Synthesis of Carboxamide-Containing Tranylcypromine Analogues as LSD1 (KDM1A) Inhibitors Targeting Acute Myeloid Leukemia

Lysine-specific demethylase 1 (LSD1/KDM1A) oxidatively removes methyl groups from histone proteins, and its aberrant activity has been correlated with cancers including acute myeloid leukemia (AML). We report a novel series of tranylcypromine analogues with a carboxamide at the 4-position of the aryl ring. These compounds, such as 5 a and 5 b with benzyl and phenethylamide substituents, respectively, had potent sub-micromolar IC₅₀ values for the inhibition of LSD1 as well as cell proliferation in a panel of AML cell lines. The dose-dependent increase in cellular expression levels of H3K4me2, CD86, CD11b and CD14 supported a mechanism involving LSD1 inhibition. The tert-butyl and ethyl carbamate derivatives of these tranylcypromines, although inactive in LSD1 inhibition, were of similar potency in cell-based assays with a more rapid onset of action. This suggests that carbamates can act as metabolically labile tranylcypromine prodrugs with superior pharmacokinetics.     

Synthesis of pH-sensitive,water-soluble paclitaxel prodrugs based on norbornene-functional polylactide by copper-free click chemistry

In this study, a novel polymeric prodrug based on norbornene-functional polylactide with functionalized paclitaxels (LEV-PTXL) covalently conjugated to water-soluble polymer carrier via a pH-sensitive hydrazone bond was developed. A series of water-soluble PTXL prodrugs with decent drug contents of 17.3 and 24.7 wt% (denoted as PTXL prodrugs 1 and 2) were prepared. The molecular structures and characteristics were confirmed by ¹H NMR, Fourier transform infrared, and gel permeation chromatography. In vitro release studies showed that PTXL release rate from polymeric prodrug were significantly accelerated under acidic medium, due to the acid-cleavable hydrazone linkage. As illustrated by cytotoxicity study, while the corresponding polymer carrier was nontoxic, the polymeric prodrug exhibited higher therapeutic efficacy toward MCF-7 and A549 (IC50 = 0.19 and 4.94 µg PTXL equiv/mL, respectively) cancer cells. The norbornene-functional polylactide-based polymeric prodrug has appeared as a novel anticancer nanomedicine for cancer therapy.     

NIR Fluorogenic Dye as a Modular Platform for Prodrug Assembly: Real‐Time in vivo Monitoring of Drug Release

The ability to monitor drug release in vivo provides essential pharmacological information. We developed a new modular approach for the preparation of theranostic prodrugs with a turn‐ON near‐infrared (NIR) fluorescence mode of action. The prodrugs release their chemotherapeutic cargo and an active cyanine fluorophore upon reaction with a specific analyte. The prodrug platform is based on the fluorogenic dye QCy7; upon removal of a triggering substrate, the dye fluoresces, and the free drug is released. The evaluated camptothecin prodrug was activated by endogenous hydrogen peroxide produced in tumor cells in vitro and in vivo. Drug release and in vitro cytotoxicity were correlated with the emitted fluorescence. The prodrug activation was effectively imaged in real time in mice bearing tumors. The modular design of the QCy7 fluorogenic platform should allow the preparation of numerous other prodrugs with various triggering substrates and chemotherapeutic agents. We anticipate that the development of real‐time in vivo monitoring tools such as that described herein will pave the way for personalized therapy.     

Quantitative Insight into the Design of Compounds Recognized by the L‐Type Amino Acid Transporter 1 (LAT1)

L ‐Type amino acid transporter 1 (LAT1) is a transmembrane protein expressed abundantly at the blood–brain barrier (BBB), where it ensures the transport of hydrophobic acids from the blood to the brain. Due to its unique substrate specificity and high expression at the BBB, LAT1 is an intriguing target for carrier‐mediated transport of drugs into the brain. In this study, a comparative molecular field analysis (CoMFA) model with considerable statistical quality (Q²=0.53, R²=0.75, Q² SE=0.77, R² SE=0.57) and good external predictivity (CCC=0.91) was generated. The model was used to guide the synthesis of eight new prodrugs whose affinity for LAT1 was tested by using an in situ rat brain perfusion technique. This resulted in the creation of a novel LAT1 prodrug with L ‐tryptophan as the promoiety; it also provided a better understanding of the molecular features of LAT1‐targeted high‐affinity prodrugs, as well as their promoiety and parent drug. The results obtained will be beneficial in the rational design of novel LAT1‐binding prodrugs and other compounds that bind to LAT1.     

