Table 1. Structure–activity relationships in the anilide ring.

Compound number	Structure R	Fzd1-GFP Internalization @12.5 μMa	Inhibition Wnt/β-catenin transcription TopFlash IC50 (μM) ± SE	ClogP	pKa NHb
1		5	0.34 ± 0.08	3.1	11.2
2		0	11.81	3.3	13.2
3		5	0.99 ± 0.32	2.7	12.1
4		0	6.42 ± 0.51	3.0	12.5
5		0	>12	1.9	12.7
6		0	7.66	2.6	11.4
7		0	15.04	4.2	12.5
8		0	32.89	4.1	12.6
9		5	0.56 ± 0.21	4.8	11.9
10		5	0.29 ± 0.06	4.5	12.4
11		5	0.89 ± 0.32	4.7	12.5
12		5	0.55 ± 0.11	4.5	11.8
13		3	1.30 ± 0.18	4.0	12.8
14		5	1.65 ±0.12	4.0	12.6
15		1	3.05 ± 0.87	3.9	12.3
16		5	0.42 ± 0.10	4.5	11.9
17		5	0.75 ± 0.13	4.4	11.7
18		5	0.70 ± 0.13	4.5	12.0
19		5	0.73 ± 0.13	4.5	12.2
20		0	>12	4.3	11.4
21		0	3.84 ± 0.29	3.9	13.1
22		2	4.02 ± 0.79	4.0	12.6
23		ND	>12	3.9	12.1
24		3	1.41 ± 0.21	4.4	12.2
25		5	1.21 ± 0.17	4.4	12.5

Internalization of Frizzled1-GFP stably expressed in U2OS cells was determined by confocal microscopy. Cells were treated with compounds for 6 h, fixed, and scored visually by the amount of punctate observed versus DMSO control. Inhibition of Wnt3A-stimulated Wnt/β-catenin transcription was determined by TOPFlash normalized to renilla control. ClogP and pK_a values were calculated in Maestro 9.3 (Schrodinger, Inc.) using Qikprops and Epik.

^aPunctate similar to control = 0, trace amount of punctate greater than control = 1, moderate = 3, strong = 5.

^bCalculated pK_a values in water are reported. The calculated value of the pK_a of the –OH group is 6.8 in each example listed. The calculated pK_a of the NH is the salicylamide NH without ionization of the –OH group. The estimated error of the pK_a calculation for the OH group is ±1 log unit, and ±2 for the NH group. ND: not determined.