Anti-influenza A virus activity and structure–activity relationship of a series of nitrobenzoxadiazole derivatives

Figures & data

Figure 1. Most representative commercially available anti-IV drugs.

Figure 2. Most relevant benzoxadiazole derivatives (A–D) previously reported as anti-IV A agents^24,25.

Figure 3. Structures of the compounds (1–45) evaluated in the study.

Scheme 1. Compound 9 preparation. Reagents and conditions: (a) succinic anhydride, DMAP, dry DCM, reflux.

Scheme 2. Reagents and conditions: (a) succinic anhydride, DMAP, dry DCM, reflux.

Scheme 3. Preparation of compounds 19, 22, and 23. Reagents and conditions: (a) appropriate thiol (R-SH), pyridine, EtOH:H₂O (0.3:1 v/v), rt.

Table 1. Antiviral activity and cytotoxicity of NBD derivatives 1–45.

Compd	X	R	IC50 (µM)	Cytotoxicity
1	S		–	Toxic^a
2	S		–	Toxic^a
3	S		–	Toxic^a
4	S		–	Toxic^a
5	S		–	Toxic^a
6	S		–	Toxic^a
7	S		–	Toxic^a
8	S		Inactive^b	NOT toxic
9	S		17.1 ± 0.9	Toxic from 30 µM
10	S		22.2 ± 1.4	NOT toxic
11	S		34.9 ± 3.6	NOT toxic
12	S		1.1 ± 0.1	Toxic from 20 µM
13	S		–	Toxic^a
14	S		Inactive^b	NOT toxic
15	S		–	Toxic^a
16	S		–	Toxic^a
17	S		4.0 ± 0.4	Toxic from 20 µM
18	S		–	Toxic^a
19	S		24.0 ± 2.3	NOT toxic
20	S		–	Toxic^a
21	S		Inactive^b	NOT toxic
22	S		–	Toxic^a
23	S		Inactive^b	NOT toxic
24	S		–	Toxic^a
25	S		6.3 ± 0.5	Toxic from 20 µM
26	S		–	Toxic^a
27	S		Inactive^b	NOT toxic
28	S		Inactive^b	NOT toxic
29	S		–	Toxic^a
30	S		Inactive^b	NOT toxic
31	S		Inactive^b	NOT toxic
32	S		–	Toxic^a
33	S		Inactive^b	NOT toxic
34	S		–	Toxic^a
35	S		–	Toxic^a
36	S		–	Toxic^a
37	S		Inactive^b	NOT toxic
38	S		Inactive^b	NOT toxic
39	S		Inactive^b	NOT toxic
40	S		18.3 ± 1.6	Toxic from 25 µM
41	S		10.8 ± 0.6	Toxic from 20 µM
42	S		17.2 ± 1.6	NOT toxic
43	SO		Inactive^b	NOT toxic
44	SO2		Inactive^b	NOT toxic
45	NH		Inactive^b	NOT toxic

^aToxic at all tested concentrations (1 − 30 µM) as evaluated by both MTT and Trypan Blue exclusion assays. ^bAt maximum tested concentration (30 µM).

Figure 4. ICW assay on A549 cells infected with PR8 and treated with NBD derivatives 12, 17 and 25 (concentration range 1–20 μM). Left panel: The integrity of cell monolayer was revealed by Cell Tag on the 680 nm channel (red); viral NP expression on the 800 nm channel (green); merged images show the overlapping between viral protein and infected cells (yellow). Right panel. Fluorescence intensities determined by the Odyssey software and the ratios betwen NP and Cell Tag signal were calculated and averaged for duplicate wells. The values are shown as a function of compounds concentration. Error bars indicate s. d. The percentage (%) of fluorescence intensity was calculated respect to untreated infected cells (considered as 100%).

Table 2. Antiviral activity and selectivity of compounds 12, 17, and 25 in A549 cells as determined by both HAU and ICW assays.

Compd	Structure	HAU – A549		ICW – A549
		IC50 (µM)	SI^a	IC50 (µM)	SI^b
12		5.1 ± 0.2	11	10.0 ± 0.5	5
17		11.7 ± 1.0	2	9.6 ± 0.8	3
25		1.9 ± 0.1	6	8.9 ± 0.6	4

^aSI: CC₅₀/IC₅₀ HAU – A549. ^bSI: CC₅₀/IC₅₀ ICW – A549.

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