Series Of Prodrugs Research Articles

Asymmetric Construction of Substituted Purine Esters with Tunable N‐9/N‐7 Regioselectivity via Enzymatic Dynamic Kinetic Resolution

AbstractWe report an enzyme‐catalyzed dynamic kinetic resolution protocol for the one‐pot asymmetric synthesis of substituted purine derivatives. The controllable N‐9/N‐7 substitution, which posed formidable challenges in organic synthesis, could be accomplished by choosing different types of enzymes as catalysts. This method was proved scalable and was used to prepare a series of prodrugs of medicinally active acids. In addition, the recyclability test of the biocatalysts indicated the potential of this strategy for applications in the pharmaceutical industry.

Discovery of NXT-10796, an orally active, intestinally restricted EP4 agonist prodrug for the treatment of inflammatory bowel disease

A property-focused optimization strategy was employed to modify the carboxylic acid head group of a class of EP4 agonists in order to minimize its absorption upon oral administration. The resulting oxalic acid monohydrazide-derived carboxylate isostere demonstrated utility as a class of prodrug showing colon-targeted delivery of parent agonist 2, with minimal exposure observed in the plasma. Oral administration of NXT-10796 demonstrated tissue specific activation of the EP4 receptor through modulation of immune genes in the colon, without modulation of EP4 driven biomarkers in the plasma compartment. Although further in depth understanding of the conversion of NXT-10796 is required for further assessment of the developability of this series of prodrugs, using NXT-10796 as a tool molecule has allowed us to confirm that tissue-specific modulation of an EP4-modulated gene signature is possible, which allows for further evaluation of this therapeutic modality in rodent models of human disease.

Design, synthesis and characterizations of prodrugs of brexanolone

A series of prodrugs of brexanolone, the synthetic version of the endogenously produced γ-aminobutyric acid A receptors positive allosteric modulator allopregnanolone, were designed, synthesized, and evaluated in vitro and in vivo. The effect of different function groups connecting to brexanolone C3 hydroxyl as well as those at the chain terminals of prodrug moieties were explored. Through these efforts, prodrugs that can efficiently release brexanolone in vitro and in vivo, and possess a potential for sustained delivery of a long acting brexanolone were discovered.

Design, synthesis and evaluation of a series of potential prodrugs of a Bruton's tyrosine kinase (BTK) inhibitor.

BTK has become a particularly attractive therapeutic target in autoimmune diseases and B-cell malignancies, making BTK inhibitors a valuable and important therapeutic option. We present the design, synthesis, and evaluation of a series of prodrugs of a BTK inhibitor with an insoluble 2,5-diaminopyrimidine structure. Tails containing different solubilizing groups were added to the parent molecule via an ester linkage. Prodrug 5a showed good aqueous solubility and could be efficiently converted to the parent in a human plasma stability study. The rational prodrug design was supported by molecular studies and a dramatically reduced BTK kinase-inhibitory potential. Taken together, the chemical, biological, and molecular studies suggest that prodrug derivatization of the 2,5-diaminopyrimidine scaffold could be a potential strategy for advancing this series of BTK inhibitors into the therapeutic arena.

Acyloxymethyl and alkoxycarbonyloxymethyl prodrugs of a fosmidomycin surrogate as antimalarial and antibacterial agents

Fosmidomycin is a natural antibiotic with potent IspC (DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in most bacteria, including A. baumanii and M. tuberculosis, and apicomplexan parasites, including Plasmodium parasites. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against A. baumannii and M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report on the expansion of the acyloxymethyl and alkoxycarbonyloxymethyl phosphonate ester prodrug series of a fosmidomycin surrogate. Prodrug promoieties were designed based on electronic, lipophilic and siderophoric properties. This investigation led to the discovery of derivatives with two-digit nanomolar and submicromolar IC50-values against P. falciparum and A. baumanii, respectively.

Discovery of Orally Bioavailable and Brain-Penetrable Prodrugs of the Potent nSMase2 Inhibitor DPTIP.

