyl ester derivative 5a–c (Table 1), which were cyclized by sodium methoxide in methanol to give the amino ester derivatives 6a–c (Table 1). Aminolysis of compounds 6a–c by action of ammonia gas afforded the corresponding aminoamide derivatives 7a–c (Scheme 1, Table 1). The IR spectra of 6a–c showed the absence of ν (C≡N) for 5a–c and the presence of broad band corresponding to ν (NH2). Also, the IR spectra of 7a–c showed the absence of ν(C=O, ester) for 6a–c and the presence of a broad band corresponding to ν(NH2).

Table 1

Melting points, crystallization solvents, yields, molecular formulae and molecular weights of compounds 3–7.

Comp. No.	X	Y	Yield (%)	M.p. (°C)	Cryst. Solv.	Molecular Formula (Mol. Wt.)
3a	F	H	86	185–187	EtOH	C16H13ClFNO2 (505.73)
3b	Cl	H	82	155–157	EtOH	C16H13Cl2NO2 (322.19)
3c	Cl	Cl	85	203–205	EtOH	C16H12Cl3NO2 (356.63)
4a	F	H	65	192–194	DMF/H2O (2:1)	C19H13ClFN3OS (385.84)
4b	Cl	H	58	206–208	AcOH/H2O (2:1)	C19H13Cl2N3OS (402.30)
4c	Cl	Cl	70	225–227	DMF/H2O (2:1)	C19H12Cl3N3OS (436.74)
5a	F	H	78	198–200	EtOH/Ether (2:1)	C23H19ClFN3O3S (471.93)
5b	Cl	H	76	189–191	EtOH/Ether (2:1)	C23H19Cl2N3O3S (488.39)
5c	Cl	Cl	69	245–257	EtOH/Ether (2:1)	C23H18Cl3N3O3S (522.83)
6a	F	H	65	176–178	Dioxane	C23H19ClFN3O3S (471.93)
6b	Cl	H	70	214–216	EtOH	C23H19Cl2N3O3S (488.39)
6c	Cl	Cl	58	235–237	DMF/EtOH (2:1)	C23H18Cl3N3O3S (522.83)
7a	F	H	86	200–202	MeOH	C21H16ClFN4O2S (442.89)
7b	Cl	H	85	228–230	AcOH	C21H16Cl2N4O2S (459.35)
7c	Cl	Cl	84	256–258	AcOH/H2O (2:1)	C21H15Cl3N4O2S (493.79)

Scheme 1

Synthetic Pathway for Compound 3–7.

Compounds 6a–c were hydrolyzed by refluxing with <span class="Chemical">ethanolic sodium hydroxiden> (<span class="Chemical">NaOH) to the corresponding <span class="Chemical">sodium salts 8a–c, which was treated in situ with refluxing acetic anhydride to give the corresponding oxazinone derivatives 9a–c (Table 2). Reaction of 9a–c with ammonium acetate in refluxing acetic acid afforded the corresponding pyrimidinone derivatives 10a–c (Table 2), which were treated with methyl iodide in N,N-dimethylformamide in the presence of anhydrous K2CO3 to yield the corresponding 3-methyl-pyrimidinone derivatives 11a–c (Scheme 2, Table 2).

Table 2

Melting points, crystallization solvents, yields, molecular formulae and molecular weights of compounds 9–11.

Comp. No.	X	Y	Yield (%)	M.p. (°C)	Cryst. Solv.	Molecular Formula (Mol. Wt.)
9a	F	H	75	195–197	EtOH	C23H15ClFN3O3S (467.90)
9b	Cl	H	68	214–216	AcOH	C23H15Cl2N3O3S (484.35)
9c	Cl	Cl	60	282–284	DMF/H2O (2:1)	C23H14Cl3N3O3S (518.80)
10a	F	H	80	178–180	AcOH/H2O (2:1)	C23H16ClFN4O2S (466.92)
10b	Cl	H	72	188–190	AcOH/H2O (2:1)	C23H16Cl2N4O2S (483.37)
10c	Cl	Cl	65	256–258	DMF/H2O (2:1)	C23H15Cl3N4O2S (517.81)
11a	F	H	78	186–188	AcOH/H2O (2:1)	C24H18ClFN4O2S (480.94)
11b	Cl	H	66	200–202	AcOH	C24H18Cl2N4O2S (497.40)
11c	Cl	Cl	72	264–266	DMF/H2O (2:1)	C24H17Cl3N4O2S (531.84)

Scheme 2

Synthetic Pathway for Compound 9–11.

2.2. Antimicrobial Activity

The antimicrobial activities of some of the synthesized compounds were determined by the <span class="Chemical">agarn> diffusion method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [20]. The compounds were evaluated for antimicrobial activity against bacteria, viz. <span class="Species">Streptomyces sp., <span class="Species">Bacillus subtilis, Streptococcus lactis, Escherichia coli, and Pseudomonas sp. and antifungal activity against various fungi, viz. Aspergillus niger, Penicillium sp and yeast Candida albican and Rhodotorula ingeniosa.

The concentrations of the tested compounds (10 µg/mL) were used according to a modified Kirby-Bauer’s disk diffusion method. The sterile discs were impregnated with 10 µg/disc of the tested compound. Each tested compound was performed in triplicate. The solvent <span class="Chemical">DMSOn> was used as a negative control and <span class="Chemical">streptomycin/<span class="Chemical">fusidic acid were used as standard calculated average diameters (for triplicates) of the zone of inhibition (in mm) for tested samples with that produced by the standard drugs. Four of the synthesized compounds 5a, 7b, 9b and 10b exhibited potent antibacterial and antifungal bioactivity compared with the standard drug used. The other tested compounds were found to exhibit a moderate to low antibacterial activity (Table 3).

Table 3

Antimicrobial activities of the newly synthesized compounds 3–11.

