Design and synthesis of prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors as anti-prostate cancer drugs

A series of 1-aryl-3,4-substituted-1H-pyrazol-5-ol derivatives was synthesized and evaluated as prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors to obtain a novel anti-prostate cancer drug. After modifying 1-(1H-benzimidazol-2-yl)-3,4-dimethyl-1H-pyrazol-5-ol (1), a hit compound found during random screening using a recombinant PCA-1/ALKBH3, 1-(1H-5-methylbenzimidazol-2-yl)-4-benzyl-3-methyl-1H-pyrazol-5-ol (35, HUHS015), was obtained as a potent PCA-1/ALKBH3 inhibitor both in vitro and in vivo. The bioavailability (BA) of 35 was 7.2% in rats after oral administration. As expected, continuously administering 35 significantly suppressed the growth of DU145 cells, which are human hormone-independent prostate cancer cells, in a mouse xenograft model without untoward effects.     

Synthesis and evaluation of prodrugs of corticotropin-releasing factor-1 (CRF1) receptor antagonist BMS-665053 leading to improved oral bioavailability

A series of phosphate and ester-based prodrugs of anilinopyrazinone 1 (BMS-665053) containing either a methylene or an (acyloxy)alkoxy linker was prepared and evaluated in rat pharmacokinetic studies with the goal of improving the oral bioavailability of the parent (1). The prodrugs, in general, had improved aqueous solubility and oral bioavailability compared to 1. Prodrug 12, which contains an (acyloxy)alkoxy linker, showed the greatest improvement in the oral bioavailability relative to the parent (1), with a seven-fold increase (from 5% to 36%) in rat pharmacokinetic studies.     

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs activated by cytochrome P450 1A1 in breast cancer cells

The role and the importance of the sulfonate moiety in phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) were assessed using its bioisosteric sulfonamide equivalent leading to new cytochrome P450 1A1 (CYP1A1)-activated prodrugs designated as 4-(3-alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs). PAIB-SAs are active in the submicromolar to low micromolar range showing selectivity toward CYP1A1-expressing MCF7 cells as compared to cells devoid of CYP1A1 activity such as MDA-MB-231 and HaCaT cells. The most potent, PAIB-SA 13, bearing a trimethoxyphenyl group on ring B blocks the cell cycle progression in G2/M phase, disrupts the microtubule dynamics and is biotransformed by CYP1A1 into CEU-638, its potent antimicrotuble counterpart. Structure-activity relationships related to PAIB-SOs and PAIB-SAs evidenced that PAIB-SOs and PAIB-SAs are true bioisosteric equivalents fully and selectively activatable by CYP1A-expressing cells into potent antimitotics.     

SAR and biological evaluation of analogues of a small molecule histone deacetylase inhibitor N-(2-aminophenyl)-4-((4-(pyridin-3-yl)pyrimidin-2-ylamino)methyl)benzamide (MGCD0103)

Analogues of the clinical compound MGCD0103 (A) were designed and synthesized. These compounds inhibit recombinant human HDAC1 with IC₅₀ values in the sub-micromolar range. In human cancer cells growing in culture these compounds induce hyperacetylation of histones, cause expression of the tumor suppressor protein p21^WAF1/CIP1, and inhibit cellular proliferation. Lead molecule of the series, compound 25 is metabolically stable, possesses favorable pharmacokinetic characteristics and is orally active in vivo in different mouse tumor xenograft models.     

New orally bioavailable 2-aminobenzamide-type histone deacetylase inhibitor possessing a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group

New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a sulfur-containing bicyclic arylmethyl moiety—a surface recognition domain introduced to increase in cellular uptake—and a substituted tert-amino group which affects physicochemical properties such as aqueous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at 45 mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot analyses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhibited the growth of cancer cells via similar mechanisms.     

