Fig 1.
Targeted modification to design new UC-112 analogs.
Fig 2.
Reagents and conditions: (a) conc. HCl, ZnCl2, HCHO(37% in H2O); (b) substituted benzyl alcohol, NaH, THF, reflux; (c) substituted benzyl alcohol, 110 oC; (d) NH4OH, H2O, Et2O, pH 8–10; (e) paraformaldehyde, pyrrolidine,EtOH, reflux.
Fig 3.
Reagents and conditions: (a) R2CH2OH, NaH, THF, reflux; (b) R2CH2OH, 110 oC; (c) NH4OH, H2O, Et2O, pH 8–10; (d) paraformaldehyde, pyrrolidine,EtOH, reflux.
Fig 4.
Reagents and conditions: (a) benzyl alcohol, 110 oC; (b) NH4OH, H2O, Et2O, pH 8–10; (c) paraformaldehyde, piperidine for 8a, morpholine for 8b,EtOH, reflux.
Fig 5.
Synthesis of compounds 10a-10b.
Reagents and conditions: (a) 2-phenylethanol for 9a, 3-phenyl-1-propanol for 9b, 110 oC; (b) NH4OH, H2O, Et2O, pH 8–10; (c) paraformaldehyde, pyrrolidine, EtOH, reflux.
Fig 6.
Synthesis of compounds 12a-12b.
Reagents and conditions: (a) benzyl mercaptan for 11a, N-Benzylmethylamine for 11b, NaH, THF, reflux; (b) paraformaldehyde, pyrrolidine,EtOH, reflux.
Table 1.
In vitro growth inhibitory effects of UC-112 analogs with C ring substitutions.
Table 2.
In vitro growth inhibitory effects of C ring modified UC-112 analogs.
Table 3.
In vitro growth inhibitory effects of D ring modified UC-112 analogs.
Table 4.
In vitro growth inhibitory effects of UC-112 analogs with different chain lengths.
Table 5.
In vitro growth inhibitory effects of UC-112 analogs with different linkers.
Fig 7.
Average GI50 data for UC-112, compound 12c, 4c and 4g tested in NCI-60 anti-proliferative screening.
Data is shown with mean ± SD as bar graph.
Fig 8.
Heat map showing the GI50 values (nM) for UC-112 and three analogs in the NCI-60 screening.
High intensity (blue) cells indicate high activity and low intensity (red) cells indicate low activity. Average GI50 values were calculated for each compound, separately.
Table 6.
Aqueous solubility and in vitro metabolism properties of compound 4g.
Table 7.
Cytochrome P450 inhibition effects of compound 4g.
Fig 9.
Western blotting assay of A375 and PC-3 cells treated with gradient increasing dose of 4f or 4g for 24 h.
Left panel is in A375 cancer cell line. Right panel is in PC-3 cancer cell line.
Fig 10.
Relative in vitro caspases 3/7 activity of human melanoma A375 or human prostate cancer PC-3 cells were evaluated after treatment of UC-112 analogs (1 μM) for 24 h (N = 3).
The luminescence unit data was adjusted according to the cell viability results read from the same well in 96-well plate. ***P < 0.001, **P < 0.01 compared with corresponded results from control group.
Fig 11.
Potential binding pose of UC-112 and compound 4g in the SMAC N-terminus tetra-peptide AVPI binding site of survivin crystal structure (PDB entry: 3UIH).
The surface of AVPI binding site in survivin was colored according to residue charge (blue for positive while red for negative). (a) UC-112 (green tube) formed hydrogen bonds with residue Asp71 and Glu68 on the survivin protein BIR domain. (b) Compound 4g (orange tube) displayed similar binding pose with UC-112 but had better occupied the grove toward the N-terminus of survivin protein. And this pose was overlapping well with the binding mode of native ligand, SMAC AVPI (green stick).
Fig 12.
Representative drug affinity responsive target stability (DARTS) results for pronase-digested A375 or M14 cell lysates.
Immunoblotting showed protection of the target protein, survivin, by incubation with compound 4g at the concentration of 20 μM, whereas digestion of the non-target proteins like GAPDH was unchanged.
Fig 13.
In vivo anti-tumor efficacy of compound 4g (N = 6).
(a) 4g effectively inhibited the growth of A375 xenograft tumor after three weeks continuous treatment (i.p. injection) in a dose-dependent manner (left panel) without causing obvious decrease of mice body weight (right panel). (b) Western blotting results on the A375 xenograft tumor tissues (each lane represents one single mice). (c) Representative images of TUNEL assay using the formalin-fixed tumor sections.
Fig 14.
Structure activity relationships of UC-112 analogs.