A novel N-heterocycles substituted oseltamivir derivatives as potent inhibitors of influenza virus neuraminidase: discovery, synthesis and biological evaluation

Figures & data

Figure 1. Structures of approved agents for the treatment of influenza viral infection.

Figure 2. Comparison of the crystal structures of representative group-1 Nas (N1, PDB code: 2HU0, N8, PDB code 2HT7), group-2 Nas (N2, PDB code 4GZP), and 09N1 (PDB code 3TI6).

Figure 3. Structures of our previously reported group-1-specific influenza NA inhibitors (JMC23d and JMC21h) and non-specific group-1 and group-2 NA inhibitors (JMC15b and JMC15c).

Figure 4. The novel designed oseltamivir analogues via targeting the 150-cavity.

Scheme 1. Reagents and conditions: (i) Corresponding bromo-substituted aromatic heterocycle, K₃PO₄, Pd(PPh₃)₄, N₂, toluene/H₂O = 25/2, 100 °C, 12 h; (ii) NaBH₃CN, CH₃OH, r.t., 6–7 h; (iii) 1 M NaOH, CH₃OH, r.t., then 3 M HCl.

Scheme 2. Reagents and conditions: (i) Corresponding amine, K₂CO₃, DMF, 100 °C, 12 h; (ii) NaBH₃CN, CH₃OH, r.t., 6–7 h; (iii) 1 M NaOH, CH₃OH, r.t., then 3 M HCl.

Table 1. Neuraminidase (NA) inhibition of oseltamivir derivatives in chemiluminescence-based assay. .

Compds.	R	NA Enzyme-Inhibitory Assay, IC50 (μM)^a
		H1N1^b	H3N2^c	H5N1^d	H5N6^e	H5N1-H274Y^f
		group-1	group-2	group-1	group-2	group-1
3a		4.50 ± 0.69	4.63 ± 0.52	0.85 ± 0.10	0.46 ± 0.061	> 40
3b		4.20 ± 0.11	9.04 ± 0.74	1.17 ± 0.11	0.67 ± 0.10	> 40
3c		12.14 ± 1.10	7.77 ± 0.72	8.25 ± 0.25	5.37 ± 0.062	> 40
3d		4.34 ± 0.36	3.74 ± 0.19	2.63 ± 0.12	1.76 ± 0.040	> 40
3e		8.06 ± 0.48	5.62 ± 0.31	3.80 ± 0.19	2.04 ± 0.15	> 40
6a		3.94 ± 0.11	0.27 ± 0.052	2.08 ± 0.18	1.36 ± 0.11	> 40
6b		0.40 ± 0.033	0.27 ± 0.034	0.39 ± 0.045	0.26 ± 0.015	20.97 ± 0.98
6c		1.40 ± 0.073	0.76 ± 0.024	0.81 ± 0.18	0.57 ± 0.022	> 40
6d		3.10 ± 0.24	1.00 ± 0.11	1.11 ± 0.065	0.59 ± 0.057	> 40
6e		1.99 ± 0.19	0.83 ± 0.034	1.10 ± 0.13	0.75 ± 0.10	> 40
6f		0.43 ± 0.036	0.21 ± 0.019	0.24 ± 0.0078	0.18 ± 0.0099	15.77 ± 0.87
6 g		0.19 ± 0.0096	0.16 ± 0.007	0.32 ± 0.015	0.25 ± 0.015	6.69 ± 0.045
6h		0.30 ± 0.0016	0.25 ± 0.013	0.49 ± 0.025	0.50 ± 0.012	6.01 ± 0.24
6i		0.15 ± 0.031	0.16 ± 0.011	0.26 ± 0.015	0.19 ± 0.043	3.76 ± 0.15
6j		0.14 ± 0.0064	0.21 ± 0.017	0.59 ± 0.041	0.27 ± 0.0077	11.61 ± 1.56
6k		0.084 ± 0.0072	0.12 ± 0.0063	0.23 ± 0.032	0.15 ± 0.015	3.05 ± 0.47
6 l		0.23 ± 0.025	0.40 ± 0.0057	0.69 ± 0.047	0.24 ± 0.018	11.47 ± 1.67
6 m		0.37 ± 0.021	0.43 ± 0.031	0.84 ± 0.093	0.18 ± 0.023	14.79 ± 0.21
6n		0.14 ± 0.010	0.21 ± 0.0061	0.42 ± 0.0085	0.18 ± 0.028	11.23 ± 0.59
6o		0.36 ± 0.018	0.56 ± 0.037	0.89 ± 0.072	0.50 ± 0.031	21.74 ± 0.72
6p		0.17 ± 0.0041	0.31 ± 0.0078	0.52 ± 0.080	0.33 ± 0.020	11.88 ± 0.72
OSC	–	0.015 ± 0.00036	0.028 ± 0.0010	0.050 ± 0.0087	0.016 ± 0.0011	2.10 ± 0.26

^aIC₅₀: concentration required to reduce NA activity to 50% of control NA activity Values derive from three experiments and are shown as the mean ± SD.