Discovery of (R)-5-(benzo[d][1,3]dioxol-5-yl)-7-((1-(vinylsulfonyl)pyrrolidin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (B6) as a potent Bmx inhibitor for the treatment of NSCLC

Described as a Btk inhibitor, ibrutinib also potently inhibits Bmx and EGFR, two good targets for lung cancer. Owing to its high CLogP (4.07) and low aqueous solubility (<0.01 mg/ml), resulting in unfavorable bioavailability, ibrutinib requires high dosages to achieve good clinical response in the treatment of non-small cell lung cancer (NSCLC). In our effort to improve the CLogP of ibrutinib by structural optimization led to the discovery of a potent anti-cancer agent B6, with beneficial physicochemical parameters (CLogP = 2.56, solubility in water ≈ 0.1 mg/ml) meeting the principles of oral drugs. B6 exhibited anti-proliferation activities against EGFR-expressing cells, especially the mutant ones, such as H1975 (L858R/T790M, IC₅₀ = 0.92 ± 0.19 μM) and HCC827 (Del119 IC₅₀ = 0.014 ± 0.01 μM). Moreover, B6 significantly slowed down H1975 tumor growth with anti-tumor rate of 73.9% (p < 0.01). Enzyme potencies assay demonstrated B6 moderately selectively inhibited Bmx (IC₅₀ = 35.7 ± 0.1 nM) over other kinases. So, as a potent Bmx inhibitor, B6 has the potential to be an efficacious treatment for NSCLC with acquired drug resistance.     

2-Benzimidazolyl-9-(chroman-4-yl)-purinone derivatives as JAK3 inhibitors

A novel class of Janus tyrosine kinase 3 (JAK3) inhibitors based on a 2-benzimidazoylpurinone core structure is described. Through substitution of the benzimidazoyl moiety and optimization of the N-9 substituent of the purinone, compound 24 was identified incorporating a chroman-based functional group. Compound 24 shows excellent kinase activity, good oral bioavailability and demonstrates efficacy in an acute mechanistic mouse model through inhibition of interleukin-2 (IL-2) induced interferon-γ (INF-γ) production.     

(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one,a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive agent

The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in Sacchrosomes? and live human HEK293 cells. The structure-activity relationship analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on non-heterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC₅₀ values of 58 and 65?nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown >10-fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 sub-family and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes. The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and selective inhibitor of CYP1A1 enzyme have a potential for development as cancer chemopreventive agent.     

Synthesis and antitumor activity of bis(hydroxymethyl)propionate analogs of pterostilbene in cisplatin-resistant human oral cancer cells

The aim of this study was to develop a new drug substance with low toxicity and effective inhibitory activity against cisplatin-resistant oral cancer. The naturally produced pterostilbene was selected as the lead compound for design and synthesis of a series of bis(hydroxymethyl)propionate-based prodrugs. All derivatives were screened for antiproliferative effects against the cisplatin-resistant oral squamous (CAR) cell line and the results indicated that several compounds demonstrated superior inhibitory activity compared with pterostilbene and resveratrol. Among them, the most promising compound, 12, was evaluated for in vivo antitumor activity in a CAR xenograft nude mouse model. Obvious antitumor activity was observed at the lowest oral dose (25?mg/kg/day). Increasing the dose of 12 to 100?mg/kg/day reduced the tumor size to 22% of the control group. Based on these findings as well as the extremely low toxicity seen in the in vivo studies, we believe that compound 12 could serve as a new lead for further development.     

New antiproliferative 7-(4-(N-substituted carbamoylmethyl)piperazin-1-yl) derivatives of ciprofloxacin induce cell cycle arrest at G2/M phase

New N-4-piperazinyl derivatives of ciprofloxacin 2a–g were prepared and tested for their cytotoxic activity. The primary in vitro one dose anticancer assay experienced promising cytotoxic activity against different cancer cell lines especially non-small cell lung cancer. Independently, compounds 2b, 2d, 2f and 2g showed anticancer activity against human non-small cell lung cancer A549 cells (IC₅₀ = 14.8, 24.8, 23.6 and 20.7 μM, respectively) compared to the parent ciprofloxacin (IC₅₀ >100 μM) and doxorubicin as a positive control (IC₅₀ = 1 μM). The flow cytometric analysis for 2b showed dose dependent G₂/M arrest in A549 cells. Also, 2b increased the expression of p53 and p21 and decreased the expression of cyclin B1 and Cdc2 proteins in A549 cells without any effect on the same proteins expression in WI-38 cells. Specific inhibition of p53 by pifithrin-α reversed the G₂/M phase arrest induced by the 2b compound, suggesting contribution of p53 to increase. Taken together, 2b induced G₂/M phase arrest via p53/p21 dependent pathway. The results indicate that 2b can be used as a lead compound for further development of new derivatives against non-small cell lung cancer.     

