N-Pyrazinylhydroxybenzamides As Biologically Active Compounds: A Hit-Expansion Study and Antimicrobial Evaluation

Figures & data

Figure 1. Design of the compounds.

MIC: Minimum inhibitory concentration; Mtb: Mycobacterium tuberculosis; SA: Staphylococcus aureus; SAR: Structure–activity relationship.

Figure 2. Representative scheme of the synthesis.

(A) Acylation, (B) hydrolysis and (C) acetylation. Analogically used for 4-substituted and 2,4-disubstituted derivatives.

DCM: Dichloromethan; DMF: N,N-dimethylformamide.

Table 1. Antibacterial activity expressed as MIC in μM.^†

						Strain
Code	Y	Z	R^1	R^2	X	Staphylococcus aureus subsp. aureus	Staphylococcus aureus subsp. aureus methicillin-resistant	Staphylococcus epidermidis	Enterococcus faecalis	Escherichia coli	Klebsiella pneumoniae	Acinetobacter baumannii	Pseudomonas aeruginosa
1	N	N	OH	H	5–Cl	125	125	125	>500	>500	>500	>500	>500
1Ac	N	N	OAc	H	5–Cl	125	125	125	500	500	250	500	>500
2	N	N	OH	H	6–Cl	250	500	500	500	>500	>500	>500	>500
2Ac	N	N	OAc	H	6–Cl	125	125	62.5	250	500	250	250	>500
3	N	N	OH	H	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
3Ac	N	N	OAc	H	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
4	N	N	OH	H	–	>500	>500	>500	>500	>500	>500	>500	>500
4Ac	N	N	OAc	H	–	>500	>500	>500	>500	>500	>500	>500	>500
5	N	N	H	OH	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
5Ac	N	N	H	OAc	5–Cl	62.5	125	125	500	500	250	>500	>500
6	N	N	H	OH	6–Cl	>125	>125	>125	>125	>125	>125	>125	>125
6Ac	N	N	H	OAc	6–Cl	>125	>125	>125	>125	>125	>125	>125	>125
7	N	N	H	OH	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
7Ac	N	N	H	OAc	3–Cl	>125	>125	125	>125	>125	>125	>125	>125
8	N	N	H	OH	–	>500	>500	>500	>500	>500	>500	>500	>500
8Ac	N	N	H	OAc	–	>500	>500	>500	>500	>500	>500	>500	>500
9	N	N	OH	OH	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
9Ac	N	N	OAc	OAc	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
10	N	N	OH	OH	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
10Ac	N	N	OAc	OAc	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
11	N	N	OH	OH	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
11Ac	N	N	OAc	OAc	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
12	N	N	OH	OH	–	>500	>500	>500	>500	>500	>500	>500	>500
13	N	CH	OH	H	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
13Ac	N	CH	OAc	H	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
14	CH	N	OH	H	6–Cl	250	500	>500	>500	>500	>500	>500	>500
14Ac	CH	N	OAc	H	6–Cl	125	125	125	500	250	>500	>500	>500
15	N	CH	OH	H	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
15Ac	N	CH	OAc	H	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
16	CH	N	OH	H	5–Cl	250	500	500	>500	>500	>500	>500	>500
16Ac	CH	N	OAc	H	5–Cl	250	500	500	>500	500	>500	>500	>500
17	N	CH	OH	H	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
17Ac	N	CH	OAc	H	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
18	CH	N	OH	H	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
18Ac	CH	N	OAc	H	3–Cl	125	500	250	500	>500	500	>500	>500
19	N	CH	H	OH	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
19Ac	N	CH	H	OAc	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
20	CH	N	H	OH	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
20Ac	CH	N	H	OAc	6–Cl	>500	500	500	500	>500	>500	>500	>500
21	N	CH	H	OH	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
21Ac	N	CH	H	OAc	5–Cl	>125	>125	>125	>125	>125	>125	>125	>125
22	CH	N	H	OH	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
22Ac	CH	N	H	OAc	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
23	N	CH	H	OH	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
23Ac	N	CH	H	OAc	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
24	CH	N	H	OH	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
24Ac	CH	N	H	OAc	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
25	N	CH	OH	OH	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
25Ac	N	CH	OAc	OAc	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
26	CH	N	OH	OH	6–Cl	>500	>500	>500	>500	>500	>500	>500	>500
26Ac	CH	N	OAc	OAc	6–Cl	500	>500	>500	>500	>500	>500	>500	>500
27	N	CH	OH	OH	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
27Ac	N	CH	OAc	OAc	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
28	CH	N	OH	OH	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
28Ac	CH	N	OAc	OAc	5–Cl	>500	>500	>500	>500	>500	>500	>500	>500
29	N	CH	OH	OH	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
29Ac	N	CH	OAc	OAc	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
30	CH	N	OH	OH	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500
30Ac	CH	N	OAc	OAc	3–Cl	>500	>500	>500	>500	>500	>500	>500	>500

† Bold text indicates signifcance of the result.

