Anticancer activities of emetine prodrugs that are proteolytically activated by the prostate specific antigen (PSA) and evaluation of in vivo toxicity of emetine derivatives.

Emetine is a small molecule protein synthesis inhibitor that is toxic to all cell types and therefore suitable for complete killing of all types of heterogeneous cancer cells within a tumor. It becomes significantly inactive (non-toxic) when derivatized at its N-2' secondary amine. This provides a strategy for targeting emetine to cancerous tumor without killing normal cells. In this report, PSA activatable peptide prodrugs of emetine were synthesized. To overcome steric hindrances and enhance protease specific cleavage, a 2-stage prodrug activation process was needed to release emetine in cancer cells. In this 2-stage process, emetine prodrug intermediates are coupled to PSA peptide substrate (Ac-His-Ser-Ser-Lys-Leu-Gln) to obtain the full prodrug. Both prodrug intermediates 10 (Ala-Pro-PABC-Emetine) and 14 (Ser-Leu-PABC-Emetine) were evaluated for kinetics of hydrolysis to emetine and potency [Where PABC = p-aminobenzyloxycarbonyl]. While both intermediates quantitatively liberate emetine when incubated under appropriate conditions, upon coupling of PSA substrate to give the full prodrugs, only prodrug 16, the prodrug obtained from 14 was hydrolyzable by PSA. Cytotoxicity studies in PSA producing LNCaP and CWR22Rv1 confirm the activation of the prodrug by PSA with an IC50 of 75 nM and 59 nM respectively. The cytotoxicity of 16 is significantly reduced in cell lines that do not produce PSA. Further, in vivo toxicity studies are done on these prodrugs and other derivatives of emetine. The results show the significance of conformational modulation in obtaining safe emetine prodrugs.

Iterative design of emetine-based prodrug targeting fibroblast activation protein (FAP) and dipeptidyl peptidase IV DPPIV using a tandem enzymatic activation strategy.

BACKGROUND: There is an urgent need to develop new agents for treating metastatic prostate cancer to overcome multiple drug resistance to the current standard targeted cancer therapy. Emetine is a highly cytotoxic natural product protein synthesis inhibitor, which is toxic to all cell types. Its cytotoxicity can be blocked by derivatizing its N-2' position. Thus emetine can be selectively delivered to cancer cells in the region of metastatic cancer as a prodrug that will be activated by an enzyme selectively overexpressed within the metastatic tumor microenvironment. In this work, we convert emetine to a prodrug activatable by the fibroblast activation protein (FAP), a serine protease overexpressed by the carcinoma associated fibroblasts. METHOD: By using an iterative structure-activity relationship strategy, several peptidyl emetine prodrug analogs (1-11) were synthesized by chemical derivatization of emetine at its N-2' position and tested for in-vitro activation by FAP. The lead prodrug 11 is made up of a DPPIV activatable prodrug precursor 10 (Ala-Pro-PABC-Emetine) coupled to FAP substrate (Ala-Ser-Gly-Pro-Ala-Gly-Pro). Activation assays of the prodrugs were performed in purified FAP, DPPIV, FBS, and human serum and were analyzed by LCMS. In vitro cytotoxicity assays of these prodrugs are carried out in prostate (LNCaP, PC3) and breast (MCF7 and MDA-MB-231) cancer cell lines. The prodrugs are also tested in normal immortalized human prostatic epithelial cell line (PrEC). RESULTS: The lead FAP activated emetine prodrug 11 is activated to emetine in tandem by FAP and DPPIV in about 70% conversion within 24 hr. In prostate and breast cancer cell lines treated with prodrug 11, it is found to be equipotent with emetine in the presence of FAP and DPPIV. However, in the PrEC cell line grown in serum free media, prodrug 11 is more than 200-fold less cytotoxic than emetine in the absence of FAP and DPPIV. CONCLUSION: This FAP activated prodrug of cytotoxic agent emetine further shows the crucial role of the N-2' position of emetine in controlling its cytotoxicity. Significantly reduced toxicity observed in the PrEC cell line in the absence of FAP and DPPIV shows that prodrug 11 could be systemically delivered to regions of metastatic prostate cancer or other solid tumor for activation by cancer selective enzymes within the cancer microenvironment, such as FAP that is overexpressed by the carcinoma-associated fibroblasts. The two-step tandem enzymatic activation of prodrug 11 by FAP and DPPIV is a strategy for overcoming steric hindrance.

Novel drug design for Chagas disease via targeting Trypanosoma cruzi tubulin: Homology modeling and binding pocket prediction on Trypanosoma cruzi tubulin polymerization inhibition by naphthoquinone derivatives.

