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1.
Cancers (Basel) ; 16(1)2023 Dec 23.
Article in English | MEDLINE | ID: mdl-38201517

ABSTRACT

Neurofibromatosis type 1 (NF1) is a disorder in which RAS is constitutively activated due to the loss of the Ras-GTPase-activating activity of neurofibromin. RAS must be prenylated (i.e., farnesylated or geranylgeranylated) to traffic and function properly. Previous studies showed that the anti-growth properties of farnesyl monophosphate prodrug farnesyltransferase inhibitors (FTIs) on human NF1 malignant peripheral nerve sheath tumor (MPNST) cells are potentiated by co-treatment with lovastatin. Unfortunately, such prodrug FTIs have poor aqueous solubility. In this study, we synthesized a series of prodrug FTI polyamidoamine generation 4 (PAMAM G4) dendrimers that compete with farnesyl pyrophosphate for farnesyltransferase (Ftase) and assessed their effects on human NF1 MPNST S462TY cells. The prodrug 3-tert-butylfarnesyl monophosphate FTI-dendrimer (i.e., IG 2) exhibited improved aqueous solubility. Concentrations of IG 2 and lovastatin (as low as 0.1 µM) having little to no effect when used singularly synergistically suppressed cell proliferation, colony formation, and induced N-RAS, RAP1A, and RAB5A deprenylation when used in combination. Combinational treatment had no additive or synergistic effects on the proliferation/viability of immortalized normal rat Schwann cells, primary rat hepatocytes, or normal human mammary epithelial MCF10A cells. Combinational, but not singular, in vivo treatment markedly suppressed the growth of S462TY xenografts established in the sciatic nerves of immune-deficient mice. Hence, prodrug farnesyl monophosphate FTIs can be rendered water-soluble by conjugation to PAMAM G4 dendrimers and exhibit potent anti-tumor activity when combined with clinically achievable statin concentrations.

2.
Biochem Pharmacol ; 86(5): 597-611, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23830822

ABSTRACT

SH2 domains are attractive targets for chemotherapeutic agents due to their involvement in the formation of protein-protein interactions critical to many signal transduction cascades. Little is known, however, about how synthetic SH2 domain ligands would influence the growth properties of tumor cells or with which intracellular proteins they would interact due to their highly charged nature and enzymatic lability. In this study, a prodrug delivery strategy was used to introduce an enzymatically stable, phosphotyrosine peptidomimetic into tumor cells. When tested in a human tumor cell panel, the prodrug exhibited a preference for inhibiting the growth of leukemia and lymphoma cells. In these cells, it was largely cytostatic and induced endoreduplication and the appearance of midbodies. Proteomic analyses identified multiple targets that included mitotic centromere-associated kinesin (MCAK). Molecular modeling studies suggested the ATP-binding site on MCAK as the likely site of drug interaction. Consistent with this, ATP inhibited the drug-MCAK interaction and the drug inhibited MCAK ATPase activity. Accordingly, the effects of the prodrug on the assembly of the mitotic spindle and alignment of chromosomes were consistent with the identification of MCAK as an important intracellular target.


Subject(s)
Kinesins/metabolism , Peptidomimetics/metabolism , Phosphotyrosine/metabolism , Cell Cycle , Cell Line, Tumor , Humans , Ligands , Phosphorylation , Protein Binding , Spectrometry, Mass, Electrospray Ionization
3.
Vaccine ; 31(40): 4362-7, 2013 Sep 13.
Article in English | MEDLINE | ID: mdl-23887038

ABSTRACT

The strongest mechanism for adsorption of antigens to aluminum adjuvants is ligand exchange, which involves the replacement of a surface hydroxyl on the adjuvant by a terminal phosphate group of the antigen. A novel phosphonate linker was developed that allows the addition of phosphonate (C-PO3) groups to proteins under controlled and chemically mild conditions. Increasing the number of linkers per protein molecule progressively increased the adsorption strength to aluminum hydroxide adjuvant (AH) as measured by elution in serum. The effect of phosphonate conjugation on the antibody response was determined with hen egg lysozyme (HEL), a protein that has the same charge as AH at neutral pH and does not adsorb to AH. The phosphonylated form of HEL (HEL-P) adsorbed to AH, indicating that the ligand exchange interaction could overcome the electrostatic repulsion. Mice injected with HEL-P/AH had a higher antibody titer to HEL than mice injected with HEL/AH, especially at lower antigen doses, suggesting that adsorption of antigen has a dose-sparing effect. Conjugation of CRM197, an antigen that adsorbs electrostatically to AH, with phosphonate linkers did not enhance the antibody response, indicating that adsorption by either electrostatic or ligand exchange to AH is sufficient to enhance the antibody response.


