Page 1 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of Medicinal Chemistry
Design, Synthesis and Biological Evaluation of a Series of anthracene-9, 10-dione dioxime -catenin Pathway Inhibitors Raffaella Soldi§, Stephen K. Horrigan≠, Marek W. Cholody±, Janak Padia∞, Venkataswamy Sorna §, Jared Bearss§, Glynn Gilcrease§, Kapil Bhalla¶, Anupam Verma£, Hariprasad Vankayalapati§*, Sunil Sharma§*. §Division
of Oncology of School of Medicine and Center for Investigational Therapeutics at Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112, USA ≠BetaCat Pharmaceuticals LLC 22 Firstfield Rd, Gaithersburg, MD; ±Selvita S.A, Park Life Sciences, 30-348 Krakow, Poland ∞PrimeTime Life Sciences, 9700, Great Seneca Highway, Rockville, MD 20850, USA ¶Cockrell Center for Advanced Therapeutics, The Methodist Hospital Research Institute, 6670 Bertner Ave., R9113, Houston, TX 77030, USA £Primary Children's Hospital, Department of Pediatric Hematology/Oncology, University of Utah, 100 Mario Capecchi Dr., Salt Lake City, UT 84113, USA.
ABSTRACT: The Wnt/β-catenin signaling pathway plays a vital role in cell growth, the regulation, cell development, and the differentiation of normal stem cells. Constitutive activation of the Wnt/β-catenin signaling pathway is found in many human cancers, and thus it is an attractive target for anti-cancer therapy. Specific inhibitors of this pathway have been keenly researched and developed. Cell based screening of compounds library, hit-to-lead optimization, computational and structure-based design strategies resulted in the design and synthesis of a series of anthracene-9, 10-dione dioxime series of compounds demonstrated potent inhibition of β-catenin in vitro (IC50 10 M) indicates the importance of a hetero-cycloalkyl moieties, size and its hydrophobic binding pocket. We then chose to preserve the most important structural features of a tricyclic central core such as; 9Hfluoren-9-one
oxime
(3),
9H-xanthen-9-one
oxime
(5)
10,11-dihydro-5H-
dibenzo[a,d][7]annulen-5-one oxime (6), acridin-9(10H)-one oxime (7) (Figure 3) and solvent binding symmetrical sulfonyl functionality substituted by hetero-cycloalkyl moieties (compound 2 and 3), while exploring the effects of alterations at the 2, 7 sites of the core scaffold molecule.17, 18 Interestingly, the compound series 6 and 7 lost its activity completely
ACS Paragon Plus Environment
Journal of Medicinal Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
where as 9H-xanthen-9-one oxime (5) series having 6, 7 or 8 membered ring heterocycloalkyl groups at 2, 7 position exhibited low micromolar IC50 in our HT-29 cancer cells and in -catenin depletion assays (540 nM to 10 M). These active mono-oxime series of compounds were not considered for further development due to its poor solubility and pharmacokinetic properties. Symmetrical anthracene-9, 10-dione dioxime series. The preliminary structureactivity relationships based on three key pharmacophores we considered in our analogue design suggested a possible hypothetical model for these dioxime series of compounds and their interactions with a binding partner depicted in Figure 4. Since there is no physical or structural binding data available during our optimization process, the tight hydrophobic and electrostatic interactions were considered for the design of novel oxime series of compounds. As a strategy to lead optimization, we considered the original 2, 7-disubstituted 9H-fluoren-9-one oxime and or 9H-xanthen-9-one oxime active hits. We then modified the central pyran ring oxygen atom of 9H-xanthen-9-one oxime 5 by substituting with second oxime functionality lead to the identification symmetrical anthracene-9, 10-dione dioxime series of compounds. On the basis of these considerations, specifically compounds with the general structure of 8 were explored (Figure 3). To reduce molecular weight, we chose the 6 or 7 ring hetero-cycloalkyl group on 2, 7-disubstituted anthracene-9, 10-dione dioxime ring A and ring B with heteroatoms such as –N or -O to increase the solvent binding affinity. Since R1 and R2 groups project toward the solvent binding site, we used a variety of basic and polar groups to explore their effects on binding, solubility, and pharmacokinetic (PK) properties. Particularly the compounds having 6, 7 membered hetero-cycloalkyl rings