Chagas disease, also called American trypanosomiasis, is a parasitic disease caused by Trypanosoma cruzi (T. cruzi). Recent findings have underscored the abundance of the causative organism, (T. cruzi), especially in the southern tier states of the US and the risk burden for the rural farming communities there. Due to a lack of safe and effective drugs, there is an urgent need for novel therapeutic options for treating Chagas disease. We report here our first scientific effort to pursue a novel drug design for treating Chagas disease via the targeting of T. cruzi tubulin. First, the anti T. cruzi tubulin activities of five naphthoquinone derivatives were determined and correlated to their anti-trypanosomal activities. The correlation between the ligand activities against the T. cruzi organism and their tubulin inhibitory activities was very strong with a Pearson's r value of 0.88 (P value <0.05), indicating that this class of compounds could inhibit the activity of the trypanosome organism via T. cruzi tubulin polymerization inhibition. Subsequent molecular modeling studies were carried out to understand the mechanisms of the anti-tubulin activities, wherein, the homology model of T. cruzi tubulin dimer was generated and the putative binding site of naphthoquinone derivatives was predicted. The correlation coefficient for ligand anti-tubulin activities and their binding energies at the putative pocket was found to be r=0.79, a high correlation efficiency that was not replicated in contiguous candidate pockets. The homology model of T. cruzi tubulin and the identification of its putative binding site lay a solid ground for further structure based drug design, including molecular docking and pharmacophore analysis. This study presents a new opportunity for designing potent and selective drugs for Chagas disease.

Design, synthesis and cytotoxicity studies of dithiocarbamate ester derivatives of emetine in prostate cancer cell lines.

A small library of emetine dithiocarbamate ester derivatives were synthesized in 25-86% yield via derivatization of the N2'- position of emetine. Anticancer evaluation of these compounds in androgen receptor positive LNCaP and androgen receptor negative PC3 and DU145 prostate cancer cell lines revealed time dependent and dose-dependent cytotoxicity. With the exception of compound 4c, all the dithiocarbamate ester analogs in this study showed appreciable potency in all the prostate cancer cell lines (regardless of whether it is androgen receptor positive or negative) with a cytotoxicity IC50 value ranging from 1.312 ± 0.032 µM to 5.201 ± 0.125 µM by day 7 of treatment. Compared to the sodium dithiocarbamate salt 1, all the dithiocarbamate ester analogs (2 and 4a-4 g) displayed lower cytotoxicity than compound 1 (PC3, IC50 = 0.087 ± 0.005 µM; DU145, IC50 = 0.079 ± 0.003 µM and LNCaP, IC50 = 0.079 ± 0.003 µM) on day 7 of treatment. Consequently, it appears that S-alkylation of compound 1 leads to a more stable dithiocarbamate ester derivative that resulted in lower anticancer activity in the prostate cancer cell lines.

N-(3-Chloro-1,4-dioxo-1,4-di-hydro-naph-thalen-2-yl)-N-propionylpropionamide.

In the title mol-ecule, C16H14ClNO4, the four essentially planar atoms of the imide group [r.m.s. deviation = 0.0286 (11) Å] form a dihedral angle of 77.36 (13)° with the naphtho-quinone group [maximun deviation = 0.111 (2) Šfor the carbonyl O atom in the naphthalene 1-position] and the two imide carbonyl groups are oriented anti with respect to each other. In the crystal, mol-ecules are connected by weak C-H⋯O hydrogen bonds, as well as π-π stacking inter-actions [centroid-centroid distance = 3.888 (3) Å], forming a three-dimensional network.

2-Chloro-N-(2-chloro-benzo-yl)-N-(2-ethyl-4-oxo-3,4-di-hydro-quinazolin-3-yl)benzamide.

In the title compound, C24H17Cl2N3O3, the quinazolinone ring system is close to planar (r.m.s. deviation = 0.0132 Å), with the imide unit almost perpendicular to it, subtending a dihedral angle of 89.1 (1)°. However, the imide unit itself is not planar, the dihedral angle between the two O=C-N components being 34.6 (1)°. The dihedral angle between the two chlorobenzene rings is 40.50 (7)°, while the angles between these rings and the imide moiety are 54.6 (1) and 58.2 (1)°, respectively. The dihedral angles between the 2-chloro-phenyl rings and the quinazolinone ring system are 48.77 (5) and 32.92 (7)° for rings A and B, respectively. In the crystal, weak C-H⋯O inter-actions link the mol-ecules into a three-dimensional array.

PSMA-targeted combination brusatol and docetaxel nanotherapeutics for the treatment of prostate cancer.