Novel electrospun nanofibers incorporating polymeric prodrugs of ketoprofen: Preparation,characterization, and in vitro sustained release

A facile and efficient protocol for the preparation of nanofibers incorporating polymeric ketoprofen prodrugs and polyvinylpyrrolidone was developed. Polymeric ketoprofen prodrugs were constructed by a two‐step chemo‐enzymatic synthetic route, and nanofibers prepared by electrospinning from dimethylformamide/ethanol (1 : 1, v/v) solutions. The morphological characteristics of the fibers were influenced by the concentration of active agent in the spinning solution; average diameters varied from 196 to 370 nm. In vitro release studies indicated that the ketoprofen release rate from the electrospun fibers was significantly higher than that from the pure polymeric prodrugs. Cumulative drug release from the electrospun fibers reached 40–70% after 3 h and 75–100% after 12 h, while the pure polymeric prodrug released only 7–9% of the active agent over 12 h. Functional nanofibers incorporating polymeric prodrugs therefore comprise potentially effective drug delivery systems for sustained release. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 1570–1577, 2013     

Liver‐targeting doxorubicin‐conjugated polymeric prodrug with pH‐triggered drug release profile

The aim of the research presented was to develop a potential liver‐targeting prolonged‐circulation polymeric prodrug of doxorubicin (Dox) with a pH‐triggered drug release profile. In particular, linear dendritic block copolymers composed of polyamidoamine dendrimer (PAMAM) and poly(ethylene glycol) (PEG; number‐average molecular weight of 2000 g mol^?1) with or without galactose (Gal) were synthesized. Dox was coupled to the copolymers via an acid‐labile hydrazone linker. These prodrugs, designated Gal‐PEG‐b‐PAMAM‐Dox_n and mPEG‐b‐PAMAM‐Dox_m, showed accelerated Dox release as the pH decreased from 8.0 to 5.6. Cytotoxicity of the prodrugs was lower than that of free Dox due to the gradual drug release nature. Compared to mPEG‐b‐PAMAM‐Dox_m, Gal‐PEG‐b‐PAMAM‐Dox_n showed rather high cytotoxicity against Bel‐7402, suggestive of its galactose receptor‐mediated enhanced tumor uptake. This galactose receptor‐mediated liver‐targeted profile was further confirmed by the prolonged retention time in hepatoma tissue monitored using magnetic resonance imaging. Gal‐PEG‐b‐PAMAM‐Dox_n showed better in vivo antitumor efficacy than free Dox, suggesting its great potential as a polymeric antitumor prodrug. Copyright © 2010 Society of Chemical Industry     

New prodrugs of the antiprotozoal drug pentamidine

Pentamidine is an effective antimicrobial agent that is approved for the treatment of African trypanosomiasis but suffers from poor oral bioavailability and central nervous system (CNS) penetration. This work deals with the development and systematic characterisation of new prodrugs of pentamidine. For this reason, numerous prodrugs that use different prodrug principles were synthesised and examined in vitro and in vivo. Another objective of the study was the determination of permeability of the different pentamidine prodrugs. While some of the prodrug principles applied in this study are known, such as the conversion of the amidine functions into amidoximes or the O-alkylation of amidoximes with a carboxymethyl residue, others were developed more recently and are described here for the first time. These newly developed methods aim to increase the affinity of the prodrug for the transporters and mediate an active uptake via carrier systems by conjugation of amidoximes with compounds that improve the overall solubility of the prodrug. The different principles chosen resulted in several pentamidine prodrugs with various advantages. The objective of this investigation was the systematic characterisation and evaluation of eight pentamidine prodrugs in order to identify the most appropriate strategy to improve the properties of the parent drug. For this reason, all prodrugs were examined with respect to their solubility, stability, enzymatic activation, distribution, CNS delivery, and oral bioavailability. The results of this work have allowed reliable conclusions to be drawn regarding the best prodrug principle for the antiprotozoal drug pentamidine.     

Use of Self-Assembled Colloidal Prodrug Nanoparticles for Controlled Drug Delivery of Anticancer,Antifibrotic and Antibacterial Mitomycin

Herein we present the synthesis of a polymeric prodrug nanomaterial capable of spontaneous, self-assembled nanoparticle formation and of the conjugation (encapsulation) of drugs with amino and/or carboxyl and/or hydroxyl groups via ester and/or amide linkage. Mitomycin C (MMC) a versatile drug with antibiotic, antibacterial and antineoplastic properties, was used to prove this concept. The in vitro drug release experiments showed a fast release for the pure MMC (k = 49.59 h⁻ⁿ); however, a significantly lower MMC dissolution rate (k = 2.25, 1.46, and 1.35 h⁻ⁿ) was obtained for the nanoparticles with increased cross-link density (3, 10, 21%). The successful modification and conjugation reactions were confirmed using FTIR and EDX measurements, while the mucoadhesive properties of the self-assembled particles synthesized in a simple one-pot reaction were proved by rheological measurement. The prepared biocompatible polymeric prodrugs are very promising and applicable as a drug delivery system (DDS) and useful in the area of cancer treatment.     