Figure 3. Visualization of structure–activity relationships for antimycobacterial activity against Mycobacterium tuberculosis H37Ra.

The heat map represents the cumulative activity (reciprocal to MIC), from blue (lowest) to red (highest). See the online version of the manuscript for a colored version of the figure.

MIC: Minimum inhibitory concentration.

Table 2. Antimycobacterial activity expressed as minimum inhibitory concentration in μg/ml.

						Strain
Code	Y	Z	R^1	R^2	X	Mycobacterium tuberculosis H37Ra	Mycobacterium tuberculosis H37Rv	Mycobacterium kansasii	Mycolicibacterium avium	Mycobacterium aurum	Mycobacterium smegmatis
1	N	N	OH	H	5–Cl	15.625	12.5	7.81	15.625	15.625	7.81
1Ac	N	N	OAc	H	5–Cl	15.625	>100	1.98	7.81	15.625	15.625
2	N	N	OH	H	6–Cl	15.625	12.5	7.81	15.625	15.625	15.625
2Ac	N	N	OAc	H	6–Cl	7.81	12.5	7.81	7.81	15.625	7.81
3	N	N	OH	H	3–Cl	62.5	>100	62.5	62.5	62.5	125
3Ac	N	N	OAc	H	3–Cl	31.25	100	31.25	62.5	62.5	250
4	N	N	OH	H	–	≥500	100	15.625	125	≥500	≥500
4Ac	N	N	OAc	H	–	250	100	62.5	125	500	≥500
5	N	N	H	OH	5–Cl	≥500	50	15.625	250	500	62.5
5Ac	N	N	H	OAc	5–Cl	15.625	12.5	3.91	7.81	7.81	15.625
6	N	N	H	OH	6–Cl	Insoluble	>100	Insoluble	Insoluble	Insoluble	Insoluble
6Ac	N	N	H	OAc	6–Cl	Insoluble	>100	Insoluble	Insoluble	Insoluble	Insoluble
7	N	N	H	OH	3–Cl	≥500	>100	125	≥500	≥500	≥500
7Ac	N	N	H	OAc	3–Cl	125	>100	125	≥500	250	500
8	N	N	H	OH	–	≥500	>100	≥500	≥500	≥500	≥500
8Ac	N	N	H	OAc	–	≥500	>100	125	≥500	≥500	≥500
9	N	N	OH	OH	5–Cl	≥500	50	31.25	31.25	≥500	≥500
9Ac	N	N	OAc	OAc	5–Cl	125	100	31.25	62.5	125	125
10	N	N	OH	OH	6–Cl	125	50	62.5	125	125	≥500
10Ac	N	N	OAc	OAc	6–Cl	125	50	15.625	62.5	125	250
11	N	N	OH	OH	3–Cl	250	>100	250	≥500	250	500
11Ac	N	N	OAc	OAc	3–Cl	125	>100	125	250	250	250
12	N	N	OH	OH	–	≥500	>100	≥500	≥500	250	≥500
12Ac	N	N	OAc	OAc	–	NA	NA	NA	NA	NA	NA
13	N	CH	OH	H	6–Cl	125	50	7.81	31.25	≥500	≥500
13Ac	N	CH	OAc	H	6–Cl	15.625	50	7.81	15.625	31.25	31.25
14	CH	N	OH	H	6–Cl	31.25	50	15.625	15.625	31.25	≥500
14Ac	CH	N	OAc	H	6–Cl	15.625	50	7.81	31.25	31.25	62.5
15	N	CH	OH	H	5–Cl	≥500	>100	7.81	≥500	≥500	≥500
15Ac	N	CH	OAc	H	5–Cl	250	>100	7.81	7.81	250	≥500
16	CH	N	OH	H	5–Cl	31.25	12.5	15.625	31.25	31.25	62.5
16Ac	CH	N	OAc	H	5–Cl	31.25	50	15.625	31.25	62.5	62.5
17	N	CH	OH	H	3–Cl	31.25	>100	7.81	31.25	62.5	250
17Ac	N	CH	OAc	H	3–Cl	125	25	62.5	31.25	62.5	250
18	CH	N	OH	H	3–Cl	62.5	12.5	15.625	31.25	125	≥500
18Ac	CH	N	OAc	H	3–Cl	31.25	12.5	15.625	31.25	62.5	62.5
19	N	CH	H	OH	6–Cl	≥500	>100	3.91	≥500	≥500	≥500
19Ac	N	CH	H	OAc	6–Cl	≥500	>100	7.81	≥500	≥500	≥500
20	CH	N	H	OH	6–Cl	≥500	50	31.25	≥500	125	62.5
20Ac	CH	N	H	OAc	6–Cl	125	50	31.25	125	125	250
21	N	CH	H	OH	5–Cl	62.5	50	3.91	31.25	250	250
21Ac	N	CH	H	OAc	5–Cl	62.5	50	7.81	62.5	125	31.25
22	CH	N	H	OH	5–Cl	62.5	50	15.625	125	125	125
22Ac	CH	N	H	OAc	5–Cl	62.5	100	15.625	125	125	125
23	N	CH	H	OH	3–Cl	≥500	>100	250	≥500	500	≥500
23Ac	N	CH	H	OAc	3–Cl	500	>100	250	≥500	≥500	≥500
24	CH	N	H	OH	3–Cl	≥500	>100	125	125	≥500	≥500
24Ac	CH	N	H	OAc	3–Cl	≥500	>100	125	≥500	500	≥500
25	N	CH	OH	OH	6–Cl	≥500	>100	250	≥500	≥500	≥500
25Ac	N	CH	OAc	OAc	6–Cl	62.5	50	15.625	62.5	62.5	125
26	CH	N	OH	OH	6–Cl	≥500	6.25	500	≥500	≥500	≥500
26Ac	CH	N	OAc	OAc	6–Cl	250	25	31.25	250	62.5	≥500
27	N	CH	OH	OH	5–Cl	125	25	3.91	31.25	125	125
27Ac	N	CH	OAc	OAc	5–Cl	62.5	50	3.91	62.5	62.5	125
28	CH	N	OH	OH	5–Cl	≥500	>100	125	≥500	≥500	≥500
28Ac	CH	N	OAc	OAc	5–Cl	125	25	31.25	125	125	250
29	N	CH	OH	OH	3–Cl	125	50	62.5	125	250	≥500
29Ac	N	CH	OAc	OAc	3–Cl	125	50	62.5	125	250	≥500
30	CH	N	OH	OH	3–Cl	≥500	>100	≤≥500	≥500	≥500	≥500
30Ac	CH	N	OAc	OAc	3–Cl	125	>100	62.5	125	125	250
Isoniazid^†	–	–	–	–	–	0.5	0.2	6.25	1000	3.91	31.25