Extracellular vesicles (EVs) can carry pathological cargo and play an active role in disease progression. Neutral sphingomyelinase-2 (nSMase2) is a critical regulator of EV biogenesis, and its inhibition has shown protective effects in multiple disease states. 2,6-Dimethoxy-4-(5-phenyl-4-thiophen-2-yl-1H-imidazol-2-yl)phenol (DPTIP) is one of the most potent (IC50 = 30 nM) inhibitors of nSMase2 discovered to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), limiting its clinical development. To overcome DPTIP's PK limitations, we synthesized a series of prodrugs by masking its phenolic hydroxyl group. When administered orally, the best prodrug (P18) with a 2',6'-diethyl-1,4'-bipiperidinyl promoiety exhibited >fourfold higher plasma (AUC0-t = 1047 pmol·h/mL) and brain exposures (AUC0-t = 247 pmol·h/g) versus DPTIP and a significant enhancement of DPTIP half-life (2 h vs ∼0.5 h). In a mouse model of acute brain injury, DPTIP released from P18 significantly inhibited IL-1β-induced EV release into plasma and attenuated nSMase2 activity. These studies report the discovery of a DPTIP prodrug with potential for clinical translation.

Design, synthesis and biological activity evaluation of a series of bardoxolone methyl prodrugs

Bardoxolone methyl (CDDO-Me) has exhibited positive therapeutic effects in clinical trials for diabetic nephropathy (DN), but serious safety risks in the heart exist because of the potential off-target response resulting from the highly active part of CDDO-Me. Herein, we reported a novel strategy to prepare Cathepsin B (CTSB) cleavable and improved water-soluble prodrugs of CDDO-Me. CTSB linkers connection to the highly active α-cyano-α, β-unsaturated ketone (CUK) part of CDDO-Me with the incorporation of polyethylene glycol (PEG) moieties, provided a series of prodrugs of CDDO-Me without CUK part exposure. Theoretically, these prodrugs shielding CUK part can be stably circulated and finally cleaved by CTSB in lysosomes to release CDDO-Me. In this paper, preliminary curative effects and safety of all prodrugs were determined. Wherein, prodrug 20 displayed relatively better activities than other prodrugs in inhibiting the release of NO from RAW264.7 cells, activating Keap1-Nrf2-ARE signaling pathway and inhibiting NF-κB signaling pathway, which were comparable to CDDO-Me. More importantly, prodrug 20 showed relatively lower human ether-a-go-go-related gene (hERG) inhibitory activity compared with CDDO-Me, which demonstrated prodrug 20 might be safer than CDDO-Me. In conclusion, the novel strategy of shielding CUK part with CTSB linkers provided a new idea for solving the limitations of CDDO-Me in clinical application.

Interrogation of the Structure-Activity Relationship of a Lipophilic Nitroaromatic Prodrug Series Designed for Cancer Gene Therapy Applications.

PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned > 2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.

Synthesis and biological evaluation of prodrugs for nitroreductase based 4-β-amino-4′-Demethylepipodophyllotoxin as potential anticancer agents

A series of prodrugs for nitroreductase (NTR) based 4-β-amino-4′- Demethylepipodophyllotoxin as potential anticancer agents were synthesized, and their antiproliferative activities in vitro showed compounds 2b (IC50 = 0.77, 0.83 and 1.19 μM) and 2d (IC50 = 0.98, 0.91 and 1.58 μM) were greatly selectively toxic to tumor cells A-549, HeLa and HepG2, respectively, and lower damage to normal WI-38 cells in comparison with positive agent Etoposide and Demethylepipodophyllotoxin, and induced cell cycle arrest in the G2/M phase with a concomitant decrease in the population of G1 phase in HeLa cells, which were accompanied by apoptosis. Furthermore, Molecular docking model showed that compounds 2b and 2d appeared to form stable bonds with NTR 1DS7. Taken together, these conjugates have the potential to be developed as antitumor drugs.