substituted with methyl, tert-butyl, and dimethyl functional groups demonstrated potent activity both in -catenin depletion assay with an IC50 between 60-190 nanomolar in HT-29 cell lines (14, 23, 35, and 51). With an exception of compound 26, the compounds having bulkier 7-12 ring cycloalkyl groups exhibited low M activity in HT-29 transcriptional inhibition. Additionally, our attempts to improve the solubility by replacing oxime to keto functionality, and introduction of second hetero atoms at the cycloalkyl rings resulted in complete loss of activity (17, 48). Incorporation piperidine sulfonyl groups at the position 6 of anthracene ring is well tolerated (54, 57), unfortunately these compounds suffered from
ACS Paragon Plus Environment
Page 4 of 23
Page 5 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of Medicinal Chemistry
poor solubility. On the basis of these considerations, compounds 2, 7-substituted dioxime series of compounds were explored. The symmetrical 3,5-dimethyl substituted piperidine analogs, particularly the compound 14 has been profiled extensively in many cancer cell lines. The compound 14 showed potent growth inhibition in HT-29 treated cells with an IC50 of 90 nM. However, the inhibition was much less in its transcriptional inhibition of HT29 cells (IC50, 630 nM). With its promising -catenin depletion and cellular activities, the compound 14 was profiled for its ADME-Tox experiments. The Log D, solubility of 14 found to be 3.3, 1 g/mL and in vitro hERG safety profile indicated no cardiovascular liability. The additional safety screening includes; CYP2C9 (5.62 M) and no P-gp activities noted. The compound 14 possessed significant plasma protein binding with 99.8% was further assessed for exposure in blood from single-dose mouse PK and in vivo efficacy experiments. The general synthesis exemplified by these compounds were shown in Figure 5 and Scheme 1. The 9,10-dioxo-9,10-dihydroanthracene-2,7-disulfonyl dichloride 9 was reacted with the substituted heterocycloalkyl 6-12 membered ring structure in presence basic or other solvents; triethylamine (TEA), diisopropylethylamine (DIPEA), pyridine in DCM, chloroform, THF or acetonitrile solvents for 30 minute to 4 hours to give the series of anthracene-9,10-dione compounds of general structure 10 in quantitative yields. In the final step, the excess of hydroxylamine hydrochloride and pyridine at 95 oC for 16-36 hours afforded the complete conversion of anthracene-9,10-dione compounds to racemic oxime compounds 11. The racemic lead oxime 14 was then prepared by treating compound 11 with hydroxylamine hydrochloride in pyridine for 16 hours at 115 oC provided 99.6 % as white solid. Compound 14 was isolated as enantiomerically pure (3R, 5S)-3,5-dimethylpiperidine compound (Scheme 2). Due to the symmetric nature of oximes it was not feasible to assign its E/Z, E/E isomerism at 9 and 10 positions. Representative compounds of 14 in this series and their biological data are reported in Table 1. To assess the potency of the new analogues as inhibitors of the β-catenin signaling pathway, we used two assays; growth inhibition in a β-catenin dependent colon cancer cell line (HT-29), and a gene-expression based biomarker profile signature that reports on β-catenin pathway activity15, 16, 19a-21. With an exception of
ACS Paragon Plus Environment
Journal of Medicinal Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
compounds 23-24, the N-methylpiperazine, morpholin and piperazine substitutions at 2, 7 disulfonamide site lost its cellular activity in both assays. The bulky hydrophobic cycloalkyl ring substitutions tolerated and were found to be very potent β-catenin pathway inhibitors with and IC50 ranging from 0.02 to 3.90 μM (Table 1) against the HT-29 colon adenocarcinoma cell line. Both oxime and sulfonyl groups at the 2 and 7 positions of the anthracene ring are well tolerated with 7 to 12 ring hydrophobic moieties. As previously discussed17, 18 these oxime22 series of compounds are stereospecific in their activity on βcatenin signaling. The absolute configuration is critical for potency, as the active enantiomer of compound 14 (3R, 5S) was about 100-fold more potent (IC50 of 7 nM) than the other enantiomers under the same assay conditions. Compound 14, was further characterized despite of its low solubility of