Comp. No.	Fungi		Yeast		Str. sp	Bacteria
	A.n	Pen. sp	C. a	R.i		Gram − ve		Gram + ve
						B.s	S.l	E.c	P. sp
3a	12	12	12	11	13	14	14	14	13
3b	12	12	10	11	9	8	7	9	11
3c	8	10	9	11	12	12	12	11	14
4a	10	12	11	11	13	11	10	12	11
4b	11	12	13	11	14	13	11	12	12
4c	10	12	12	13	13	12	10	12	11
5a	17	16	16	17	22	23	24	23	21
5b	4	5	4	3	7	8	7	9	8
5c	13	12	12	13	11	13	12	10	9
6a	10	13	10	11	21	20	21	23	23
6b	8	8	6	7	11	12	13	13	12
6c	12	13	13	12	13	11	13	12	13
7a	13	12	12	13	11	13	12	10	9
7b	7	5	8	9	6	12	13	13	12
7c	12	13	11	13	12	8	8	6	7
9a	12	10	11	11	20	20	21	19	20
9b	19	20	19	19	11	13	12	10	9
9c	10	11	11	12	10	11	10	12	11
10a	15	16	13	14	11	11	12	12	13
10b	23	22	22	20	11	23	22	24	23
10c	11	10	12	11	11	10	12	11	11
11a	13	12	12	13	11	13	12	10	9
11b	10	12	11	11	13	11	10	12	11
11c	13	11	10	12	11	10	12	11	11
Streptomycin	-	-	-	-	21	22	21	22	21
Fusidic acid	17	17	18	18	-	-	-	-	-

A.n: Aspergillus niger; Pen. sp: Penicillium sp; C. a: Candida albican; Str. sp: Streptomyces sp; R.i: Rhodotorula ingeniosa; B.s: Bacillus subtilis; S.l: Streptococcus lactis; E.c: Escherichia coli; P. sp: Pseudomonas sp.

On the other hand, when different concentrations of compound 9a were used, it was exhibited a moderate antibacterial activity, but it exhibited very good antibacterial activity at higher concentrations (3× and 4×) (Table 4), while different concentrations of compounds 5a and 10a exhibited very good antifungal activities (2× and 3×) (Table 5).

Table 4

Antibacterial activity of compound 9a at different concentrations.

Comp. No.	Conc.	Strep. sp	Bacteria
			Gram − ve		Gram + ve
			B.s	S.l	E.c	Ps
9a	1×	20	20	21	19	20
	2×	23	23	22	23	22
	3×	25	24	24	24	26
	4×	25	25	27	25	26

Where × = 10 μg.

Table 5

Antifungal activity of compounds 5a and 10a at different concentrations.

Comp. No.	Conc.	Fungi
		A.n	Pen. sp	C. a	R.i
5a	1×	17	16	16	17
	2×	18	18	19	19
	3×	19	20	20	21
	4×	20	22	20	21
10a	1×	15	16	13	14
	2×	16	18	18	17
	3×	18	20	20	20
	4×	20	22	20	21

Where × = 10 μg.

3. Experimental

3.1. Chemistry

Melting points were measured using Electrothermal 9100 digital melting point apparatus (Electrothermal, Essex, UK) and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1600 FTIR (Perkin-Elmer, Downers Grove, IL, USA) in <n class="Chemical">span class="Chemical">KBr discs. <spn>an class="Chemical">1H- and 13C-NMR spectra were measured on a Jeol 5000 MHz spectrometer (Jeol, Tokyo, Japan) in DMSO-d6, and chemical shifts were recorded in δ ppm relative to the internal standard TMS. The Mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer (Madison, WI, USA) using EI and the values of m/z are indicated in Dalton. Elemental analyses were performed on a Perkin-Elmer 2400 analyzer (Perkin-Elmer) and were found within ±0.4% of the theoretical values. All reactions were followed by TLC (Silica gel, Aluminum Sheets 60 F254, Merck, Darmstadt, Germany). Starting material 2 was prepared from citrazinic acid (1) according to published procedures [1,19]. Antimicrobial screening was carried out in Department of Microbial Chemistry, National Research Center, Cairo, Egypt.

<span class="Chemical">1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(substituted phenyl)prop-2-en-1-ones 3a–c. A mixture of <span class="Chemical">2-chloro-6-ethoxy-4-acetylpyridine (2) [19] (1 mmol) and an aromatic aldehyde, namely, 4-flouro-, 4-chloro- or 2,4-dichlorobenzaldehyde (1 mmol) in absolute <span class="Chemical">ethanol (30 mL) was refluxed in the presence of a mixture of TEA/DEA (3 mL, 1:1 v:v) for 6 h. The reaction mixture was concentrated under reduced pressure, the obtained solid was filtered off, washed with ether, dried and crystallized from the proper solvents to afford the corresponding acryloyl derivatives 3a–c, respectively.

<span class="Chemical">1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(4-fluorophenyl)prop-2-en-1-onen> (3a). IR (<span class="Chemical">KBr, cm−1): ν 1679 (C=O), 1607 (C=C); <span class="Chemical">1H-NMR: δ 1.32 (t, 3H, CH3, J = 6.95 Hz), 3.81 (q, 2H, CH2, J = 6.95 Hz), 6.65 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 6.98 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.28–7.96 (m, 6H, 4 Ph-H + 2 pyr-H); 13C-NMR: 13.68, 64.32, 104.95, 109.56, 114.72, 121.30, 129.86, 130.05, 144.65, 145.84, 146.50, 160.95, 164.96, 186.50; MS, m/z (%): 306 (M+, 15), 184 (100); Elemental analysis for C16H13ClFNO2 (305.73): calcd.: C, 62.86; H, 4.29; Cl, 11.60; N, 4.58. found: C, 62.80; H, 4.26; Cl, 11.55; N, 4.52.

