Synthesis and structure–activity relationships of pyrazole-based inhibitors of meprin α and β

Figures & data

Figure 1. Examples of known inhibitors of meprin α and β.

Figure 2. Protease domains of meprin α and β. Possible modifications of the pyrazole scaffold to address and modulate interactions with the S1, S1′, or S2′ pocket of meprin α and β are depicted.

Scheme 1. Synthesis of 3,5-diarylpyrazole hydroxamic acid derivatives with variation of one arylmoiety. Reagents and conditions: (a) Bn-ONH₂*HCl, TBTU, DIPEA, DMF, RT (66%); (b) R²-COCl or acid anhydride, LiHMDS, toluene, 0 °C to RT (for 13a–f,q, 26–74%); (c) i: R²-COOH, CDI, THF, RT, ii: LiHMDS, toluene, 0 °C to RT (for 13g–p,r–t, 16–90%); (d) N₂H₄*H₂O, AcOH, toluene, EtOH, THF, RT to 50 °C; (e): H₂, 4 bar, Pd/C, MeOH/THF (1:1, v/v), RT (for 14d–g, 41–82%); (f) BBr₃, DCM, 0 °C to RT (for 14a–c and h–t, 2–45%).

Scheme 2. Synthesis of functionalised 3,5-diarylpyrazole hydroxamic acid derivatives with variation of both aryl moieties. Reagents and conditions: (a) BrCH₂COOMe for 10a, BrCH₂COOtBu for 10b–d, DMPU, LiHMDS, THF, −60 °C to RT (60–100%); (b) LiOH*H₂O, THF/H₂O (3:1, v/v), RT (for 11b, 99%); (c) TFA/DCM (1:1, v/v), RT (for 11c–e, 71–99%); (d) Bn-ONH₂*HCl, TBTU, DIPEA, DMF, RT (48–78%); (e) R²-COCl or acid anhydride, LiHMDS, toluene, 0 °C to RT (for 15a,k); (f) i: R²-COOH, CDI, THF, RT, ii: LiHMDS, toluene, 0 °C to RT (for 15b–j,l,m); (g) N₂H₄*H₂O, AcOH, toluene, EtOH, THF, RT to 50 °C (7–56%); (h): H₂, 4 bar, Pd/C, MeOH/THF (1:1, v/v, for 16a,e,f,k, 10–69%), RT; (i) BBr₃, DCM, 0 °C to RT (for 16b–d, g–j, l, m, 1–26%).

Table 1. Inhibition of meprin α and β by 3,4,5-substituted pyrazoles (structural variation of one aryl moiety).

	R^2	Ki^(app) (nM)^a		SF
		Meprin α	Meprin β
7a		1.3 (1.19)	115.8 (1.08)	116
14a		302.0 (1.12)	2110.8 (1.10)	7
14b		69.7 (1.05)	1733.4 (1.11)	25
14c		5.6 (1.11)	319.7 (1.06)	53
14d		1.1 (1.10)	116.0 (1.14)	105
14e		1.3 (1.14)	62.0 (1.04)	48
14f		1.1 (1.09)	109.1 (1.09)	99
14g		0.7 (1.31)	83.2 (1.05)	117
14h		4.8 (1.10)	334.7 (1.06)	67
14i		13.6 (1.04)	995.0 (1.14)	71
14j		7.5 (1.33)	47.5 (1.16)	6
14k		17.2 (1.16)	116.0 (1.15)	7
14l		2.2 (1.23)	60.6 (1.10)	31
14m		0.1 (1.62)	0.4 (1.14)	3
14n		15.9 (1.26)	13.9 (1.08)	1
14o		1.6 (1.14)	88.4 (1.12)	55
14p		0.7 (1.09)	6.7 (1.15)	10
14q		0.3 (1.35)	32.6 (1.16)	127
14r		1.4 (1.25)	54.5 (1.15)	39
14s		11.6 (1.15)	97.7 (1.09)	8
14t		9.9 (1.18)	34.9 (1.15)	4

SF: selectivity factor (K_i^(app) meprin β/K_i^(app) meprin α).

^aGeometric mean of three independent experiments with standard deviation factor.

Table 2. Inhibition of meprin α and β by 3,4,5-substituted pyrazoles (structural variation of both aryl moieties).