Prodrug strategies in anticancer chemotherapy

The majority of clinically approved anticancer drugs are characterized by a narrow therapeutic window that results mainly from a high systemic toxicity of the drugs in combination with an evident lack of tumor selectivity. Besides the development of suitable galenic formulations such as liposomes or micelles, several promising prodrug approaches have been followed in the last decades with the aim of improving chemotherapy. In this review we elucidate the two main concepts that underlie the design of most anticancer prodrugs: drug targeting and controlled release of the drug at the tumor site. Consequently, active and passive targeting using tumor-specific ligands or macromolecular carriers are discussed as well as release strategies that are based on tumor-specific characteristics such as low pH or the expression of tumor-associated enzymes. Furthermore, other strategies such as ADEPT (antibody-directed enzyme prodrug therapy) and the design of self-eliminating structures are introduced. Chemical realization of prodrug approaches is illustrated by drug candidates that have or may have clinical importance.     

Xylitol as a Hydrophilization Moiety for a Biocatalytically Synthesized Ibuprofen Prodrug

Biocatalyzed synthesis can be exploited to produce high-value products, such as prodrugs. The replacement of chemical approaches with biocatalytic processes is advantageous in terms of environmental prevention, embracing the principles of green chemistry. In this work, we propose the covalent attachment of xylitol to ibuprofen to produce an IBU-xylitol ester prodrug. Xylitol was chosen as a hydrophilizer for the final prodrug, enhancing the water solubility of ibuprofen. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) extensively used as an analgesic, anti-inflammatory, and antipyretic. Despite being the third-most-prescribed medicine in the world, the aqueous solubility of ibuprofen is just 21 mg/L. This poor water solubility greatly limits the bioavailability of ibuprofen. We aimed to functionalize ibuprofen with xylitol using the reusable immobilized N435 biocatalyst. Instead of a biphasic media, we proposed a monophasic reaction environment. The characterization of the IBU-xylitol ester was performed by ¹H, ¹³C-NMR, DEPT, COSY, HMQC, HMBC, FTIR, and MS spectroscopy. Preliminary in vitro tests showed that this enzymatically synthesized prodrug of ibuprofen reduced the expression of the interleukin 8 genes in human bronchial epithelial cells (IB3-1) from cystic fibrosis (CF) patients.     

含5-氟尿嘧啶的氨基甲酸聚乙二醇酯前药的合成与表征

采用异氰酸-α-氯乙酰酯与不同相对分子质量的聚乙二醇反应得到双氯乙酰氨基甲酸聚乙二醇酯,由于端基的α-氯具有良好的反应活性,将它与抗肿瘤药物5-氟尿嘧啶结合制备不同相对分子质量的高分子前药。对产物进行红外、核磁、紫外表征,证明5-氟尿嘧啶已经成功接到聚乙二醇链的末端,由紫外分光光度法测得最大载药量为22.6%。与5-氟尿嘧啶相比,前药的水溶性得到增强,而且具有长效缓释的功能。前药在不同pH的缓冲液中可以释放5-氟尿嘧啶或其衍生物,当聚乙二醇相对分子质量为2000时水解速度最快,20h的最大释药率为71.5%。     

Using Theory To Extend the Scope of Azobenzene Drugs in Chemotherapy: Novel Combinations for a Specific Delivery

Gut microorganisms metabolize azobenzene compounds (Ph¹−N=N−Ph²) into free aniline products (Ph¹−NH₂+H₂N−Ph²), a process that has been largely investigated to reduce dyes residues in the textile industry. However, the action of bacterial core enzymes such as azoreductases (AzoR) might also help to deliver prodrugs that become active when they reach the colonic region, a mechanism with potential applications for the treatment of inflammatory bowel disease (IBD) and colorectal cancer. So far, three azo-bonded prodrugs of 5-aminosalicylic acid (5-ASA), for example, sulfasalazine, olsalazine and balsalazide, have been used for colon-targeted delivery. The present contribution describes the first rational design of a novel azobenzene prodrug thanks to a computational approach, with a focus on linking 5-ASA to another approved anti-inflammatory drug. The resulting prodrugs were assessed for their degradation upon AzoR action. Replacing the original carriers by irsogladine is found to improve action.     