<span class="Chemical">1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(4-chlorophenyl)prop-2-en-1-onen> (3b). IR (<span class="Chemical">KBr, cm−1): ν 1682 (C=O), 1610 (C=C); <span class="Chemical">1H-NMR: δ 1.33 (t, 3H, CH3, J = 6.95 Hz), 3.92 (q, 2H, CH2, J = 6.95 Hz), 6.58 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.05 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.12–7.88 (m, 6H, 4 Ph-H + 2 pyr-H); 13C-NMR: 13.86, 64.26, 105.78, 109.62, 121.12, 126.86, 128.25, 132.85, 132.96, 144.68, 145.78, 146.65, 164.84, 186.86; MS, m/z (%): 322 (M+, 8), 165 (100); Elemental analysis for C16H13Cl2NO2 (322.18): calcd.: C, 59.65; H, 4.07; Cl, 22.01; N, 4.35. found: C, 59.60; H, 4.00; Cl, 21.96; N, 4.30.

<span class="Chemical">1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(2,4-dichlorophenyl)prop-2-en-1-onen> (3c). IR (<span class="Chemical">KBr, cm−1): ν 1678 (C=O), 1612 (C=C); <span class="Chemical">1H-NMR: δ 1.28 (t, 3H, CH3, J = 6.95 Hz), 3.86 (q, 2H, CH2, J = 6.95 Hz), 6.46 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.10 (d, 1H, CH-olefinic-H, J = 14.60 Hz), 7.25–7.76 (m, 5H, 3 Ph-H + 2 pyr-H); 13C-NMR: 13.92, 64.30, 105.96, 109.46, 121.21, 125.69, 128.78, 129.56, 130.85, 132.05, 133.65, 144.86, 145.88, 146.54, 164.78, 187.05; MS, m/z (%): 356 [M+,10], 199 [100, base peak]; Elemental analysis for C16H12Cl3NO2 (356.63): calcd.: C, 53.89; H, 3.39; Cl, 29.82; N, 3.93. found: C, 53.83; H, 3.34; Cl, 29.80; N, 3.88.

6-(2-Chloro-6-ethoxypyridin-4-yl)-4-(substituted phenyl)-1,2-dihydro-2-<span class="Chemical">thioxopyridine-3-carbonitriles 4a–c. A mixture of 3a–c (1 mmol), <span class="Chemical">2-cyanothioacetamide (0.10 g, 1 mmol) and <span class="Chemical">ammonium acetate (0.6 g, 8 mmol) in absolute ethanol (30 mL) was refluxed for 5 h. After cooling, the formed product was collected by filtration, washed with ethanol, dried and crystallized from the proper solvents to give the corresponding thioxopyridine derivatives 4a–c, respectively.

<span class="Chemical">6-(2-Chloro-6-ethoxypyridin-4-yl)-4-(4-fluorophenyl)-1,2-dihydro-2-thioxopyridine-3-carbonitrilen> (4a). IR (<span class="Chemical">KBr, cm−1): ν 3330 (NH), 2210 (CN), 1218 (C=S); <span class="Chemical">1H-NMR: δ 1.30 (t, 3H, CH3, J = 6.95 Hz), 3.90 (q, 2H, CH2, J = 6.95 Hz), 6.95–7.78 (m, 6H, 4 Ph-H + 2 pyr-H), 8.46 (s, 1H, pyr-5'-H), 9.24 (s, 1H, NH exchangeable with D2O); 13C-NMR: 13.66, 63.98, 103.66, 103.88, 107.89, 108.55, 114.58, 116.02, 127.50, 128.04, 145.48, 148.60, 160.56, 161.76, 164.65, 167.47, 168.05; MS, m/z (%): 386 [M+,24], 135 [100, base peak]; Elemental analysis for C19H13ClFN3OS (385.84): calcd.: C, 59.14; H, 3.40; Cl, 9.19; N, 10.89; S, 8.31. found: C, 59.10; H, 3.35; Cl, 9.14; N, 10.85; S, 8.28.

<span class="Chemical">6-(2-Chloro-6-ethoxypyridin-4-yl)-4-(4-chlorophenyl)-1,2-dihydro-2-thioxopyridine-3-carbonitrilen> (4b). IR (<span class="Chemical">KBr, cm−1): ν 3356 (NH), 2215 (CN), 1210 (C=S); <span class="Chemical">1H-NMR: δ 1.34 (t, 3H, CH3, J = 6.95 Hz), 3.86 (q, 2H, CH2, J = 6.95 Hz), 7.12–7.80 (m, 6H, 4 Ph-H + 2 pyr-H), 8.52 (s, 1H, pyr-5'-H), 9.18 (s, 1H, NH exchangeable with D2O); 13C-NMR: 13.92, 64.12, 103.96, 104.04, 108.14, 108.86, 115.82, 127.66, 128.10, 129.68, 132.67, 145.56, 148.72, 160.77, 164.58, 167.55, 167.86; MS, m/z (%): 402 [M+,32], 211 [100, base peak]; Elemental analysis for C19H13Cl2N3OS (402.29): calcd.: C, 56.73; H, 3.26; Cl, 17.63; N, 10.45; S, 7.97. found: C, 56.68; H, 3.20; Cl, 17.60; N, 10.40; S, 7.92.