	R^1	R^2	Ki^(app) (nM)^a		SF
			Meprin α	Meprin β
16a			0.6 (1.08)	112.9 (1.12)	177
16b			9.9 (1.00)	14.5 (1.22)	1
16c			55.8 (1.03)	116.3 (1.01)	2
16d			4.2 (1.08)	13.9 (1.12)	3
16e			3.1 (1.14)	44.4 (1.04)	15
16f			10.5 (1.05)	92.8 (1.02)	9
16g			20.3 (1.02)	32.5 (1.17)	2
16h			9.6 (1.05)	11.2 (1.06)	1
16i			5.1 (1.02)	4.0 (1.51)	1
16j			0.1 (1.35)	0.2 (1.13)	2
16k			0.4 (1.21)	5.2 (1.16)	13
16l			19.8 (1.10)	20.9 (1.16)	1
16m			21.0 (1.14)	14.5 (1.08)	0.7

SF: selectivity factor (K_i^(app) meprin β/K_i^(app) meprin α).

^aGeometric mean of three independent experiments with standard deviation factor.

Scheme 3. Synthesis of N-substituted 3,5-diarylpyrazole hydroxamic acid derivatives. Reagents and conditions: (a) i: NaH, DMF, 0 °C, ii: R³-Hal, 0 °C to RT (19a–c,f and 20, 71–98%); (b) TFA/DCM (1:1, v/v), RT (for 19d,e, 94–99%); (c) NH₂OH*HCl, NaOCH₃, MeOH, microwave, 80 °C (for 21a,e–h, 5–53%); (d) BBr₃, DCM, 0 °C to RT (for 21i and 22, 23–54%); (e) R³-B(OH)₂, Cu(OAc)₂, TEA, molecular sieves 3 Å, DCM, RT (25–30%); (f) TFA/DCM (1:1, v/v), TIS, RT (for 21b–d, 39–55%).

Table 3. Inhibition of meprin α and β by N-substituted 3,5-diphenylpyrazoles.

	R^3	n	Ki^(app) (nM)^a		SF
			Meprin α	Meprin β
7a		1	1 (1.19)	116 (1.08)	116
21a		1	5 (1.02)	437 (1.08)	87
21b		1	4 (1.04)	667 (1.09)	167
21c		1	4 (1.08)	212 (1.06)	53
21d		1	4 (1.12)	182 (1.09)	46
21e		1	1 (1.08)	291 (1.12)	291
21f		1	0.6 (1.23)	305.0 (1.02)	508
21g		1	0.3 (1.12)	10.6 (1.14)	35
21h		1	0.4 (1.36)	9.4 (1.16)	24
21i		1	0.08 (1.30)	9.3 (1.04)	116
22		2	0.15 (1.26)	1183.8 (1.09)	7892

SF: selectivity factor (K_i^(app) meprin β/K_i^(app) meprin α).

^aGeometric mean of three independent experiments with standard deviation factor.

Figure 3. Putative binding mode of 14m found by docking to the active site of meprin α (A) and meprin β (B).

Figure 4. Docking solutions of 21i (A) and 22 (B) to the active site of meprin α in comparison to 14m (light blue).

Table 4. Inhibition of off-target metalloproteases by selected 3,4,5-substituted pyrazole inhibitors.

	Residual activity (%)^a					Cell viability (%)^b
	MMP2	MMP9	MMP13	ADAM10	ADAM17	Hep-G2	SY5Y
7a	104	81	75	73	61	100	92
14d	78	80	85	68	52	104	84
14f	41	72	71	59	51	96	90
14g	83	90	101	87	65	99	85
14m	66	86	88	104	68	103	86
16a	29	63	56	62	57	n.d.	n.d.
16j	95	94	105	90	99	98	110
21f	77	74	78	69	21	103	88
21g	89	83	83	92	84	99	85
21h	85	80	86	91	67	103	88
21i	69	70	84	85	72	107	79
22	102	100	98	117	90	100	98

n.d.: not determined.

^aResidual activity (%) @ 10 µM inhibitor concentration; mean of at least two independent experiments.

^bInhibitor concentration 30 µM.

Table 5. Inhibitory activities (IC₅₀) against MMP2, MMP13, ADAM10, ADAM17 of selected compounds.

IC50 (µM)^a	MMP2	MMP13	ADAM10	ADAM17
14d	n.d.	n.d.	n.d.	11.6 (1.05)
14f	5.3 (1.11)	n.d.	13.9^b	7.4 (1.10)
16a	1.4 (1.34)	15.7 (1.53)	12.0 (1.02)	9.6 (1.06)
21f	n.d.	n.d.	n.d.	2.3 (1.22)

n.d.: not determined.

^aIC₅₀ (µM) as geometric mean of two independent experiments with standard deviation factor.

^bOnly single measurement.