Active targeting to cancer involves exploiting specific interactions between receptors on the surface of cancer cells and targeting moieties conjugated to the surface of vectors such that site-specific delivery is achieved. Prostate specific membrane antigen (PSMA) has proved to be an excellent target for active targeting to prostate cancer. We report the synthesis and use of a PSMA-specific ligand (Glu-NH-CO-NH-Lys) for the site-specific delivery of brusatol- and docetaxel-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles to prostate cancer. The PSMA targeting ligand covalently linked to PLGA-PEG3400 was blended with methoxyPEG-PLGA to prepare brusatol- and docetaxel-loaded nanoparticles with different surface densities of the targeting ligand. Flow cytometry was used to evaluate the impact of different surface densities of the PSMA targeting ligand in LNCaP prostate cancer cells at 15 min and 2 h. Cytotoxicity evaluations of the targeted nanoparticles reveal differences based on PSMA expression in PC-3 and LNCaP cells. In addition, levels of reactive oxygen species (ROS) were measured using the fluorescent indicator, H2DCFDA, by flow cytometry. PSMA-targeted nanoparticles loaded with docetaxel and brusatol showed increased ROS generation in LNCaP cells compared to PC-3 at different time points. Furthermore, the targeted nanoparticles were evaluated in male athymic BALB/c mice implanted with PSMA-producing LNCaP cell tumors. Evaluation of the percent relative tumor volume show that brusatol-containing nanoparticles show great promise in inhibiting tumor growth. Our data also suggest that the dual drug-loaded targeted nanoparticle platform improves the efficacy of docetaxel in male athymic BALB/c mice implanted with PSMA-producing LNCaP cell tumors.

Synthesis and characterization of novel unsymmetrical and symmetrical 3-halo- or 3-methoxy-substituted 2-dibenzoylamino-1,4-naphthoquinone derivatives.

Symmetrical and unsymmetrical 3-halo- or 3-methoxy- substituted 2-dibenzoylamino- 1,4-naphthoquinone analogs were synthesized with an average yield of 45% via sodium hydride promoted bis-acylation of 2-amino-3-chloro-1,4-naphthoquinone, 2-amino-3-bromo-1,4-naphthoquinone and 2-amino-3-methoxy-1,4-naphthoquinone.

N-Butanoyl-N-(3-chloro-1,4-dioxonaph-thalen-2-yl)butanamide.

In the title compound, C18H18ClNO4, the imide group with its two alkyl substituents is approximately perpendicular to the plane of the naphtho-quinone ring system [dihedral angle = 78.5 (1)°]. Further, the imide carbonyl groups are oriented in an anti sense. In the crystal, the substituted naphtho-quinone rings form π-π stacks in the a-axis direction [perpendicular centroid-centroid distance = 3.209 (2) Šand slippage = 4.401 Å].

Synthesis and cytotoxic activities of some 2-arylnaphtho[2,3-d]oxazole-4,9-dione derivatives on androgen-dependent (LNCaP) and androgen-independent (PC3) human prostate cancer cell lines.

The synthesis of five 2-arylnaphtho[2,3-d]oxazole-4,9-dione derivatives was accomplished by refluxing 2-amino-3-bromo-1,4-naphthoquinone with appropriate benzoyl chloride analogs at elevated temperatures. In vitro anticancer evaluation of these compounds was performed on androgen-dependent, LNCaP, and androgen-independent, PC3, human prostate cancer cell lines. In general, these compounds displayed slightly stronger cytotoxicity on the androgen-dependent LNCaP than on the androgen-independent PC3 prostate cancer cell lines. The meta-substituted 2-(3-Chloro-phenyl)-naphtho[2,3-d]oxazole-4,9-dione (10) appear to display the best cytotoxicity on both cell lines with an IC(50) of 0.03 µM on LNCaP and 0.08 µM on PC3 after 5 days of exposure.

N-Benzoyl-N-(1,4-dioxonaphthalen-2-yl)benzamide.

The title mol-ecule, C(24)H(15)NO(4), crystallizes with two mol-ecules in the asymmetric unit (Z' = 2). For both mol-ecules, the two amide groups are not coplanar, as the dihedral angles of the respective NCO groups are similar at 50.37 (14) and 51.22 (13)°. However, the orientations of the substituent phenyl rings with the central naphthalene system are significantly different for the two mol-ecules; for one mol-ecule, these dihedral angles are 80.29 (3) and 80.95 (4)°, while for the second mol-ecule they are 86.63 (3) and 72.82 (4)°. The crystal packing shows the mol-ecules to be linked by weak C-H⋯O inter-actions.