Bold text indicates significant results. Results are considered significant, if the MIC is ≤ 62.5 µg/ml.

† Standard drug: isoniazid. Results for other standards can be found in Supplementary Table 1.

MIC: Minimum inhibitory concentration; NA: Not available.

Figure 4. Results of macromolecular assay.

Inhibition of biosynthetic pathway is indicated by lower incorporation of radioactively labelled precursors (A) [³H] N-acetylglucosamine, (B) [³H] uridine, (C) [³H] thymidine and (D) [³H] Leu by methicillin-resistant Staphylococcus aureus (ATCC 43300) strain treated for 2 h at 4 × MIC of VAN, RIF, CIP, CHF, CHX, compound 1 or 5Ac and UCRL. Results are expressed as the percentage of untreated controls. The values shown are means of two independent experiments prepared in duplicates ± SEM. Dotted lines represent 100% incorporation of labelled macromolecules (UCRL). Significant reduction in biosynthetic pathway compared with UCRL is indicated by p-value where p < 0.05 was accepted as statistically significant determined by nonparametric one-way ANOVA test (Kruskal–Wallis test).

*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

ANOVA: Analysis of variance; ATCC: American Type Collection Cultures; CHF: Chloramphenicol; CHX: Chlorhexidine; CIP: Ciprofloxacin; RIF: Rifampicin; SEM: Standard error of the mean; UCRL: Untreated control; VAN: Vancomycin.

Figure 5. Membrane depolarization effect of compounds 1 and 5Ac on methicillin-resistant Staphylococcus aureus.

Measured by DiSC₃(5)-based kinetic fluorescence. The graph depicts the mean and SD of six replicates. The black arrow indicates the time of addition of tested compounds or positive control.

CHX: Chlorhexidine; SD: Standard deviation; RFU: Relative fluorescence units.

Table 3. Comparison of experimentally determined solubility expressed as log S for chosen compounds.

Code	Solubility μg/ml		Method^†	Experimental log S (mean ± SD)
	First run	Second run
1	9.84	8.85	Kinetic	-4.43 ± 0.02
15	2.17	2.45	Kinetic	-5.03 ± 0.03
16	20.89	20.25	Kinetic	-4.08 ± 0.01
1Na	13581	–	Thermodynamic	-1.30

† For methodology, see Supplementary materials.