An injectable superior depot of Telratolimod inhibits post-surgical tumor recurrence and distant metastases

The clinical success of Toll-like receptor (TLR) agonists is based on their capacity to efficiently mobilize both innate and adaptive immunity. However, rapid distribution of TLR agonists into the systemic circulation may result in systemic cytokine storms. Telratolimod (Tel) is a TLR 7/8 agonist whose structure has a hydrophobic long chain that helps to prolong its release. Despite this, the phase I study of Tel showed cytokine release syndromes in 3/35 patients. Herein, we designed an injectable phase transition gel (PGE) that served as a superior drug depot for fatty acid-modified drugs. PGE further minimized the systemic drug exposure of Tel and the possible cytokine storms. In vivo studies demonstrated that Tel@PGE facilitated the recruitment of effector CD8+ T lymphocytes (T cells) and the polarization of myeloid-derived suppressor cells (MDSCs) and immunosuppressive M2-like macrophages to tumoricidal antigen-presenting cells. The reshaping of the tumor microenvironment (TME) by Tel@PGE elicited systematic immune responses to significantly prevent B16F10 or 4T-1 tumor postoperative recurrence and metastasis. Therefore, this platform of Tel is expected to provide a clinically available option for effective postoperative combined therapy. Statement of significanceA series of prodrugs or conjugates containing hydrophobic blocks were designed to achieve sustained release at the injection site by reducing the water solubility. However, this strategy sometimes failed short of expectations. Thus, we constructed a biocompatible and biodegradable injectable phase transition gel (PGE) with superior release properties that can be injected subcutaneously into the surgery site. In the long-lasting treatment, the melanoma and breast cancer immunotherapeutic effect significantly enhanced and the risk of cancer metastasis and relapse was reduced. Crucially, for some immune agonists, a superior release control can significantly reduce adverse effects which was decisive for the availability of the drugs.

Inhibition of phospholipase A2 by Virasole derivation

Kinetics of phosphatidylcholine (PC) hydrolysis under the action of pancreatic phospholipase A2 IB, (EC 3.1.1.4, PLA2) in the presence of a lipophilic derivative of the antiviral drug Virazole 1-(3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5- (((4-methoxyphenyl)diphenylmethoxy)methyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide (Virazole2ЗГ) was studied. The both steps of phospholipolysis were quantitatively characterized: the binding of the enzyme to the lipid-water interface (Ks) and directly the catalytic act (Km) with the determination of the maximum reaction rate (Vmax). It was found that Virazole2ЗГ at a concentration of 0.5 μmol/ml does not affect the Ks value; on the contrary, the Michaelis constant, Km, increases by a factor of 1.8 along with the constancy of the parameter Vmax. Based on the constancy of the Ks values, it seems to be assumed that there is no inhibition of the disintegration of the enzyme-micelle complex in the presence of the effector under the studied reaction conditions. The kinetic parameters of the reaction (the increase in Km and the constancy of Vmax in the presence of Virazole2ЗГ) testify in favor of a moderate competitive inhibition of pancreatic PLA2, Ki = 65 mM, which indicates the possibility of searching for the biological activity of the anti-pancreatitis action in the series of pro-drugs of nucleoside nature.

Design and synthesis of water-soluble grifolin prodrugs for DNA methyltransferase 1 (DNMT1) down-regulation.

DNA methylation and gene silencing play indispensable roles in the epigenetic landscape and gene expression. DNA methyltransferase 1 (DNMT1), a member of the DNMT family, which catalyzes the addition of methyl groups on DNA has been identified to have a close relationship with tumorigenesis. But DNMT1 inhibitors are rare except for the highly toxic nucleoside derivates. Grifolin is a unique natural product which down-regulates DNMT1 and has low toxicity. However, the poor solubility and stability of grifolin limit its application. Herein, we synthesized PEG5-Grifolin as a water-miscible prodrug of grifolin. The half-life of PEG5-Grifolin at 25 °C was considerably extended, revealing excellent stability. Meanwhile, PEG5-Grifolin suppressed tumor growth of by downregulating DNMT1 and reactivating the expression of several tumor suppressor genes in vivo. PEG5-Grifolin might be a promising demethylation agent for DNMT1 associated diseases and benefit much against various types of DNMT1 associated cancer.