Subject(s)
Adjuvants, Immunologic/chemistry , Aluminum Hydroxide/chemistry , Antibody Formation/immunology , Muramidase/immunology , Organophosphonates/chemistry , Adjuvants, Immunologic/chemical synthesis , Adsorption , Aluminum Hydroxide/immunology , Animals , Antibodies/blood , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Female , Humans , Mice , Mice, Inbred BALB C , Muramidase/chemistry , Protein Binding/immunology , Rats
4.
Chem Biol ; 19(6): 764-71, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22726690

ABSTRACT

Many protein-protein interactions in cells are mediated by functional domains that recognize and bind to motifs containing phosphorylated serine and threonine residues. To create small molecules that inhibit such interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. For this study, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. The prodrug is cytotoxic at low micromolar concentrations when applied to cancer cells and induces caspase activation resulting in apoptosis. The prodrug reverses the 14-3-3-mediated inhibition of FOXO3a resulting from its phosphorylation by Akt1 in a concentration-dependent manner that correlates well with its ability to inhibit cell growth. This methodology can be applied to target a variety of proteins containing phosphoserine and other phosphoamino acid binding domains.


Subject(s)
14-3-3 Proteins/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Molecular Mimicry , Phosphoserine/chemistry , Prodrugs/chemical synthesis , Prodrugs/pharmacology , 14-3-3 Proteins/metabolism , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Ligands , Molecular Structure , Prodrugs/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured
5.
Tetrahedron ; 67(52): 10216-10221, 2011 Dec 30.
Article in English | MEDLINE | ID: mdl-22199405

ABSTRACT

Synthesis of a potential Src family SH2 domain inhibitor incorporating a 1,4-cis-enediol scaffold is reported. The synthetic route offers straightforward and highly selective access to the enediol and its associated chiral centers. Key steps include stereocontrolled syn-aldol coupling, amide alkynylation, and asymmetric ketone reduction.

6.
Antioxid Redox Signal ; 14(8): 1387-401, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-20874257

ABSTRACT

APE1 is a multifunctional protein possessing DNA repair and redox activation of transcription factors. Blocking these functions leads to apoptosis, antiangiogenesis, cell-growth inhibition, and other effects, depending on which function is blocked. Because a selective inhibitor of the APE redox function has potential as a novel anticancer therapeutic, new analogues of E3330 were synthesized. Mass spectrometry was used to characterize the interactions of the analogues (RN8-51, 10-52, and 7-60) with APE1. RN10-52 and RN7-60 were found to react rapidly with APE1, forming covalent adducts, whereas RN8-51 reacted reversibly. Median inhibitory concentration (IC(50) values of all three compounds were significantly lower than that of E3330. EMSA, transactivation assays, and endothelial tube growth-inhibition analysis demonstrated the specificity of E3330 and its analogues in blocking the APE1 redox function and demonstrated that the analogues had up to a sixfold greater effect than did E3330. Studies using cancer cell lines demonstrated that E3330 and one analogue, RN8-51, decreased the cell line growth with little apoptosis, whereas the third, RN7-60, caused a dramatic effect. RN8-51 shows particular promise for further anticancer therapeutic development. This progress in synthesizing and isolating biologically active novel E3330 analogues that effectively inhibit the APE1 redox function validates the utility of further translational anticancer therapeutic development.


