Table 1.

Data collection and refinement statistics

Data collection	ALDH3A1-CB29 complex
Space group	P1
Cell dimensions
a,b,c [Å]	89.1, 95.4, 117.2
α,β,γ [°]	112.4, 91.7, 91.0
R merge	0.067 (0.25)
I/σI	10.4 (3.1)
Completeness	93.6
Redundancy	2.2
Refinement
Resolution [Å]	50-2.5
No. of reflections	109,547
Rwork/Rfree	0.23/0.25
No of atoms
Protein	28003
Ligand	192
Water	218
B-factor (overall)	25.2
RMSD Bond angles [Å]	1.15
RMSD Bond Length [A]	0.008

Our results showed that an aniline at the R2 position was required for inhibition of ALDH3A1 [Table 3, compare 15 (NI), 16 (NI) with CB29 (IC₅₀ = 16 μM), 17 (IC₅₀ = 27 μM) and 18 (IC₅₀ = 30 μM)]. Even the substitution of an ether linkage greatly reduced potency [Table 3, compare CB29 (IC₅₀ = 16 μM) with 20 (IC₅₀ = 100 μM)]. We evaluated a series of anilines at the R2 position with substitutions at the ortho, meta and para positions. Compounds with substituents at the ortho position (R4/R8) lost all activity toward ALDH3A1 [Table 2, 7 (NI)]. Compounds with substitutions at the meta positions (R5/R7) showed similar potencies to CB29 [Table 2, 8 (IC₅₀ = 10 μM) and 9 (IC₅₀ = 26 μM)]. Finally, we examined substitutions at the para (R6) position. Since our parent compound CB29 had an acetamide at this position, we looked for analogs having ether instead of amine linkage at the corresponding position (10 and 11). This substitution yielded compounds with similar potencies [10 (IC₅₀ = 31 μM), 11 (IC₅₀ = 24 μM)], which is consistent with the structural data that shows no hydrogen bonding from this nitrogen to the enzyme. We next looked larger amide substitutions at the R6 position. Here a surprising pattern was seen, when the acetamide was substituted with isobutyramide (12) or isopentanamide (13), these two compounds were inhibitory towards both ALDH1A1 and ALDH3A1 (Table 2). In addition, analogs with larger amides at the R6 position and larger substitutions at the R1 position (14) showed greater potency toward ALDH1A1 than toward ALDH3A1 (Table 2). In contrast, as mentioned above when the acetamide group was held constant and the larger morpholine was introduced at the R1 position (1), the compound lost all inhibitory potency toward either ALDH1A1or ALDH3A1. Interestingly, none of the compounds tested, except for 13, showed any inhibitory effect on ALDH1A2, ALDH1A3 and ALDH1B1 activity. Compound 13 showed some inhibitory effect on ALDH1A2 activity (~50% at 100 μM concentration) with no effect on ALDH1A3, ALDH1B1, or ALDH2. Based on SAR study, we chose compound CB29, 17, 2, 18, 19, 11, 8, 9 and 10 for phenotypic studies since these compounds were selective toward ALDH3A1 and showed no inhibition toward ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1 and ALDH2 activity in vitro.