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A series of COX-2 selective inhibitor nimesulide derivatives were synthesized. Their anti cell proliferation activities were evaluated with a long term estrogen deprived MCF-7aro (LTEDaro) breast cancer cell line, which is the biological model of aromatase inhibitor resistance for hormone dependent breast cancer. Compared to nimesulide which inhibited LTEDaro cell proliferation with an IC50 at 170.30μM, several new compounds showed IC50 close to 1.0 μM.
About two-thirds of breast cancers are hormone-dependent, which contain estrogen receptors (ER) and require estrogen for tumor growth. These patients are therefore suitable candidates for hormonal therapy, which aims to block estrogen stimulation of breast cancer cell growth.1 Tamoxifen has been the mainstay of hormonal therapy in both early and advanced breast cancer patients for approximately three decades. However, aromatase inhibitors (AIs) are now proving to be more effective and to increase survival more than antiestrogens.2-5 Over recent years AIs have become the first-line endocrine therapy for ER positive patients with advanced breast cancer.6 However, after prolonged endocrine therapy, acquired resistance to AIs is expected to occur in a majority of breast cancer patients.7-10 The possible resistance mechanism has been investigated in preclinical models in our laboratory and others.8-11
The long-term estrogen deprivation (LTED) system has been used as a model for AI resistance in several laboratories, mainly due to its lack of a hormone environment that mimics the aromatase inhibition effect.8;12-15 It has been reported that the activation of the growth factor signaling pathways in LTED cell lines such as HER2 and insulin-like growth factor I receptor, which crosstalk with the ER signaling pathway resulting in an activation of various MAPKs and PI3K/AKT, is responsible for the cell survival and proliferation.8-10 Although ER is still functional in LTEDaro cells, the transactivation potential of ER is altered which suggests that ER transcriptional regulation function was partially lost. LTEDaro cell line is more like a late stage endocrine resistance model. Nevertheless, from drug discovery point of view, LTEDaro is a good model for the evaluation of potential compounds to overcome AI resistance. 15
Nonsteroidal anti-inflammatory drugs (NSAIDs) are beneficial in breast cancer treatment.16 It has been reported that COX-2 inhibitor nimesulide suppressed the development of 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP)-induced mammary gland carcinogenesis in rats.17 Other research demonstrated that nimesulide also suppressed aromatase activity and expression in several breast cancer cell lines.18 Nimesulide derivatives which do not have COX-2 inhibitory activity were more active than nimesulide to target aromatase.19;20 Further study reveals that several nimesulide analogs were able to selectively inhibit Her2 overexpressing breast cancer cell proliferation, which suggests that they are potentially able to overcome AI resistant breast cancer cell growth.21 Consequent investigations demonstrated that the compounds induce LTEDaro cell apoptosis, which exhibited that they can overcome AI resistance for hormone dependent breast cancer. Because of the unique character of nimesulide derivatives, we propose that the modification of the structure might change the drug from a COX-2 inhibitor to an anti-cancer agent.20 Furthermore, these new analogs selectively target Her2 overexpressing breast cancer cells which makes them good candidates to overcome AI resistance.
We try to further optimize the structure of nimesulide using the combinatorial strategies to modify the four positions depicted in Figure 1. Previous study demonstrated that B position as proton, or methyl group, is the best fit for the analogs to inhibit cancer cell growth. For C position, small methyl sulfonamide or acetyl groups is the best fit. Bulky groups will decrease the pharmacological activity.20 For A position, methyl group substituted benzyl is better for the activity.20 For D position, we will try pyridine group in this study. Since nitrogen containing heterocyclics can increase aromatase inhibition activity, according to several other reports.22;23 In the newly designed derivatives, we will keep B position as methyl group and C position as methyl sulfonamide. A was modified by using different positions and numbers of methyl group substituted benzyls. D will be kept as pyridine or hydrophobic groups (Scheme 1). These compounds and their biological activity will enable us to identify the key pharmacophore of this scaffold on the suppression of LTEDaro breast cancer cell growth.
The results suggest that A position as 2, 5 dimethyl or dichloro benzyl is the best fit. Compounds 33-36 and 58-61 are relatively more active, except compound 37 and 62 (Table 1). It seems that D position as a picolinyl group harms the biological activity. Only one methyl group substituted benzyl group at A position definitely decreases the activity. Compounds 38-47 show much lower activity compared with compounds 33-36. Compounds 48-57 are not as active as compounds 33-36, which suggests that the methyl group at 2, 5 position of benzyl at A position is very critical for the activity. Tri methyl groups clearly do not increase the activity, which has been demonstrated by relatively low activity of compounds 53-57. 4-isopropyl benzyl group or hexyl group at A position does not help the activity based on the biological results of compounds 63-72. However, 2, 5 dichloro benzyl group at A position can slightly increase the activity. Compounds 58 and 59 show better activity compared with compounds 33 and 34.24 Overall, nitrogen containing aromatic group at D position does not increase the biological activity, even though compound 36 is slightly more potent than compounds 33 and 34.
In brief, we optimized nimesulide structure and developed several more potent analogs, such as compounds 36, 58 and 59, which inhibit LTEDaro cell growth with IC50 of 1.69 ± 0.25 μM, 1.00 ± 0.39 μM and 2.15 ± 0.54μM, respectively. Compared with nimesulide with IC50 of 173.30 ± 20.30 μM, the new derivatives have much more potent pharmacological activity against LTEDaro breast cancer cell growth. Structure activity relationship study suggests that A position needs 2,5-dimethyl or dichloro benzyl group to increase the biological activity. The exact biological mechanism of the compound is still under investigation in our laboratory.
This work was supported by grants from the National Institutes of Health CA44735 (SC), ES08528 (SC).
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