Subject(s)
Antineoplastic Agents/pharmacology , Benzoquinones/pharmacology , DNA-(Apurinic or Apyrimidinic Site) Lyase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Propionates/pharmacology , Signal Transduction/drug effects , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Benzoquinones/chemical synthesis , Benzoquinones/chemistry , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cloning, Molecular , DNA-(Apurinic or Apyrimidinic Site) Lyase/isolation & purification , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Molecular Structure , Organization and Administration , Oxidation-Reduction/drug effects , Propionates/chemical synthesis , Propionates/chemistry , Structure-Activity Relationship
7.
J Pharmacol Exp Ther ; 333(1): 23-33, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20086055

ABSTRACT

Prenylation inhibitors have gained increasing attention as potential therapeutics for cancer. Initial work focused on inhibitors of farnesylation, but more recently geranylgeranyl transferase inhibitors (GGTIs) have begun to be evaluated for their potential antitumor activity in vitro and in vivo. In this study, we have developed a nonpeptidomimetic GGTI, termed GGTI-2Z [(5-nitrofuran-2-yl)methyl-(2Z,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl 4-chlorobutyl(methyl)phosphoramidate], which in combination with lovastatin inhibits geranylgeranyl transferase I (GGTase I) and GGTase II/RabGGTase, without affecting farnesylation. The combination treatment results in a G(0)/G(1) arrest and synergistic inhibition of proliferation of cultured STS-26T malignant peripheral nerve sheath tumor cells. We also show that the antiproliferative activity of drugs in combination occurs in the context of autophagy. The combination treatment also induces autophagy in the MCF10.DCIS model of human breast ductal carcinoma in situ and in 1c1c7 murine hepatoma cells, where it also reduces proliferation. At the same time, there is no detectable toxicity in normal immortalized Schwann cells. These studies establish GGTI-2Z as a novel geranylgeranyl pyrophosphate derivative that may work through a new mechanism involving the induction of autophagy and, in combination with lovastatin, may serve as a valuable paradigm for developing more effective strategies in this class of antitumor therapeutics.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Autophagy , Diterpenes/pharmacology , Lovastatin/pharmacology , Organophosphorus Compounds/pharmacology , Transferases/antagonists & inhibitors , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Drug Synergism , G1 Phase/drug effects , GTP Phosphohydrolases/metabolism , Humans , Mice , Protein Prenylation , Resting Phase, Cell Cycle/drug effects , Schwann Cells/cytology , Schwann Cells/drug effects
8.
J Med Chem ; 53(3): 1200-10, 2010 Feb 11.
Article in English | MEDLINE | ID: mdl-20067291

ABSTRACT

The multifunctional enzyme apurinic endonuclease 1/redox enhancing factor 1 (Ape1/ref-1) maintains genetic fidelity through the repair of apurinic sites and regulates transcription through redox-dependent activation of transcription factors. Ape1 can therefore serve as a therapeutic target in either a DNA repair or transcriptional context. Inhibitors of the redox function can be used as either therapeutics or novel tools for separating the two functions for in vitro study. Presently there exist only a few compounds that have been reported to inhibit Ape1 redox activity; here we describe a series of quinones that exhibit micromolar inhibition of the redox function of Ape1. Benzoquinone and naphthoquinone analogues of the Ape1-inhibitor E3330 were designed and synthesized to explore structural effects on redox function and inhibition of cell growth. Most of the naphthoquinones were low micromolar inhibitors of Ape1 redox activity, and the most potent analogues inhibited tumor cell growth with IC(50) values in the 10-20 microM range.


Subject(s)
Benzoquinones/chemical synthesis , Benzoquinones/pharmacology , DNA-(Apurinic or Apyrimidinic Site) Lyase/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Methacrylates/chemical synthesis , Methacrylates/pharmacology , Naphthoquinones/chemical synthesis , Naphthoquinones/pharmacology , Propionates/chemistry , Benzoquinones/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Electrophoretic Mobility Shift Assay , Enzyme Inhibitors/chemistry , Female , Humans , Magnetic Resonance Spectroscopy , Methacrylates/chemistry , Naphthoquinones/chemistry , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Oxidation-Reduction
9.
Antioxid Redox Signal ; 10(11): 1853-67, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18627350

ABSTRACT

The DNA base excision-repair pathway is responsible for the repair of DNA damage caused by oxidation/alkylation and protects cells against the effects of endogenous and exogenous agents. Removal of the damaged base creates a baseless (AP) site. AP endonuclease1 (Ape1) acts on this site to continue the BER-pathway repair. Failure to repair baseless sites leads to DNA strand breaks and cytotoxicity. In addition to the repair role of Ape1, it also functions as a major redox-signaling factor to reduce and activate transcription factors such as AP1, p53, HIF-1alpha, and others that control the expression of genes important for cell survival and cancer promotion and progression. Thus, the Ape1 protein interacts with proteins involved in DNA repair, growth-signaling pathways, and pathways involved in tumor promotion and progression. Although knockdown studies with siRNA have been informative in studying the role of Ape1 in both normal and cancer cells, knocking down Ape1 does not reveal the individual role of the redox or repair functions of Ape1. The identification of small-molecule inhibitors of specific Ape1 functions is critical for mechanistic studies and translational applications. Here we discuss small-molecule inhibition of Ape1 redox and its effect on both cancer and endothelial cells.