6-(2-Chloro-6-ethoxypyridin-4-yl)-4-(2,4-dichlorophenyl)-1,2-dihydro-2-<span class="Chemical">thioxopyridinen>-3-carbonitrile (4c). ν 3348 (NH), 2218 (CN), 1212 (C=S); <span class="Chemical">1H-NMR: δ 1.32 (t, 3H, CH3, J = 6.95 Hz), 3.78 (q, 2H, CH2, J = 6.95 Hz), 6.98–7.68 (m, 5H, 3 Ph-H + 2 pyr-H), 8.64 (s, <span class="Chemical">1H, pyr-5'-H), 9.34 (s, 1H, NH exchangeable with D2O); 13C-NMR: 14.14, 64.18, 103.88, 104.08, 108.22, 108.92, 115.76, 125.98, 128.56, 129.16, 131.86, 132.15, 134.86, 145.64, 148.80, 161.24, 164.32, 167.45, 168.18; MS, m/z (%): 436 [M+,14], 279 [100, base peak]; Elemental analysis for C19H12Cl3N3OS (436.74): calcd.: C, 52.25; H, 2.77; Cl, 24.35; N, 9.62; S, 7.34. found: C, 52.20; H, 2.71; Cl, 24.30; N, 9.57; S, 7.28.

<span class="Chemical">Ethyl 2-(6-(2-chloro-6-ethoxypyridin-4-yl)-3-cyano-4-(substituted phenyl)pyridin-2-ylthio)acetatesn> 5a–c. To a mixture of 4a–c (1 mmol) and anhydrous <span class="Chemical">K2CO3 (0.18 g, 1 mmol) in <span class="Chemical">N-dimethylformamide (25 mL) was stirred at room temperature for 2 h, ethyl chloroacetate (0.18 g, 1.5 mmol) was added with stirring. The reaction mixture was heated at 60 °C for 2 h and after cooling poured into ice. The solid formed was collected by filtration, washed with water, dried and crystallized from the proper solvents to afford the corresponding pyridinethioacetate derivatives 5a–c, respectively.

<span class="Chemical">Ethyl 2-(6-(2-chloro-6-ethoxypyridin-4-yl)-3-cyano-4-(4-fluorophenyl)pyridin-2-ylthio)acetaten> (5a). IR (<span class="Chemical">KBr, cm−1): ν 2219 (CN), 1735 (C=O, <span class="Chemical">ester); 1H-NMR: δ 1.28, 1.32 (2t, 6H, 2 CH3), 3.68, 3.86 (2q, 4H, 2 CH2), 4.38 (s, 2H, S–CH2), 7.16–7.82 (m, 6H, 4 Ph-H + 2 pyr-H), 8.18 (s, 1H, pyr-5'-H); 13C-NMR: 13.65, 14.05, 32.04, 59.86, 64.08, 101.36, 101.57, 102.85, 115.02, 116.75, 117.02, 128.74, 132.58, 145.65, 151.56, 153.65, 157.08, 162.15, 163.56, 163.94, 168.90; MS, m/z (%): 472 [M+,12], 426 [100, base peak]; Elemental analysis for C23H19ClFN3O3S (471.93): calcd.: C, 58.54; H, 4.06; Cl, 7.51; N, 8.90; 17; S, 6.79. found: C, 58.48; H, 4.00; Cl, 7.45; N, 8.84; 17; S, 6.72.

<span class="Chemical">Ethyl 2-(6-(2-chloro-6-ethoxypyridin-4-yl)-3-cyano-4-(4-chlorophenyl)pyridin-2-ylthio)acetaten> (5b). IR (<span class="Chemical">KBr, cm−1): ν 2222 (CN), 1732 (C=O, <span class="Chemical">ester); 1H-NMR: δ 1.29, 1.32 (2t, 6H, 2 CH3), 3.56, 3.84 (2q, 4H, 2 CH2), 4.42 (s, 2H, S–CH2), 7.10–7.72 (m, 6H, 4 Ph-H + 2 pyr-H), 8.64 (s, 1H, pyr-5'-H); 13C-NMR: 13.78, 14.15, 32.18, 60.05, 64.18, 101.48, 101.68, 102.74, 116.88, 117.02, 127.54, 128.12, 129.57, 133.45, 145.56, 150.96, 153.64, 157.18, 163.72, 164.05, 170.04; MS, m/z (%): 488 [M+,32], 120 [100, base peak]; Elemental analysis for C23H19Cl2N3O3S (488.38): calcd.: C, 56.56; H, 3.92; Cl, 14.52; N, 8.60; S, 6.57. found: C, 56.50; H, 3.88; Cl, 14.47; N, 8.55; S, 6.51.

<span class="Chemical">Ethyl 2-(6-(2-chloro-6-ethoxypyridin-4-yl)-3-cyano-4-(2,4-dichlorophenyl)pyridin-2-ylthio)acetaten> (5c). IR (<span class="Chemical">KBr, cm−1): ν 2218 (CN), 1735 (C=O, <span class="Chemical">ester); 1H-NMR: δ 1.26, 1.30 (2t, 6H, 2 CH3), 3.58, 3.78 (2q, 4H, 2 CH2), 4.36 (s, 2H, S–CH2), 7.12–7.65 (m, 5H, 3 Ph-H + 2 pyr-H), 8.56 (s, 1H, pyr-5'-H); 13C-NMR: 13.84, 14.18, 32.18, 59.92, 64.18, 100.98, 101.59, 102.66, 116.82, 117.06, 125.86, 128.48, 129.24, 131.92, 132.24, 134.74, 145.58, 151.08, 153.72, 157.22, 163.88, 164.15, 168.84; MS, m/z (%): 523 [M+,8], 247 [100, base peak]; Elemental analysis for C23H18Cl3N3O3S (522.83): C, 52.84; H, 3.47; Cl, 20.34; N, 8.04; S, 6.13. found: C, 52.78; H, 3.40; Cl, 20.28; N, 8.00; S, 6.07.

