148
Z. Zhang et al. / European Journal of Medicinal Chemistry 59 (2013) 141e149
19H16N2O, calcd for 288.1263, found 288.1269. 13C NMR (400 MHz,
16
14
12
10
8
C
CDCl3): d 160.25,153.60,147.97,137.18,133.57,132.45,130.91,129.10,
128.90, 128.35, 127.91, 123.06, 115.83, 105.51, 44.42. TOF MS (EIþ):
C19H16N2O, calcd for 288.1263, found 288.1269. HPLC system:
purity ¼ 99.75%, tR ¼ 17.20 min.
Bcl-xL
inhibitors
y = 0.0196x + 8.4383
Mcl-1
inhibitors
4.2. Isothermal titration calorimetry
6
y = 0.1697x + 1.8817
R² = 0.7235
Isothermal titration calorimetry (ITC) was performed using
iTC200 (Microcal). Experiments were performed in 20 mM Tris pH
8.0, 150 mM NaCl, 1% DMSO at 25 ꢂC Titrations consisted of
4
2
12 ꢃ 3
mL injections of compound at 300 mM into Mcl-1 (30 mM). All
0
sample data obtained after control data corrections were analyzed
to fit to a one-site model. For control ITC experiments, the sample
cells were filled with assay buffer and the compound solution was
added. This process was identical to that for protein samples.
0
10
20
30
40
50
60
HAC(heavy atom count)
Fig. 8. Free energy of binding (kcal molꢀ1) for all the reported Bcl-xL and Mcl-1
inhibitors plotted as a function of the number of heavy atoms in the compounds.
Acknowledgments
2.7. Comparing the contribution per atom of Mcl-1 inhibitors and
Bcl-xL inhibitors
The authors would like to thank Prof. Zhongjun Li (State Key
Laboratory of Natural and Biomimetic Drugs, Peking University,
Beijing, People’s Republic of China) for the discussion. The work
was supported by the Fundamental Research Funds for the
Central Universities (DUT11SM08) and partly supported by the
National Natural Science Foundation of China (81141099,
81272876).
The very high LE of fragment 4 toward Mcl-1 and the losing Bcl-
2 of 4g raised a possibility that the character of the hotspot of Mcl-1
might be divergent than that of Bcl-2/Bcl-xL, which may reflect on
the LE of their specific ligands. We then surveyed all the reported
Bcl-xL inhibitors and Mcl-1 inhibitors since Bcl-xL inhibitors are
more reported than Bcl-2 inhibitors. All these compounds were
Appendix A. Supplementary material
listed in Table S1 and S2. Their
DG against HAC was plotted as
shown in Fig. 8. From Fig. 8, we got two lines with different slopes.
Additionally, in the case of the Bcl-xL/Mcl-1 dual inhibitors (circled
in Fig. 8), we found that they showed a higher affinity to Mcl-1 than
to Bcl-xL. It suggested that the BH3 groove of Mcl-1 might be more
druggable than those of Bcl-xL.
Supplementary material associated with this article can be
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4. Experimental
4.1. Compound synthesis
The synthesis of 4g is shown in Supplementary material Scheme
S2. Yield: 339 mg, 59%. 1H NMR (400 MHz, CDCl3):
d 8.94 (br, t,
J ¼ 6.0 Hz, 1H), 8.02 (d, J ¼ 11.6, 1H), 7.71 (d, J ¼ 4.2 Hz, 1H), 7.69 (s,
1H), 7.16e7.51 (m, 10H), 4.40 (d, J ¼ 6.0 Hz, 2H). TOF MS (EIþ):