Subject(s)
DNA Repair/physiology , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Endothelial Cells/physiology , Amino Acid Substitution , Animals , Benzoquinones/chemical synthesis , Benzoquinones/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , DNA-(Apurinic or Apyrimidinic Site) Lyase/antagonists & inhibitors , Endothelial Cells/cytology , Endothelial Cells/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/physiopathology , Neovascularization, Physiologic/drug effects , Oxidation-Reduction/drug effects , Propionates/chemical synthesis , Propionates/pharmacology , Retina/cytology , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcriptional Activation/drug effects , Transfection , Zebrafish
10.
J Pharmacol Exp Ther ; 326(1): 1-11, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18367665

ABSTRACT

Neurofibromatosis type 1 (NF1) is a genetic disorder that is driven by the loss of neurofibromin (Nf) protein function. Nf contains a Ras-GTPase-activating protein domain, which directly regulates Ras signaling. Numerous clinical manifestations are associated with the loss of Nf and increased Ras activity. Ras proteins must be prenylated to traffic and functionally localize with target membranes. Hence, Ras is a potential therapeutic target for treating NF1. We have tested the efficacy of two novel farnesyl transferase inhibitors (FTIs), 1 and 2, alone or in combination with lovastatin, on two NF1 malignant peripheral nerve sheath tumor (MPNST) cell lines, NF90-8 and ST88-14. Single treatments of 1, 2, or lovastatin had no effect on Ras prenylation or MPNST cell proliferation. However, low micromolar combinations of 1 or 2 with lovastatin (FTI/lovastatin) reduced Ras prenylation in both MPNST cell lines. Furthermore, this FTI/lovastatin combination treatment reduced cell proliferation and induced an apoptotic response as shown by morphological analysis, procaspase-3/-7 activation, loss of mitochondrial membrane potential, and accumulation of cells with sub-G(1) DNA content. Little to no detectable toxicity was observed in normal rat Schwann cells following FTI/lovastatin combination treatment. These data support the hypothesis that combination FTI plus lovastatin therapy may be a potential treatment for NF1 MPNSTs.


Subject(s)
Apoptosis/drug effects , Farnesyltranstransferase/antagonists & inhibitors , Lovastatin/administration & dosage , Nerve Sheath Neoplasms/drug therapy , Nerve Sheath Neoplasms/pathology , Neurofibromatosis 1/drug therapy , Neurofibromatosis 1/pathology , Animals , Animals, Newborn , Apoptosis/physiology , Cell Line , Cell Line, Transformed , Cell Line, Tumor , Drug Therapy, Combination , Enzyme Inhibitors/administration & dosage , Farnesyltranstransferase/metabolism , Lovastatin/chemistry , Neurofibromatosis 1/enzymology , Rats , Rats, Sprague-Dawley
11.
J Med Chem ; 50(15): 3743-6, 2007 Jul 26.
Article in English | MEDLINE | ID: mdl-17602464

ABSTRACT

A gemcitabine (2',2'-difluorodeoxycytidine, dFdC) phosphoramidate prodrug designed for the intracellular delivery of gemcitabine 5'-monophosphate was synthesized. The prodrug was about an order of magnitude less active than gemcitabine against wild-type cells, and the nucleoside transport inhibitor dipyridamole reduced prodrug activity. The prodrug was more active than gemcitabine against two deoxycytidine kinase-deficient cell lines. The results suggest that the prodrug is a potent growth inhibitor that can bypass dCK deficiency at higher drug concentrations.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cytidine Monophosphate/analogs & derivatives , Deoxycytidine/analogs & derivatives , Prodrugs/pharmacology , Antimetabolites, Antineoplastic/chemical synthesis , Antimetabolites, Antineoplastic/chemistry , Cell Line, Tumor , Cytidine Monophosphate/chemical synthesis , Cytidine Monophosphate/chemistry , Cytidine Monophosphate/pharmacology , Deoxycytidine/pharmacology , Deoxycytidine Kinase/genetics , Dipyridamole/pharmacology , Drug Screening Assays, Antitumor , Humans , Nucleoside Transport Proteins/antagonists & inhibitors , Prodrugs/chemical synthesis , Prodrugs/chemistry , Structure-Activity Relationship , Gemcitabine
12.
J Med Chem ; 50(14): 3274-82, 2007 Jul 12.
Article in English | MEDLINE | ID: mdl-17555307