Ethyl 3-amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(substituted phenyl)<span class="Chemical">thieno[2,3-b]pyridine-2-carboxylates 6a–c. A mixture of 5a–c (1 mmol) in <span class="Chemical">sodium methoxide solution (20 mL, 2%) was refluxed for 1 h on a <span class="Chemical">water bath at 70 °C with stirring. The reaction mixture was evaporated under reduced pressure, the obtained residue was dissolved in CH2Cl2, washed with H2O, 10 mL 1 N HCl and then with water. The solvent was dried over anhydrous CaCl2, evaporated under reduced pressure, and the obtained product was crystallized to afford from the proper solvents to afford the corresponding ethyl thienopyridinecarboxylates 6a–c, respectively.

Ethyl 3-amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(4-fluorophenyl)<span class="Chemical">thieno[2,3-b]pyridinen>-2-carboxyl-ate (6a). IR (<span class="Chemical">KBr, cm−1): ν 3443 (NH2), 1742 (C=O, <span class="Chemical">ester); 1H-NMR: δ 1.30, 1.34 (2t, 6H, 2 CH3), 3.72, 4.06 (2q, 4H, 2 CH2), 4.36 (s, 2H, NH2 exchangeable with D2O), 7.24–7.75 (m, 6H, 4 Ph-H + 2 pyr-H), 8.35 (s, 1H, pyr-5'-H); 13C-NMR: 13.95, 14.16, 60.24, 64.18, 101.58, 103.02, 115.16, 118.35, 120.76, 122.15, 128.66, 132.64, 134.12, 145.72, 149.65, 151.64, 154.57, 155.75, 160.12, 162.65, 164.12; MS, m/z (%): 472 [M+,26], 317 [100, base peak]; Elemental analysis for C23H19ClFN3O3S (471.93): calcd.: C, 58.54; H, 4.06; Cl, 7.51; N, 8.90; S, 6.79. found: C, 58.48; H, 4.00; Cl, 7.45; N, 8.86; S, 6.71.

Ethyl 3-amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(4-chlorophenyl)<span class="Chemical">thieno[2,3-b]pyridinen>-2-carboxyl-ate (6b). IR (<span class="Chemical">KBr, cm−1): ν 3452 (NH2), 1737 (C=O, <span class="Chemical">ester); 1H-NMR: δ 1.26, 1.31 (2t, 6H, 2 CH3), 3.78, 4.10 (2q, 4H, 2 CH2), 4.48 (s, 2H, NH2 exchangeable with D2O), 7.24–7.82 (m, 6H, 4 Ph-H + 2 pyr-H), 8.72 (s, 1H, pyr-5'-H); 13C-NMR: 14.08, 14.25, 60.15, 64.10, 100.42, 103.64, 118.05, 121.16, 122.25, 127.66, 128.44, 133.45, 133.95, 134.50, 146.02, 149.75, 151.18, 154.65, 156.05, 159.64, 164.15; MS, m/z (%): 488 [M+,8], 332 [100, base peak]; Elemental analysis for C23H19Cl2N3O3S (488.38): calcd.: C, 56.56; H, 3.92; Cl, 14.52; N, 8.60; S, 6.57. found: C, 56.50; H, 3.88; Cl, 14.46; N, 8.55; S, 6.50.

Ethyl 3-amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(2,4-dichlorophenyl)<span class="Chemical">thieno[2,3-b]pyridinen>-2-carboxylate (6c). IR (<span class="Chemical">KBr, cm−1): ν 3456 (NH2), 1735 (C=O, <span class="Chemical">ester); 1H-NMR: δ 1.30, 1.33 (2t, 6H, 2 CH3), 3.82, 4.15 (2q, 4H, 2 CH2), 4.56 (s, 2H, NH2 exchangeable with D2O), 7.08–7.68 (m, 5H, 3 Ph-H + 2 pyr-H), 8.62 (s, 1H, pyr-5'-H); 13C-NMR: 14.12, 14.26, 59.98, 64.32, 100.86, 102.72, 118.02, 121.06, 122.00, 126.16, 128.87, 129.36, 132.18, 134.05, 135.44, 136.74, 145.64, 149.85, 151.38, 154.72, 155.43, 160.04, 164.25; MS, m/z (%): 523 [M+,6], 177 [100, base peak]; Elemental analysis for C23H18Cl3N3O3S (522.83): calcd.: C, 52.84; H, 3.47; Cl, 20.34; N, 8.04; S, 6.13. found: C, 52.77; H, 3.42; Cl, 20.30; N, 7.97; S, 6.08.

3-Amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(substituted-phenyl)<span class="Chemical">thieno[2,3-b]pyridinen>-2-carbox-amides 7a–c. A current of <span class="Chemical">ammonia gas was passed through a suspension of 6a–c (1 mmol) in absolute <span class="Chemical">ethanol (100 mL), at 0 °C till saturation. The reaction mixture was left overnight at −4 °C, evaporated under reduced pressure, the residue obtained was triturated with n-hexane, the formed solid was filtered off, washed with water and crystallized from the proper solvents to give the corresponding thienopyridine carboxamides 7a–c, respectively.

<span class="Chemical">3-Amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(4-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamiden> (7a). IR (<span class="Chemical">KBr, cm−1): ν 3460–3380 (NH2), 1675 (C=O, amide); <span class="Chemical">1H-NMR: δ 1.32 (t, 3H, CH3), 3.85 (q, 2H, CH2), 4.46, 6.85 (2s, 4H, 2 NH2 exchangeable with D2O), 7.12–7.68 (m, 6H, 4 Ph-H + 2 pyr-H), 8.56 (s, 1H, pyr-5'-H); 13C-NMR: 14.06, 64.28, 100.96, 102.22, 115.36, 120.82, 122.45, 128.37, 128.46, 132.84, 137.15, 145.82, 149.65, 152.00, 154.74, 157.75, 161.55, 162.76, 164.30; MS, m/z (%): 443 [M+,8], 332 [100, base peak]; Elemental analysis for C21H16ClFN4O2S (442.89): calcd.: C, 56.95; H, 3.64; Cl, 8.00; N, 12.65; S, 7.24. found: C, 56.90; H, 3.60; Cl, 7.940; N, 12.60; S, 7.19.