N-(1,4-Dioxo-1,4-dihydro-naphthalen-2-yl)benzamide.

The title compound, C(17)H(11)NO(3), was an inter-mediate synthesized during bis-acyl-ation of 2-amino-1,4-naphtho-quinone with benzoyl chloride. A mixture of block- and needle-shaped crystals were obtained after column chromatography. The block-shaped crystals were identified as the imide and the needles were the title amide. The naphtho-quinone scaffold is roughly planar (r.m.s. deviation = 0.047 Šfor the C atoms). The N-H and C=O bonds of the amide group are anti to each other. A dihedral angle between the naphtho-quinone ring system and the amide group of 3.56 (3)°, accompanied by a dihedral angle between the amide group and the phenyl group of 9.51 (3)°, makes the naphtho-quinone ring essentially coplanar with the phenyl ring [dihedral angle = 7.12 (1)°]. In the crystal, molecules are linked by a weak N-H⋯O hydrogen bond and by two weak C-H⋯O interactions leading to the formation of zigzag chains along [010].

2,3-Dichloro-5,8-dimeth-oxy-1,4-naphtho-quinone.

In the crystal structure of the title compound, C(12)H(8)Cl(2)O(4), mol-ecules crystallize in planes parallel to (-204) with an inter-planar distance of 3.288 (2) Š[centroid-centroid distance = 3.819 (2) and slippage = 1.932 (2) Å]. The structure features C-H⋯O inter-actions involving meth-oxy and aromatic H atoms and the carbonyl O atoms as well as a C-H⋯Cl inter-action involving an aromatic H atom. In addition there are short inter-halogen contacts between adjoining mol-ecules [Cl⋯Cl = 3.3709 (5) Å].

Synthesis of 3-[(N-carboalkoxy)ethylamino]-indazole-dione derivatives and their biological activities on human liver carbonyl reductase.

A series of indazole-dione derivatives were synthesized by the 1,3-dipolar cycloaddition reaction of appropriate substituted benzoquinones or naphthoquinones and N-carboalkoxyamino diazopropane derivatives. These compounds were evaluated for their effects on human carbonyl reductase. Several of the analogs were found to serve as substrates for carbonyl reductase with a wide range of catalytic efficiencies, while four analogs display inhibitory activities with IC(50) values ranging from 3-5 microM. Two of the inhibitors were studied in greater detail and were found to be noncompetitive inhibitors against both NADPH and menadione with K(I) values ranging between 2 and 11 microM. Computational studies suggest that conformation of the compounds may determine whether the indazole-diones bind productively to yield product or nonproductively to inhibit the enzyme.

Biological evaluation of 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone as an anti-breast cancer agent.

BACKGROUND: Breast cancer is the most frequent cancer and the second leading cause of cancer deaths in women today. A number of 1,4-naphthoquinone derivatives have been found to possess significant pharmacological effects associated with marked antimicrobial and antitumor activities. In the present study, the in vitro effect of 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone (DCDMNQ) was evaluated on estrogen-positive MCF-7 and estrogen-negative MDA-MB-436 and Hs-578T human breast cancer cell lines. Moreover, the in vitro activity of this compound on cell cycle regulation and apoptosis were evaluated. MATERIALS AND METHODS: Established methods of cell viability, cell cycle, Western blot and apoptosis were used. RESULTS: The effect of DCDMNQ on MCF-7, MDA-MB-436 and Hs-578T cells revealed significant antitumor activities with IC(50)s, of 0.6 +/- 0.02, 1.4 +/- 0.25 and 3.1 +/- 0.4 microM respectively. Cell cycle analysis showed that DCDMNQ inhibited progression through the cell cycle in MCF-7 and MDA-MB-436 cell lines in a time-dependent manner. DCDMNQ arrested cells in the S-phase of the cell cycle with the greatest proportion of cells in the S-phase by day 5. This cell-cycle arrest was corroborated by inhibition of topoisomerase I induced by DCDMNQ. These findings were further validated using Western blot analysis of retinoblastoma protein time-dependent phosphorylation. Furthermore, DCDMNQ induced apoptosis in both estrogen-positive and -negative cell lines in a time-dependent manner. However, the highest percentages of apoptotic cells were observed in the MDA-MB-436 cell line. CONCLUSION: Although the mechanism of action of DCDMNQ has not been completely elucidated, it appears that this compound can inhibit topoisomerase I in a concentration-dependent manner. These promising results to explore novel naphthoquinone analogues as potential breast cancer agents. This study suggests that DCDMNQ may have an impact on treatment of estrogen-positive and -negative breast cancer while protecting the bone marrow.