ABSTRACT

Certain farnesyl diphosphate (FPP) analogs are potent inhibitors of the potential anticancer drug target protein farnesyltransferase (FTase), but these compounds are not suitable as drug candidates. Thus, phosphoramidate prodrug derivatives of the monophosphate precursors of FPP-based FTase inhibitors have been synthesized. The monophosphates themselves were significantly more potent inhibitors of FTase than the corresponding FPP analogs. The effects of the prodrug 5b (a derivative of 3-allylfarnesyl monophosphate) have been evaluated on prenylation of RhoB and on the cell cycle in a human malignant schwannoma cell line (STS-26T). In combination treatments, 1-3 microM 5b plus 1 microM lovastatin induced a significant inhibition of RhoB prenylation, and a combination of these drugs at 1 microM each also resulted in significant cell cycle arrest in G1. Indeed, combinations as low as 50 nM lovastatin + 1 microM 5c or 250 nM lovastatin + 50 nM 5c were highly cytostatic in STS-26T cell culture.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase/antagonists & inhibitors , Polyisoprenyl Phosphates/chemical synthesis , Polyisoprenyl Phosphates/pharmacology , Prodrugs/pharmacology , Cell Line , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Prodrugs/chemical synthesis , Spectrometry, Mass, Electrospray Ionization
13.
Org Lett ; 9(2): 215-8, 2007 Jan 18.
Article in English | MEDLINE | ID: mdl-17217268

ABSTRACT

Both enantiomers of protected and unprotected 2-hydroxymethylaziridines are efficiently and enantiospecifically synthesized by using a combination of enzymatic and synthetic methods. PPL was used for lipase-catalyzed desymmetrization of N-protected serinol. [reaction: see text].


Subject(s)
Aziridines/chemical synthesis , Lipase/chemistry , Animals , Aziridines/chemistry , Catalysis , Magnetic Resonance Spectroscopy , Molecular Conformation , Pancreas/enzymology , Sensitivity and Specificity , Stereoisomerism , Swine
14.
J Med Chem ; 50(4): 856-64, 2007 Feb 22.
Article in English | MEDLINE | ID: mdl-17249650

ABSTRACT

Our laboratory recently reported the development of novel prodrug chemistry for the intracellular delivery of phosphotyrosine mimetics. This chemistry has now been adapted for the synthesis of a prodrug that delivers the nonhydrolyzable difluoromethylphosphonate moiety intracellularly. Activation of the prodrug generates a difluoromethylphosphonamidate anion that undergoes subsequent cyclization and hydrolysis with a t1/2 = 44 min. A highly potent and selective inhibitor of protein tyrosine phosphatase 1B (PTP1B) with a nanomolar Ki has been reported, but this bis(difluoromethylphosphonate) lacks potential utility due to its exceedingly low membrane permeability at physiological pH. A prodrug of this inhibitor has been synthesized and evaluated in a cell-based assay. The prodrug exhibits nanomolar PTP1B inhibitory activity in this assay, confirming the efficacy of intracellular phosphonate delivery using this prodrug approach.