<span class="Chemical">3-Amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(4-chlorophenyl)thieno[2,3-b]pyridine-2-carboxamiden> (7b). ν 3456–3378 (NH2), 1672 (C=O, amide); <span class="Chemical">1H-NMR: δ 1.30 (t, 3H, CH3), 3.82 (q, 2H, CH2), 4.44, 6.88 (2s, 4H, 2 NH2 exchangeable with D2O), 7.32–7.68 (m, 6H, 4 Ph-H + 2 pyr-H), 8.68 (s, <span class="Chemical">1H, pyr-5'-H); 13C-NMR: 13.68, 64.12, 100.00, 102.04, 121.24, 122.12, 127.85, 128.38, 128.55, 134.05, 135.15, 137.05, 146.12, 149.65, 151.00, 154.36, 156.14, 161.42, 164.04; MS, m/z (%): 459 [M+,25], 287 [100, base peak]; Elemental analysis for C21H16Cl2N4O2S (459.34): calcd.: C, 54.91; H, 3.51; Cl, 15.44; N, 12.20; S, 6.98. found: C, 54.86; H, 3.45; Cl, 15.39; N, 12.16; S, 6.92.

<span class="Chemical">3-Amino-6-(2-chloro-6-ethoxypyridin-4-yl)-4-(2,4-dichlorophenyl)thieno[2,3-b]pyridine-2-carboxamiden> (7c). IR (<span class="Chemical">KBr, cm−1): ν 3456 (NH2), 1735 (C=O, <span class="Chemical">ester); 1H-NMR: δ 1.28 (t, 3H, CH3), 3.86 (q, 2H, CH2), 4.54, 6.76 (2s, 4H, 2 NH2 exchangeable with D2O), 7.12–7.73 (m, 5H, 3 Ph-H + 2 pyr-H), 8.48 (s, 1H, pyr-5'-H); 13C-NMR: 14.12, 64.33, 101.04, 102.84, 121.32, 122.40, 126.24, 128.65, 128.75, 129.72, 132.32, 135.12, 136.66, 137.22, 145.56, 149.55, 151.22, 154.44, 157.12, 161.26, 164.57; MS, m/z (%): 494 [M+,12], 320 [100, base peak]; Elemental analysis for C21H15Cl3N4O2S (493.79): calcd.: C, 51.08; H, 3.06; Cl, 21.54; N, 11.35; S, 6.49. found: C, 51.00; H, 3.00; Cl, 21.50; N, 11.30; S, 6.44.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(substituted-phenyl)-2-methyl-4H-pyrido[3',2':4,5]thieno[3,2-d]-[1,3]-oxazin-4-ones 9a–c. A mixture of 6a–c (1 mmol) in <span class="Chemical">ethanoln>ic <span class="Chemical">NaOH (100 mL, 5%) was heated under reflux for 4 h. The solvent was evaporated under reduced pressure, the obtained <span class="Chemical">sodium salt 8a–c was dissolved in acetic anhydride (100 mL) and refluxed it for 6 h. The reaction mixture was concentrated and allowed to cool, poured onto ice water, the obtained solid was collected by filtration, washed with water, dried and crystallized from the proper solvents to afford the corresponding thienooxazinopyridine derivatives 9a–c, respectively.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-fluorophenyl)-2-methyl-4H-pyrido[3',2':4,5]thieno[3,2-d]-[1,3]oxazin-4-one (9a). IR (<span class="Chemical">KBrn>, cm−1): ν 1750 (C=O); <span class="Chemical">1H-NMR: δ 1.30 (t, 3H, CH3), 2.03 (s, 3H, CH3), 3.78 (q, 2H, CH2), 7.04–7.58 (m, 6H, 4 Ph-H + 2 pyr-H), 8.62 (s, <span class="Chemical">1H, pyr-5'-H); 13C-NMR: 14.10, 18.98, 64.30, 100.86, 101.68, 116.02, 120.80, 125.85, 128.42, 132.78, 134.46, 135.35, 145.72, 150.05, 151.75, 154.90, 155.25 158.62, 162.70, 164.08, 165.25; MS, m/z (%): 468 [M+,6], 217 [100, base peak]; Elemental analysis for C23H15ClFN3O3S (467.89): calcd.: C, 59.04; H, 3.23; Cl, 7.58; N, 8.98; S, 6.85. found: C, 58.96; H, 3.18; Cl, 7.52; N, 8.90; S, 6.80.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-chlorophenyl)-2-methyl-4H-pyrido[3',2':4,5]thieno[3,2-d]-[1,3]oxazin-4-one (9b). IR (<span class="Chemical">KBrn>, cm−1): ν 1745 (C=O); <span class="Chemical">1H-NMR: δ 1.28 (t, 3H, CH3), 2.01 (s, 3H, CH3), 3.89 (q, 2H, CH2), 7.23–7.65 (m, 6H, 4 Ph-H + 2 pyr-H), 8.42 (s, <span class="Chemical">1H, pyr-5'-H); 13C-NMR: 14.08, 21.60, 64.18, 100.55, 101.45, 121.12, 125.68, 128.12, 128.96, 133.05, 134.58, 135.32, 135.72, 145.92, 149.75, 151.84, 154.86, 155.14, 158.70, 164.12, 165.18; MS, m/z (%): 484 [M+,15], 156 [100, base peak]; Elemental analysis for C23H15Cl2N3O3S (484.35): calcd.: C, 57.03; H, 3.12; Cl, 14.64; N, 8.68; S, 6.62. found: C, 56.95; H, 3.10; Cl, 14.60; N, 8.63; S, 6.58.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methyl-4H-pyrido[3',2':4,5]thieno[3,2-d]-[1,3]oxazin-4-one (9c). IR (<span class="Chemical">KBrn>, cm−1): ν 1750 (C=O); <span class="Chemical">1H-NMR: δ 1.30 (t, 3H, CH3), 2.00 (s, 3H, CH3), 3.82 (q, 2H, CH2), 7.21–7.68 (m, 5H, 3 Ph-H + 2 pyr-H), 8.54 (s, <span class="Chemical">1H, pyr-5'-H); 13C-NMR: 14.10, 19.18, 64.22, 100.28, 101.15, 121.16, 125.56, 126.46, 128.58, 129.80, 132.44, 134.34, 135.18, 135.45, 136.73, 145.88, 149.72, 151.69, 154.78, 155.18, 159.06, 164.18, 165.32; MS, m/z (%): 519 [M+,8], 320 [100, base peak]; Elemental analysis for C23H14Cl3N3O3S (518.79): calcd.: C, 53.25; H, 2.72; Cl, 20.50; N, 8.10; S, 6.18. found: C, 53.18; H, 2.68; Cl, 20.45; N, 8.00; S, 6.12.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(substituted-phenyl)-2-methylpyrido[3',2':4<span class="Chemical">,5]thieno[3,2-d]-pyrimidin-4(3H)-onen>s 10a–c