Bis(1,10-phenanthroline-5,6-dione-κN,N')silver(I) tetra-fluoridoborate.

In the structure of the title compound, [Ag(C(12)H(6)N(2)O(2))(2)]BF(4) or [AgL(2)]BF(4) (L = phendione), the Ag and B atoms are located on twofold rotation axes. The dihedral angle between the two phendione ligands is 36.7 (2)°. The coordination about the Ag(I) center is distorted tetra-hedral (τ(4) = 0.546). The crystal structure is consolidated by weak C-H⋯O(phendione) and C-H⋯F(BF(4) (-)) inter-actions. The BF(4) (-) counter-anion is strongly disordered and was modelled with two sets of idealized F atoms.

N-(3-Bromo-1,4-dioxo-1,4-dihydro-2-naphth-yl)-4-fluoro-N-(4-fluoro-benzo-yl)benzamide.

In the title compound, C(24)H(12)BrF(2)NO(4), synthesized from 2-amino-3-bromo-1,4-naphthoquinone and 4-fluoro-benzoyl chloride, the two p-fluoro-phenyl rings are inclined at 73.9 (1) and 73.6 (1)° to the naphthoquinone ring system. The two imido carbonyl O atoms are anti to each other, while the fluoro-phenyl rings are located opposite each other, connected to the imide group in a funnel-like arrangement. This conformation allows the fluorine groups be oriented slightly away from each other. An examination of the packing shows a close inter-molecular F⋯O contact of 2.982 (5) Šand a Br⋯O contact of 2.977 (4) Å. In addition, the mol-ecules are linked by weak inter-molecular C-H⋯O and C-H⋯F inter-actions.

Rational design of novel antiandrogens for neutralizing androgen receptor function in hormone refractory prostate cancer.

BACKGROUND: The standard hormonal therapy with currently available antiandrogens and the leutinizing hormone releasing hormone (LHRH) analogs is not effective in the hormone-refractory stage of prostate cancer due to changes in androgen receptor (AR) signaling axis. In this refractory stage, AR continues to play a significant role in the growth of cancer cells even though the cancer cells are no longer dependent on the level of circulating androgens. METHODS: A series of 11beta-Delta(9)-19 nortestosterone compounds were designed through structure-based rationale and tested for their binding affinity against AR and glucocorticoid receptor (GR) using fluorescence polarization assays, their agonistic ability to induce AR dependent transcription using PSA-driven report gene assays, and their growth inhibitory affects against a series of AR positive (LAPC4, LNCap, and CWR22R) and negative human prostate cancer cell lines (PC3) using MTT cell proliferation assays. RESULTS: This study proposes the design of novel bifunctional antiandrogens based on the conjugation of 11beta and/or 7alpha-Delta(9)-19 nortestosterone class of steroidal compounds to the synthetic ligand for FK506-binding proteins. As a critical step towards the development of bifunctional antiandrogens, highly potent and AR-specific lead compounds were identified using in vitro data. The lead compounds identified in this study possessed low binding affinity for GR, indicating the absence of undesirable antiglucocorticoid activity. CONCLUSIONS: The results of this study validate our drug discovery rationale based on the structural biology of AR and pave the pay for future development of bifunctional compounds in order to block AR function in hormone refractory stage of prostate cancer.

N-(3-Bromo-1,4-dioxo-1,4-dihydro-2-naphth-yl)-2-chloro-N-(2-chloro-benzoyl)benzamide.

The title compound, C(24)H(12)BrCl(2)NO(4), was synthesized from 2-amino-3-bromo-1,4-naphthoquinone and 2-chloro-benzoyl chloride. The crystal structure shows that each of the chloro-phenyl rings is inclined at about 60° to the naphthoquinone ring system. The two chloro-phenyl rings adopt a conformation that ensures that chlorine substituents are anti so as to reduce electronic repulsion. An examination of the packing shows close O⋯Br and Cl⋯Cl contacts of 2.947 (2) and 3.346 (1) Å, respectively. In addition, the molecules are linked by weak intermolecular C-H⋯O and C-H⋯Cl interactions.

2-Chloro-3-(4-chloro-benzamido)-1,4-naphthoquinone.

The naphthoquinone ring is almost perpendicular [dihedral angle 71.02 (3)°] to the phenyl group of the title compound, C(17)H(9)Cl(2)NO(3), while the dihedral angle between the amide group and the 4-chloro-phenyl ring is 21.9 (2)°. The conformation of the N-H and C=O bonds are anti to each other. N-H⋯Cl hydrogen bonds link the mol-ecules into chains in the a-axis direction. In addition, these chains are linked by weak inter-molecular C-H⋯O inter-actions.