Subject(s)
Dipeptides/chemical synthesis , Organophosphorus Compounds/chemical synthesis , Prodrugs/chemical synthesis , Protein Tyrosine Phosphatases/antagonists & inhibitors , Cell Line, Tumor , Cell Membrane Permeability , Dipeptides/chemistry , Dipeptides/pharmacokinetics , Humans , Insulin/physiology , Magnetic Resonance Spectroscopy , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacokinetics , Phosphorylation , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Receptor, Insulin/metabolism , Signal Transduction , Structure-Activity Relationship
15.
Mol Pharm ; 3(4): 451-6, 2006.
Article in English | MEDLINE | ID: mdl-16889439

ABSTRACT

N-2,3-dihydroxypropyl-N-4-chlorobutyl phosphoramidate prodrugs of thymidine and 5-fluoro-2'-deoxyuridine (5-FdU) 2-4 were synthesized as analogues of the known prodrugs 1a,b to assess the effects of the dihydroxypropyl moiety on prodrug activation, log P, and growth inhibitory activity in vitro. The thymidine analogues 2 and 3 were prepared as model compounds for kinetics and log P studies. 31P NMR kinetics following hydrogenolysis of 2 showed that the thymidine N-dihydroxypropyl phosphoramidate released thymidine monophosphate with a half-life of 212 min under model physiologic conditions compared to approximately 2 min for the corresponding N-methyl phosphoramidate reported previously. The measured log P for compound 3 was 1.1 log units lower than that of the analogous 1b, confirming that the dihydroxypropyl group significantly decreased prodrug lipophilicity. The dihydroxypropyl prodrug 4 showed cell growth inhibition activity in the NCI 60 cell line panel similar to that of the N-methyl analogue 1a previously reported.


Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Floxuridine/pharmacology , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Thymidine/analogs & derivatives , Amides/chemical synthesis , Amides/chemistry , Amides/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Floxuridine/analogs & derivatives , Floxuridine/chemical synthesis , Floxuridine/chemistry , Humans , Magnetic Resonance Spectroscopy , Phosphoric Acids/chemical synthesis , Phosphoric Acids/chemistry , Phosphoric Acids/pharmacology , Prodrugs/chemistry , Structure-Activity Relationship , Thymidine/chemical synthesis , Thymidine/chemistry , Thymidine/pharmacology
16.
Biochem Pharmacol ; 72(11): 1485-92, 2006 Nov 30.
Article in English | MEDLINE | ID: mdl-16797490

ABSTRACT

Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome. NF1 patients present with a constellation of clinical manifestations and have an increased risk of developing certain benign and malignant tumors. This disease results from mutation within the gene encoding neurofibromin, a GTPase activating protein (GAP) for Ras. Functional loss of this protein compromises Ras inactivation, which leads to the aberrant growth and proliferation of neural crest-derived cells and, ultimately, tumor formation. Current management of NF1-associated malignancy involves radiation, surgical excision, and cytotoxic drugs. The limited success of these strategies has fueled researchers to further elucidate the molecular changes that drive tumor formation and progression. This discussion will highlight how intracellular signaling molecules, cell-surface receptors, and the tumor microenvironment constitute potential therapeutic targets, which may be relevant not only to NF1-related malignancy but also to other human cancers.


Subject(s)
Genes, Neurofibromatosis 1 , Genetic Predisposition to Disease/genetics , Genetic Therapy , Neurofibromatosis 1/genetics , Neurofibromin 1/genetics , Animals , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Humans , Mutation , Neurofibromatosis 1/metabolism , Neurofibromin 1/metabolism , Signal Transduction
17.
J Med Chem ; 49(11): 3368-76, 2006 Jun 01.
Article in English | MEDLINE | ID: mdl-16722656

ABSTRACT

A novel approach to the intracellular delivery of aryl phosphates has been developed that utilizes a phosphoramidate-based prodrug approach. The prodrugs contain an ester group that undergoes reductive activation intracellularly with concomitant expulsion of a phosphoramidate anion. This anion undergoes intramolecular cyclization and hydrolysis to generate aryl phosphate exclusively with a t(1/2) = approximately 20 min. Phosphoramidate prodrugs (8-10) of phosphate-containing peptidomimetics that target the SH2 domain were synthesized. Evaluation of these peptidomimetic prodrugs in a growth inhibition assay and in a cell-based transcriptional assay demonstrated that the prodrugs had IC50 values in the low micromolar range. Synthesis of phosphorodiamidate analogues containing a P-NH-Ar linker (16-18) was also carried out in the hope that the phosphoramidates released might be phosphatase-resistant. Comparable activation rates and cell-based activities were observed for these prodrugs, but the intermediate phosphoramidate dianion underwent spontaneous hydrolysis with a t(1/2) = approximately 30 min.