A mixture of 9a–c (1 mmol) and <span class="Chemical">ammonium acetaten> (0.6 g, 8 mmol) in glacial <span class="Chemical">acetic acid (100 mL) was heated under reflux for 6 h. The reaction mixture was evaporated under reduced pressure, the residue was triturated with cooled <span class="Chemical">water, the solid formed was collected by filtration, washed with water, dried and crystallized from the proper solvents to afford the corresponding thienopyrimidino-pyridine 0.30 g (70%) 10a–c, respectively.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-fluorophenyl)-2-methylpyrido[3',2':4<span class="Chemical">,5]thieno[3,2-d]-pyrimidin-4(3H)-onen> (10a). IR (<span class="Chemical">KBr, cm−1): ν 3420 (NH), 1650 (C=O); <span class="Chemical">1H-NMR: δ 1.28 (t, 3H, CH3), 2.32 (s, 3H, CH3), 3.86 (q, 2H, CH2), 7.12–7.64 (m, 6H, 4 Ph-H + 2 pyr-H), 8.58 (s, 1H, pyr-5'-H), 9.26 (s, 1H, NH exchangeable with D2O); 13C-NMR: 14.08, 24.98, 64.42, 101.02, 102.54, 116.15, 121.04, 126.14, 128.85, 132.86, 136.76, 137.05, 145.88, 150.15, 151.98, 154.10, 154.86, 157.25, 160.03, 162.99, 164.28; MS, m/z (%): 467 [M+,18], 156 [100, base peak]; Elemental analysis for C23H16ClFN4O2S (466.91): calcd.: C, 59.16; H, 3.45; Cl, 7.59; N, 12.00; S, 6.87. found: C, 59.10; H, 3.38; Cl, 7.52; N, 11.94; S, 6.83.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-chlorophenyl)-2-methylpyrido[3',2':4<span class="Chemical">,5]thieno[3,2-d]-pyrimidin-4(3H)-onen> (10b). IR (<span class="Chemical">KBr, cm−1): ν 3438 (NH), 1649 (C=O); <span class="Chemical">1H-NMR: δ 1.31 (t, 3H, CH3), 2.24 (s, 3H, CH3), 3.78 (q, 2H, CH2), 7.33–7.72 (m, 6H, 4 Ph-H + 2 pyr-H), 8.62 (s, 1H, pyr-5'-H), 9.32 (s, 1H, NH exchangeable with D2O); 13C-NMR: 13.98, 25.04, 64.53, 100.12, 101.36, 121.13, 126.45, 128.15, 129.05, 133.76, 135.99, 136.88, 145.76, 146.05, 149.85, 151.90, 154.16, 154.92, 157.48, 159.73, 164.36; MS, m/z (%): 483 [M+,18], 326 [100, base peak]; Elemental analysis for C23H16Cl2N4O2S (483.36): calcd.: C, 57.15; H, 3.34; Cl, 14.67; N, 11.59; S, 6.63. found: C, 57.10; H, 3.28; Cl, 14.62; N, 11.53; S, 6.58.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methylpyrido[3',2':4<span class="Chemical">,5]thieno[3,2-d]-pyrimidin-4(3H)-onen> (10c). IR (<span class="Chemical">KBr, cm−1): ν 3465 (NH), 1653 (C=O); <span class="Chemical">1H-NMR: δ 1.26 (t, 3H, CH3), 2.12 (s, 3H, CH3), 3.80 (q, 2H, CH2), 7.21–7.70 (m, 5H, 3 Ph-H + 2 pyr-H), 8.72 (s, 1H, pyr-5'-H), 9.48 (s, 1H, NH exchangeable with D2O); 13C-NMR: 13.92, 24.87, 64.42, 99.96, 101.02, 120.33, 126.32, 126.64, 128.36, 129.72, 132.88, 135.09, 136.64, 136.84, 145.86, 146.13, 149.77, 151.92, 153.96, 154.66, 157.68, 160.02, 164.48; MS, m/z (%): 518 [M+,5], 145 [100, base peak]; Elemental analysis for C23H15Cl3N4O2S (517.81): calcd.: C, 53.35; H, 2.92; Cl, 20.54; N, 10.82; S, 6.19. found: C, 53.30; H, 2.87; Cl, 20.50; N, 10.79; S, 6.14.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-fluorophenyl)-2,3-dimethylpyrido[3',2':4<span class="Chemical">,5]thieno[3,2-d]pyrimidin-4(3H)-onesn> 11a–c. A solution of 10a–c (1 mmol) in <span class="Chemical">DMF (20 mL) was stirred with anhydrous <span class="Chemical">K2CO3 (0.19 g, 1 mmol) for 10 min at room temperature, then methyl iodide (0.28 g, 2 mmol) in DMF (5 mL) were added. The reaction mixture was heated at 60 °C for 4 h, after cooling, poured into ice water, and the formed precipitate was filtered off, washed with water, dried and crystallized from the proper solvents to afford the corresponding thieno-N-methylpyrimidinopyridines 11a–c, respectively.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-fluorophenyl)-2,3-dimethylpyrido[3',2':4<span class="Chemical">,5]thieno[3,2-d]pyrimidin-4(3H)-onen> (11a). IR (<span class="Chemical">KBr, cm−1): ν 1668 (C=O); <span class="Chemical">1H-NMR: δ 1.28 (t, 3H, CH3), 2.32, 3.10 (2s, 6H, 2 CH3), 3.78 (q, 2H, CH2), 7.08–7.68 (m, 6H, 4 Ph-H + 2 pyr-H), 8.62 (s, 1H, pyr-5'-H); 13C-NMR: 14.00, 22.14, 26.06, 64.15, 100.10, 101.32, 116.18, 120.34, 126.34, 128.95, 132.90, 136.42, 145.76, 146.15, 149.85, 151.80, 154.02, 154.77, 157.36, 159.63, 162.76, 164.30; MS, m/z (%): 481 [M+,4], 98 [100, base peak]; Elemental analysis for C24H18ClFN4O2S (480.94): calcd.: C, 59.94; H, 3.77; Cl, 7.37; N, 11.65; S, 6.67. found: C, 59.88; H, 3.72; Cl, 7.33; N, 11.60; S, 6.61.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(4-chlorophenyl)-2,3-dimethylpyrido[3',2':4<span class="Chemical">,5]thieno[3,2-d]-pyrimidin-4(3H)-onen> (11b). IR (<span class="Chemical">KBr, cm−1): ν 1670 (C=O); <span class="Chemical">1H-NMR: δ 1.29 (t, 3H, CH3), 2.18, 3.06 (2s, 6H, 2 CH3), 3.82 (q, 2H, CH2), 7.28–7.68 (m, 6H, 4 Ph-H + 2 pyr-H), 8.78 (s, 1H, pyr-5'-H); 13C-NMR: 13.86, 22.00, 26.28, 64.14, 99.58, 100.12, 120.56, 126.76, 128.00, 128.95, 133.45, 135.85, 136.56, 145.32, 146.75, 149.80, 150.87, 153.78, 154.65, 157.45, 160.02, 164.28; MS, m/z (%): 497 [M+,19], 162 [100, base peak]; Elemental analysis for C24H18Cl2N4O2S (497.39): calcd.: C, 57.95; H, 3.65; Cl, 14.26; N, 11.26; S, 6.45. found: C, 57.90; H, 3.59; Cl, 14.22; N, 11.20; S, 6.40.