Nanostructured nanoparticles of self-assembled lipid pro-drugs as a route to improved chemotherapeutic agents

We demonstrate that oral delivery of self-assembled nanostructured nanoparticles consisting of 5-fluorouracil (5-FU) lipid prodrugs results in a highly effective, target-activated, chemotherapeutic agent, and offers significantly enhanced efficacy over a commercially available alternative that does not self-assemble. The lipid prodrug nanoparticles have been found to significantly slow the growth of a highly aggressive mouse 4T1 breast tumour, and essentially halt the growth of a human MDA-MB-231 breast tumour in mouse xenografts. Systemic toxicity is avoided as prodrug activation requires a three-step, enzymatic conversion to 5-FU, with the third step occurring preferentially at the tumour site. Additionally, differences in the lipid prodrug chemical structure and internal nanostructure of the nanoparticle dictate the enzymatic conversion rate and can be used to control sustained release profiles. Thus, we have developed novel oral nanomedicines that combine sustained release properties with target-selective activation.     

Synthesis and Evaluation of Water‐Soluble Prodrugs of Ursodeoxycholic Acid (UDCA), an Anti‐apoptotic Bile Acid

Ursodeoxycholic acid (UDCA) is a bile acid with demonstrated anti‐apoptotic activity in both in vitro and in vivo models. However, its utility is hampered by limited aqueous solubility. As such, water‐soluble prodrugs of UDCA could have an advantage over the parent bile acid in indications where intravenous administration might be preferable, such as decreasing damage from stroke or acute kidney injury. Five phosphate prodrugs were synthesized, including one incorporating a novel phosphoryloxymethyl carboxylate (POMC) moiety. These prodrugs were highly water‐soluble, but showed significant differences in chemical stability, with oxymethylphosphate prodrugs being the most unstable. In a series of NMR experiments, the POMC prodrug was bioactivated to UDCA by alkaline phosphatase (AP) faster than a prodrug containing a phosphate directly attached to the alcohol at the 3‐position of UDCA. Both of these prodrugs showed significant anti‐apoptotic activity in a series of in vitro assays, although the POMC prodrug required the addition of AP for activity, while the other compound was active without exogenous AP.     

Emerging Glycation-Based Therapeutics—Glyoxalase 1 Inducers and Glyoxalase 1 Inhibitors

The abnormal accumulation of methylglyoxal (MG) leading to increased glycation of protein and DNA has emerged as an important metabolic stress, dicarbonyl stress, linked to aging, and disease. Increased MG glycation produces inactivation and misfolding of proteins, cell dysfunction, activation of the unfolded protein response, and related low-grade inflammation. Glycation of DNA and the spliceosome contribute to an antiproliferative and apoptotic response of high, cytotoxic levels of MG. Glyoxalase 1 (Glo1) of the glyoxalase system has a major role in the metabolism of MG. Small molecule inducers of Glo1, Glo1 inducers, have been developed to alleviate dicarbonyl stress as a prospective treatment for the prevention and early-stage reversal of type 2 diabetes and prevention of vascular complications of diabetes. The first clinical trial with the Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP)—a randomized, double-blind, placebo-controlled crossover phase 2A study for correction of insulin resistance in overweight and obese subjects, was completed successfully. tRES-HESP corrected insulin resistance, improved dysglycemia, and low-grade inflammation. Cell permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a prospective treatment for cancer—particularly for high Glo1 expressing-related multidrug-resistant tumors. The prototype Glo1 inhibitor is prodrug S-p-bromobenzylglutathione cyclopentyl diester (BBGD). It has antitumor activity in vitro and in tumor-bearing mice in vivo. In the National Cancer Institute human tumor cell line screen, BBGD was most active against the glioblastoma SNB-19 cell line. Recently, potent antitumor activity was found in glioblastoma multiforme tumor-bearing mice. High Glo1 expression is a negative survival factor in chemotherapy of breast cancer where adjunct therapy with a Glo1 inhibitor may improve treatment outcomes. BBGD has not yet been evaluated clinically. Glycation by MG now appears to be a pathogenic process that may be pharmacologically manipulated for therapeutic outcomes of potentially important clinical impact.     