Subject(s)
Organophosphates/chemical synthesis , Peptides/chemistry , Phosphotyrosine/analogs & derivatives , Phosphotyrosine/chemical synthesis , Prodrugs/chemical synthesis , Amides/chemical synthesis , Amides/pharmacology , Cell Membrane Permeability , Cell Proliferation/drug effects , Drug Design , Humans , Hydrolysis , Jurkat Cells , Kinetics , Molecular Mimicry , Organophosphates/chemistry , Organophosphates/pharmacology , Phosphoric Acids/chemical synthesis , Phosphoric Acids/pharmacology , Phosphotyrosine/chemistry , Phosphotyrosine/pharmacology , Prodrugs/chemistry , Prodrugs/pharmacology , Structure-Activity Relationship , src Homology Domains
18.
Org Lett ; 6(13): 2257-60, 2004 Jun 24.
Article in English | MEDLINE | ID: mdl-15200334

ABSTRACT

[reaction: see text] A novel method for the preparation of nucleoside triphosphates has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes efficient coupling with tris(tetra-n-butylammonium) hydrogen pyrophosphate to generate nucleoside triphosphate.


Subject(s)
Nucleotides/chemical synthesis , Amides/chemical synthesis , Amides/chemistry , Diphosphates/chemistry , Phosphoric Acids/chemical synthesis , Phosphoric Acids/chemistry , Pyrrolidines/chemistry , Thymidine/chemistry , Thymine Nucleotides/chemistry
19.
Mol Pharm ; 1(2): 112-6, 2004.
Article in English | MEDLINE | ID: mdl-15832507

ABSTRACT

Recently, we reported a novel approach for the intracellular delivery of the anti-cancer nucleotide 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) using phosphoramidate-based prodrugs. These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation, liberating phosphoramidate anion, which in turn undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quantitatively. This approach has now been extended to cytarabine [1-beta-D-arabinofuranosylcytosine (Ara-C)], an anti-cancer nucleoside that is limited in its utility because of poor intracellular transport characteristics and weak activity as a substrate for tumor cell kinases. The cytarabine phosphoramidate prodrug 1 has been synthesized and evaluated in comparison with cytarabine for growth inhibitory activity against wild-type, nucleoside transport-deficient, and nucleoside kinase-deficient CEM leukemia cell lines. The prodrug was comparable in growth inhibitory activity (IC50 = 32 nM) to cytarabine (IC50 = 16 nM) in wild-type CCRF-CEM cells following drug treatment for 72 h. The nucleoside transport-deficient CEM/AraC8C exhibited a high level of resistance (6400-fold) to cytarabine but was more sensitive (210-fold resistant vs CCRF-CEM cells) to prodrug 1. Similarly, the deoxycytidine kinase-deficient cell line (CEM/dCK-) was highly resistant to cytarabine (13900-fold) but more sensitive (106-fold resistant vs CCRF-CEM cells) to prodrug 1. These results indicate that prodrug 1 is significantly more potent than cytarabine against transport- and kinase-deficient cell lines and are consistent with a mechanism involving intracellular delivery of cytarabine 5'-monophosphate.


Subject(s)
Amides/chemical synthesis , Cytarabine/chemical synthesis , Phosphoric Acids/chemical synthesis , Prodrugs/chemical synthesis , Amides/pharmacology , Biological Transport , Cell Division , Cell Line, Tumor , Cytarabine/pharmacology , Humans , Magnetic Resonance Spectroscopy , Phosphoric Acids/pharmacology , Prodrugs/pharmacology
20.
Org Lett ; 5(3): 341-4, 2003 Feb 06.
Article in English | MEDLINE | ID: mdl-12556187

ABSTRACT

[reaction: see text] A novel method for the immobilization of nucleotides has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes nucleophilic attack by long-chain alkylamine-controlled pore glass (LCAA-CPG) to generate an immobilized nucleotide. Quantification of nucleotide loading was accomplished by acidic hydrolysis of the P-N bond and subsequent HPLC analysis of TMP in the presence of an internal standard. Typical nucleotide loadings of 51-59 micromol/g of support were observed.


Subject(s)
Nucleotides/chemistry , Amides/chemistry , Chromatography, High Pressure Liquid , Hydrolysis , Oligonucleotide Array Sequence Analysis/methods , Phosphoric Acids/chemistry
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