7-(2-Chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2,3-dimethylpyrido[3',2':4<span class="Chemical">,5]thieno[3,2-d]-pyrimidin-4(3H)-onen> (11c). IR (<span class="Chemical">KBr, cm−1): ν 1667 (C=O); <span class="Chemical">1H-NMR: δ 1.28 (t, 3H, CH3), 2.22, 2.96 (2s, 6H, 2 CH3), 3.80 (q, 2H, CH2), 7.24–7.70 (m, 5H, 3 Ph-H + 2 pyr-H), 8.65 (s, 1H, pyr-5'-H); 13C-NMR: 13.90, 22.01, 26.48, 64.08, 99.86, 100.09, 120.60, 126.45, 126.58, 128.25, 129.52, 132.66, 135.14, 136.60, 136.78, 145.42, 146.78, 149.82, 151.14, 153.88, 154.72, 157.48, 159.72, 164.20; MS, m/z (%): 532 [M+,19], 252 [100, base peak]; Elemental analysis for C24H17Cl3N4O2S (531.84): calcd.: C, 54.20; H, 3.22; Cl, 20.00; N, 10.53; S, 6.03. found: C, 54.15; H, 3.16; Cl, 19.85; N, 10.48; S, 6.00.

3.2. Antimicrobial Screening Media

The following media were used:

PDA medium: this medium was used for fungi cultivation. It consists of 4 g <span class="Chemical">dextrosen>/L <span class="Species">potatoes extract.

<span class="Chemical">Czapekn> <span class="Chemical">Dox medium: it consists of 10 g <span class="Chemical">glucose, 2 g KNO3, 1g K2HPO4, 0.5 g KCl, 0.5 g MgSO4, and 0.05 g ferrous sulphate/L distilled water. This medium is specialized for bacteria cultivation.

Medium 3: it consists of 10 <span class="Chemical">glucosen>, 5 g peptone, 3 <span class="Species">yeast extract, and 3 malt extract. It was used for <span class="Species">yeast cultivation.

4. Conclusions

A series of newly compounds 3–11 were prepared using <span class="Chemical">citrazinic acidn> (<span class="Chemical">2,6-dihydroxyisonicotinic acid) as a starting material. The obtained derivatives were screening as antimicrobial and antifungal agents. Four of the synthesized compounds 5a, 7b, 9b and 10b exhibited potent antibacterial and antifungal bioactivity compared with <span class="Chemical">streptomycin and fusidic acid used as reference drugs. The other tested compounds were found to exhibit moderate to low antibacterial activity. On the other hand when higher concentrations (3× and 4×) of compound 9a, which exhibited a moderate antibacterial activity, were used, this compound exhibited very good antibacterial activity. While different concentrations of compounds 5a and 10a exhibited a very good antifungal activity (2× and 3×).