Development and Structural Evaluation of N-Alkylated trans-2-Phenylcyclopropylamine-Based LSD1 Inhibitors

Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the demethylation of histone H3 and regulates gene expression. Because it is implicated in the regulation of diseases such as acute myeloid leukemia, potent LSD1-specific inhibitors have been pursued. Trans-2-phenylcyclopropylamine (2-PCPA)-based inhibitors featuring substitutions on the amino group have emerged, with sub-micromolar affinities toward LSD1 and high selectivities over monoamine oxidases (MAOs). We synthesized two N-alkylated 2-PCPA-based LSD1 inhibitors, S2116 and S2157, based on the previously developed S2101. S2116 and S2157 exhibited enhanced potency for LSD1 by 2.0- to 2.6-fold, as compared with S2101. In addition, they exhibited improved selectivity over MAOs. Structural analyses of LSD1 co-crystallized with S2101, S2116, S2157, or another N-alkylated inhibitor (FCPA-MPE) confirmed that the N-substituents enhance the potency of a 2-PCPA-based inhibitor of LSD1, without constituting the adduct formed with FAD.     

Dipeptide derivatives of AZT: synthesis, chemical stability, activation in human plasma, hPEPT1 affinity, and antiviral activity

5'-O-Dipeptide ester prodrugs of antiviral zidovudine (AZT) were designed to target the human intestinal oligopeptide transporter, hPEPT1, and were evaluated for their stability at pH 7.4 in buffer and in human plasma, affinity toward hPEPT1, cytotoxicity, and antiretroviral activity. The dipeptide esters of AZT undergo cyclization in buffer at pH 7.4 to release the parent drug at a rate that depends on the size of the side chains of the peptide carrier; the prodrug is considerably more stable if bulky beta-branched amino acids such as Ile and Val are present, particularly as C-terminal residues. Incubation in human plasma showed that most of the dipeptide esters of AZT release the parent drug through two aminopeptidase-mediated pathways: 1) stepwise cleavage of each of the amino acids and 2) direct cleavage of the dipeptide-drug ester bond. However, the plasma hydrolysis of Gly-Gly-AZT and Phe-Gly-AZT showed only direct cleavage of the dipeptide-drug ester bond. Substrate half-lives in plasma were again remarkably high when hydrophobic beta-branched amino acids (Val, Ile) were present. The esters were also good substrates for the intestinal oligopeptide transporter hPEPT1 in vitro, with Val-Gly-AZT and Val-Ala-AZT presenting the highest affinity toward the transporter (IC(50): 0.20 and 0.15 mM, respectively). The AZT dipeptide esters were assayed against the IIIB and ROD strains of HIV, and their cytotoxicity was evaluated in MT-4 cells. The selectivity index of the prodrugs was two- to threefold higher than that of AZT for all compounds analyzed. These results point to the potential of dipeptide-based carriers for the development of effective antiviral drug-delivery systems. Val-Ala-AZT appears to combine chemical stability with good affinity for the hPEPT1 transporter and an improved cytotoxicity/antiretroviral index relative to AZT.     

Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine

Glutathione transferases (GSTs) form a family of detoxication enzymes instrumental in the inactivation and elimination of electrophilic mutagenic and carcinogenic compounds. The Pi class GST P1-1 is present in most tissues and is commonly overexpressed in neoplastic cells. GST P1-1 in the dog, Canis lupus familiaris, has merits as a marker for tumors and as a target for enzyme-activated prodrugs. We produced the canine enzyme CluGST P1-1 by heterologous bacterial expression and verified its cross-reactivity with antihuman-GST P1-1 antibodies. The catalytic activity with alternative substrates of biological significance was determined, and the most active substrate found was benzyl isothiocyanate. Among established GST inhibitors, Cibacron Blue showed positive cooperativity with an IC₅₀ value of 43 nM. Dog GST P1-1 catalyzes activation of the prodrug Telcyta, but the activity is significantly lower than that of the human homolog.     

Bisamidate Prodrugs of 2‐Substituted 9‐[2‐(Phosphonomethoxy)ethyl]adenine (PMEA,adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

Novel small‐molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2‐substituted derivatives of 9‐[2‐(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L ‐phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2‐fluoro PMEA derivative (IC₅₀=0.145 μM ). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.     

The Emerging Role of Tetrazines in Drug-Activation Chemistries

Traditionally, prodrug activation has been limited to enzymatic triggers or gross physiological aberrations, such as pH, that offer low selectivity and control over dosage. In recent years, the field of prodrug activation chemistry has been transformed by the use of bioorthogonal reactions that can be carried out under biological conditions at sub-millimolar concentrations, with the tetrazine-mediated inverse electron demand Diels–Alder reaction amongst the most recognised. Their high reaction rates, chemoselectivity and excellent biocompatibility make tetrazines ideal small molecules for activating prodrugs. Recently the tetrazine moiety has been used as a prodrug for a pyridazine thus broadening the scope of prodrug systems. This article discusses the concept of using tetrazines as small-molecule activators for prodrugs, and provides an overview of tetrazine-based prodrug systems, with a particular focus on the recently reported prodrug–prodrug activation strategy.