Development of selective inhibitors for the treatment of rheumatoid arthritis: (R)-3-(3-(Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile as a JAK1-selective inhibitor

A series of 3(R)-aminopyrrolidine derivatives were designed and synthesized for JAK1-selective inhibitors through the modification of tofacitinib’s core structure, (3R,4R)-3-amino-4-methylpiperidine. From the new core structures, we selected (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine as a scaffold for further SAR studies. From biochemical enzyme assays and liver microsomal stability tests, (R)-3-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile (6) was chosen for further in vivo test through oral administration. Compound 6 showed improved selectivity for JAK1 compared to that of tofacitinib (IC₅₀ 11, 2.4?×?10², 2.8?×?10³, and 1.1?×?10²?nM for JAK1, JAK2, JAK3, and TYK2, respectively). In CIA and AIA model tests, compound 6 exhibited similar efficacy to tofacitinib citrate.     

Studies on non-steroidal inhibitors of aromatase enzyme; 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives

Steroidal and non-steroidal aromatase inhibitors target the suppression of estrogen biosynthesis in the treatment of breast cancer. Researchers have increasingly focused on developing non-steroidal derivatives for their potential clinical use avoiding steroidal side-effects.Non-steroidal derivatives generally have planar aromatic structures attached to the azole ring system. One part of this ring system comprises functional groups that inhibit aromatization through the coordination of the haem group of the aromatase enzyme. Replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase selectivity over aromatase enzyme inhibition.In this study, 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives were synthesized and physical analyses and structural determination studies were performed. The IC₅₀ values were determined by a fluorescence-based aromatase inhibition assay and compound 1 (4-(2-hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole) were found potent inhibitor of enzyme (IC₅₀:0.42?nM). Then, their antiproliferative activity over MCF-7 and HEK-293 cell lines was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds 1, 7, 8, 13, 15, 18, 21 were active against MCF-7 breast cancer cells. Lastly, a series of docking experiments were undertaken to analyze the crystal structure of human placental aromatase and identify the possible interactions between the most active structure and the active site.     

Synthesis and biological evaluation of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides as antimitotic agents

A library of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides (7a–al) have been designed, synthesized and screened for their anti-proliferative activity against some selected human cancer cell lines namely DU-145, A-549, MCF-7 and HeLa. Most of them have shown promising cytotoxicity against lung cancer cell line (A549), amongst them 7f was found to be the most potent anti-proliferative congener. Furthermore, 7f exhibited comparable tubulin polymerization inhibition (IC₅₀ value 2.04 µM) to the standard E7010 (IC₅₀ value 2.15 µM). Moreover, flow cytometric analysis revealed that this compound induced apoptosis via cell cycle arrest at G₂/M phase in A549 cells. Induction of apoptosis was further observed by examining the mitochondrial membrane potential and was also confirmed by Hoechst staining as well as Annexin V-FITC assays. Furthermore, molecular docking studies indicated that compound 7f binds to the colchicine binding site of the β-tubulin. Thus, 7f exhibits anti-proliferative properties by inhibiting the tubulin polymerization through the binding at the colchicine active site and by induction of apoptosis.     

Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

Diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of 5-(4-carboxymethylphenyl)-1-(4-methanesulfonylphenyl)-3-trifluoromethyl-1H-pyrazole and its aminosulfonyl analog: Synthesis,biological evaluation and nitric oxide release studies

A new class of hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrugs (NONO-coxibs) wherein an O²-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (13a–b), or O²-acetoxymethyl-1-(2-methylpyrrolidin-1-yl)diazen-1-ium-1,2-diolate (16a–b), NO-donor moiety was covalently coupled to the COOH group of 5-(4-carboxymethylphenyl)-1-(4-methane(amino)sulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (11a–b) was synthesized. The percentage of NO released from these diazen-1-ium-1,2-diolates was significantly higher (59.6–74.6% of the theoretical maximal release of 2 molecules of NO/molecule of the parent hybrid ester prodrug) upon incubation in the presence of rat serum, relative to incubation with phosphate buffer (PBS) at pH 7.4 (5.0–7.2% range). These incubation studies suggest that both NO and the AI compound would be released from the parent NONO-coxib upon in vivo cleavage by non-specific serum esterases. All compounds were weak inhibitors of the COX-1 isozyme (IC₅₀ = 8.1–65.2 μM range) and modest inhibitors of the COX-2 isozyme (IC₅₀ = 0.9–4.6 μM range). The most potent parent aminosulfonyl compound 11b exhibited AI activity that was about sixfold greater than that for aspirin and threefold greater than that for ibuprofen. The ester prodrugs 13b, 16b exhibited similar AI activity to that exhibited by the more potent parent acid 11b when the same oral μmol/kg dose was administered. These studies indicate hybrid ester AI/NO donor prodrugs of this type (NONO-coxibs) constitute a plausible drug design concept targeted toward the development of selective COX-2 inhibitory AI drugs that are devoid of adverse cardiovascular effects.     