^bA/PuertoRico/8/1934.

^cA/Babol/36/2005.

^dA/Goose/Guangdong/SH7/2013.

^eA/Duck/Guangdong/674/2014.

^fA/Anhui/1/2005.

Table 2. Anti-influenza virus activity and cytotoxicity of selected compounds in CEFs.

Compds.	R	EC50^a values (μM) towards influenza viruses		CC50 (μM)^b
		H5N1^c	H5N6^d
		group-1	group-2
6g		2.81 ± 0.027	4.06 ± 0.22	>200
6i		2.99 ± 0.44	5.14 ± 0.85	>200
6j		2.34 ± 0.42	5.41 ± 0.51	>200
6k		0.98 ± 0.071	9.29 ± 1.40	>200
OSC	–	0.85 ± 0.16	1.44 ± 0.13	>200

^aEC₅₀: concentration of compound required to achieve 50% protection of CEF cultures against influenza virus-induced cytopathic effect, presented as the mean ± SD.

^bCC₅₀: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the CCK-8 method.

^cA/Goose/Guangdong/SH7/2013.

^dA/Duck/Guangdong/674/2014.

Table 3. Anti-influenza virus activity and cytotoxicity of selected compounds in MDCK cells.

		EC50^a values (μM) towards influenza viruses
Compounds.	R	H1N1^c	H3N2^d	CC50 (μM) ^b
group-1	group-2
6g		0.03 ± 0.03	0.16 ± 0.08	>250
6i		0.03 ± 0.03	0.32 ± 0.08	>250
6j		0.03 ± 0.02	0.80 ± 0.003	>250
6k		0.02 ± 0.01	0.12 ± 0.03	>250
OSC	–	0.02 ± 0.01	0.36 ± 0.16	>250
ZA	–	0.009 ± 0.002	0.27 ± 0.03	>250

^aEC₅₀: compound concentration that inhibits 50% of virus plaque formation in MDCK cells infected with influenza A viral strains, presented as the mean ± SD and determined by the PRA method.

^bCC₅₀: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method.

^cA/PR/8/34.

^dA/Wisconsin/67/05.

Figure 5. Docking of compound 6k in the binding site on NA of N1 (PDB ID: 3BEQ), N1 (PDB ID: 2HU0) and N2 (PDB ID: 4K1K). (A, B, C) Superposition of 6k and OSC with N1 NA and N2 NA, respectively; (D, E, F) The key interactions formed by OSC with N1 and N2 NA, respectively; (G, H, I) The key interactions formed by 6k with N1 and N2 NA, respectively. Hydrogen bonds are shown as dashed lines (yellow).

Figure 6. The result plots of molecular dynamics simulations. (A) The protein backbone RMSD plot of the NA with 6k binding throughout the MD trajectory of 200 ns; (B) The ligand heavy atom RMSD plot when bound to the NA; (C) RMSF plot of the protein chain in 6k bound state.

Figure 7. The neuraminidase inhibitory activity assay of 6k and OSC with different incubation time with NA. (A) The dose–response–inhibition curve of 6k at different incubation time; (B) The dose-response–inhibition curve of OSC at different incubation time.

Table 4. Physicochemical properties of some representative compounds.

Parameter items^a	6g	6i	6j	6k
Fsp^3 (>0.42)	0.62	0.66	0.58	0.62
MW (<500 Da)	455.60	497.68	471.60	457.62
nON (≤10)	7	7	8	7
nOHNH (≤5)	3	3	3	3
nrotb (≤10)	10	10	10	10
TPSA (<140 Å^2)	90.90	90.90	100.13	90.90
MV	439.75	490.16	448.74	450.33
miLog p (<5)	3.23	4.29	2.22	3.00

^aMW: molecular weight; nON: number of hydrogen bond acceptors; nOHNH: number of hydrogen bond donors; nrotb: number of rotatable bonds; TPSA: topological polar surface area;

MV: molar volume; miLog P: molinspiration predicted Log P.

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