Design, Synthesis And Evaluation Of Novel Small Molecules To Target Estrogen Receptor And Histone Deacetylases For Breast Cancer Therapy

The estrogen receptor(ER) is a ligand-regulated transcription factor and regulates many physiological and pathological processes, which also plays a predominant role in breast cancer growth. Breast cancer is the leading cause of cancer death in women. Therefore, ER is regarded as important pharmaceutical target for the treatment of breast cancer.Selective estrogen receptor modulators(SERMs), which act as agonist activity in some tissues while being antagonist activity in others, are the most recently approved class of first line drugs for the treatment of breast cancer such as tamoxifen is the first clinically useful SEMR for the prevention and treatment of breast cancer. Although current SERMs have clear advantages in the treatment of breast cancer, they retain some disadvantages, for instance, tamoxifen therapy is ineffective for ER(-) and roughly half of ER(+) tumors are insensitive or lose response to continued tamoxifen therapy and gain resistance. Thus, much effort has been made to develop an "ideal SERM" that is more effective or does not develop resistance. In recent years, our group has been engaged in the search of breast cancer drug candidates, some potential SERMs with three-dimensional core scaffolds have been designed and synthesized. Among them, exo-5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfo-nic acid phenyl ester(OBHS) was the best compound.On the other hand, accumulating evidence suggests that inappropriate or overexpression of estrogen receptor or Histone deacetylase has been linked to breast cancer. Therefore, to address the need for more effective drugs for breast cancer, we chose the clinically effective HDACi(SAHA) as the second component, and successfully synthesized three series of dual-action conjugates to target ER and HDAC.As part of our ongoing interest in the development of ER ligands having therapeutic efficacy on breast cancer, we have focused on the preparation and evaluation of novel ER ligands having a more three-dimensional character. Among them, exo-5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester(OBHS). Analysis of the X-ray crystal structure of complex of ERa-LBD with OBHS, we discovered that OBHS could yield overall partial antagonist by modulate helix 12 indirectly by dislocation of the C-terminus of helix 11. The phenyl sulfonate moiety plays an important role in forming partial antagonist activity of OBHS, and the region of the phenyl sulfonate moiety directed towards ER helix 11 actually has enough room to contain much larger, bulky side group. Furthermore, in contrast to the mechanism of action of OBHS, tamoxifen directly relocates helix 12 by the bulky side group, and OBHS has two 4-hydroxyphenyl substituents, one mimics the A-ring of estradiol, the other which can be replaced with the bulky side group and mimics the basic side chain of tamoxifen. Thus, we chose the clinically effective HDACi(SAHA) as the second component of our conjugate, and synthesis of novel OBHS-HDACi conjugates. These bifunctional OBHS-HDACi conjugates had good ER binding affinity and the compound 22a has the highest binding affinity for ERa, the RBA value is 12.2%. Compound 22a and 24q have the highest potency as an ERa antagonist, and the IC50s are 0.05μM and 0.13μM, respectively. Molecular modeling shows that these two compounds represent different antagonistic mechanism:the bulkier sulfonate side chain of OBHS-HDACi conjugate 22a accentuates this clash with helix 11, thus giving 22a potent ERa antagonist activity; compound 24q mimics the binding orientation of 4-hydroxytamoxifen, with the SAHA group not directly interacting with any helix 12 residues. Instead, the SAHA side chain of 24q forms hydrogen bond contacts with helix 3 which can induce subtle shifts in helix 3 that destabilize helix 12 and destroy the transcriptional coactivator-binding site, and again thereby reducing the AF2-meditated activity of ERa by an indirect mechanism. Therefore, contact with helix 3 may represent a novel epitope to generate a full ERa antagonist.Compared with the approved drug 4OHT, these conjugates exhibited higher antitumor potency aganist MCF-7 cells, and had no toxicity towards normal cells. Moreover, and also potently inhibited HDAC.2) Base on previous works in our lab, we found that replacing the sulfonate of OBHS with secondary or tertiary sulfonamides group gave OBHS-sulfonamide analogues(OBHSA), which show the activity of full antagonists. To enhance the antagonist properties of these OBHS-HDACi conjugates, which are partial antagonists on both ERs, we have synthesized of OBHS-sulfonamide-HDACi conjugates. These conjugates had moderate ERa affinity and compounds 28a and 28g have the highest binding affinity for ERa, the RBA values are 13.07% and 11.60%, respectively; moreover, these two compounds are the ERa full antagonists. In cell proliferation assays, N-methyl substituted compounds show significant antiproliferative effects on MCF-7 breast cancer cells, and compounds 28a and 28g exhibit more potent antiproliferative activity than 4OHT, and the IC50s are 8.4μM and 3.5μM, respectively. Additionally, all conjugates are nontoxic to healthy VERO cells, while 4OHT showed considerable toxicity3) Breast cancer is the most common cancer among women. One of the drugs most frequently used to treat hormone-dependent(ER+) breast cancer is the antiestrogen agent SERMs, while SERMs were not effective independent(ER-) breast cancer cells. Therefore, there is great interest in the discovery of new molecules that would be effective on ER+ and SERMs nonresponsive or resistant ER+breast cancer. We have synthesized a series of FcOBHS-HDACi conjugates containing ferrocene groups with inhibitory activity in both hormone-dependem(ER+) and independent(ER). Among them, suberic acid derivatives demonstrate better binding affinity for ERa than the pimelic acid and azelaic acid analogues. The compound 16b has the highest binding affinity for ERa, the RBA value is 3.28%, and which also are the ERa full antagonist with high potency(IC50=110 nM) and efficacious(Eff(%E2)=3.91). In cell proliferation assays, it was found that FcOBHS-HDACi conjugates showed not only inhibition effect in hormone-dependent breast cancer MCF-7cells but also in hormone-independent MDA-MB-231 cells, the best compound 16b with an IC50 value of 11.6μM for the ER-negative MDA-MB-231 cells.