Synthesis and CYP26A1 inhibitory activity of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates

The role of all-trans-retinoic acid (ATRA) in the development and maintenance of many epithelial and neural tissues has raised great interest in the potential of ATRA and related compounds (retinoids) as pharmacological agents, particularly for the treatment of cancer, skin, neurodegenerative and autoimmune diseases. The use of ATRA or prodrugs as pharmacological agents is limited by a short half-life in vivo resulting from the activity of specific ATRA hydroxylases, CYP26 enzymes, induced by ATRA in liver and target tissues. For this reason retinoic acid metabolism blocking agents (RAMBAs) have been developed for treating cancer and a wide range of other diseases.The synthesis, CYP26A1 inhibitory activity and molecular modeling studies of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates are presented. From this series of compounds clear SAR can be derived for 4-substitution of the phenyl ring with electron-donating groups more favourable for inhibitory activity. Both the methylenedioxyphenyl imidazole (17, IC₅₀ = 8 nM) and triazole (18, IC₅₀ = 6.7 nM) derivatives were potent inhibitors with additional binding interactions between the methylenedioxy moiety and the CYP26 active site likely to be the main factor. The 6-bromo-3-pyridine imidazole 15 (IC₅₀ = 5.7 nM) was the most active from this series compared with the standards liarozole (IC₅₀ = 540 nM) and R116010 (IC₅₀ = 10 nM).     

1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea: A novel cyclophilin A allosteric activator

Cyclophilin A (CypA) plays an important role in many physiology processes and its overexpression has been involved in many diseases including immune disease, viral infection, neuro-degenerative disease, and cancer. However, the actual role of CypA in the diseases is still far from clear, and a complete understanding of CypA is necessary in order to direct more specific and effective therapeutic strategies. Based on the screening of our in-house library through the isomer-specific proteolysis method, we find a CypA activator (1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea), compound 1a, which can increase CypA’s PPIase activity and give allosteric behavior. The binding affinity of compound 1a to CypA has been confirmed by Fortebio’s Octet RED system and the increased phosphorylation of ERK in H446 cells is observed by treatment with both compound 1a and CsA. In order to further evaluate the binding mode between the activator and CypA, the allosteric binding site and allosteric mechanism of CypA are investigated by molecular dynamics (MD) simulations in combination with mutagenesis experiments. The results show that the allosteric binding site of CypA is 7 Å away from its catalytic site and is composed of Cys52, His70, His54, Lys151, Thr152 and Lys155. Compound 1a binds to the allosteric site of CypA, stabilizing the active conformation of catalytic residues, and finally promotes the catalytic efficiency of CypA. We believe our finding of the CypA allosteric activator will be used as an effective chemical tool for further studies of CypA mechanisms in diseases.     

5-((1-Aroyl-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-diones as potential anticancer agents with anti-inflammatory properties

A series of novel 5-((1-aroyl-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-diones (3a–z) have been evaluated for in vitro cytotoxicity against a panel of 60 human tumor cell lines. Compound 3k exhibited the most potent growth inhibition against melanoma MDA-MB-435 cells (GI₅₀ = 850 nM), against leukemia SR cancer cells (GI₅₀ = 1.45 μM), and OVCAR-3 (GI₅₀ = 1.26 μM) ovarian cancer cell lines. The structurally related compound 3s had a GI₅₀ value of 1.77 μM against MDA-MB-435 cells. The N-naphthoyl analogue 3t had GI₅₀ values of 1.30 and 1.91 μM against HOP-92 non-small cell lung cancer and MDA-MB-435 melanoma cell lines, respectively. The related analogue 3w had GI₅₀ values of 1.09 μM against HOP-92 non-small cell lung cancer cell lines. Interestingly, docking of the two active molecules 3k and 3w into the active site of COX-2 indicates that these compounds are COX-2 ligands with strong hydrophobic and hydrogen bonding interactions. Thus, compounds 3k, 3t, 3s, and 3w constitute a new class of anticancer/anti-inflammatory agents that may have unique potential for cancer therapy.