Targeted delivery of lopinavir to HIV reservoirs in the mesenteric lymphatic system by lipophilic ester prodrug approach

The combined antiretroviral therapy (cART) can efficiently suppress HIV replication, but the cessation of cART usually results in viral rebound, mostly due to the presence of viral reservoirs. The mesenteric lymphatic system, including mesenteric lymph nodes (MLNs), is an important viral reservoir into which antiretroviral drugs poorly penetrate. In this work, we proposed a novel lipophilic ester prodrug approach, combined with oral lipid-based formulation, to efficiently deliver lopinavir (LPV) to the mesenteric lymph and MLNs. A series of prodrugs was designed using an in-silico model for prediction of affinity to chylomicrons (CMs), and then synthesized. The potential for mesenteric lymphatic targeting and bioconversion to LPV in physiologically relevant media was assessed in vitro and ex vivo. Subsequently, LPV and selected prodrug candidates were evaluated for their in vivo pharmacokinetics and biodistribution in rats. Oral co-administration of lipids alone could not facilitate the delivery of unmodified LPV to the mesenteric lymphatic system and resulted in undetectable levels of LPV in these tissues. However, a combination of the lipophilic prodrug approach with lipid-based formulation resulted in efficient targeting of LPV to HIV reservoirs in mesenteric lymph and MLNs. The maximum levels of LPV in mesenteric lymph were 1.6- and 16.9-fold higher than protein binding-adjusted IC90 (PA-IC90) of LPV for HIV-1 (140 ng/mL) following oral administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Moreover, the concentrations of LPV in MLNs were 1.1- and 7.2-fold higher than PA-IC90 following administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Furthermore, the bioavailability of LPV was also substantially increased following oral administration of activated ester prodrug compared to unmodified LPV. This approach, especially if can be translated to other antiretroviral drugs, has potential for reducing the size of HIV reservoirs within the mesenteric lymphatic system.

Hydrolytic Stability of New Amino Acids Analogues of Memantine

In the present work, the hydrolytic stability of new memantine analogues modified with amino acids, at different pH corresponding to the human biological liquids and organs, was evaluated. Memantine is an uncompetitive N-methyl-d-aspartate receptor antagonist with low-to moderate-affinity. In addition, it is the first representative of a novel class of Alzheimer’s disease (AD) medications acting on the glutamatergic system by blocking N-methyl-D-aspartate receptors. Generally, prodrugs are compounds aiming to improve stability of active fragment and to facilitate transportation across the cell membranes or lipid barriers. The investigated series of prodrugs include modified memantine with the following amino acids: alanine, β-alanine, glycine, phenylalanine, and valine. Hydrolytic stability was determined at two different pH values 2.0 and 7.4 at 37 °C, similar to those in the human stomach and blood plasma. Specially developed UV-VIS spectrophotometric method for quantification of the concentrations of unchanged compounds was applied in the kinetic studies. Val-MEM is the most stable in neutral medium and at 37 °C compound with t1/2 = 50.2 h. The compound Phe-MEM has also very good hydrolytic stability with t1/2 = 29.6 h. The order of other compounds is: Val-MEM ≫ Phe-MEM ≫ Ala-MEM ≈ Val-MEM > β-Ala-MEM. Ala-MEM and Gly-MEM are the most stable compounds at acid condition with almost identical values for t1/2 = 17.8 h and t1/2 = 16.3 h, respectively. The stability of tested compounds in acid conditions are relatively less than in neutral one. They are ordered as follows: Ala-MEM ≈ Gly-MEM > Val-MEM ≈ Phe-MEM ≈ β-Ala-MEM. All compounds have relatively good hydrolytic stability of more than 10 h at both neutral and acid conditions, which is quite enough in order to pass in the blood circulation and to be used as a potential antimicrobial agent.

Functionalization of amino acids with aryl fluorosulfate for prodrug construction by SuFEx chemistry

Sulfur (VI) Fluoride Exchange (SuFEx) chemistry is proposed as a new generation of click chemistry with potential in drug discovery and biological study. Herein we report a simple and convenient approach to synthesize amino acid derivatives functionalized with aryl fluorosulfonyl group from a simple building block. Promoted by 1,1′-Carbonyldiimidazole (CDI), methyl protected amino acids and other amines were efficiently functionalized with SO2F by the reaction with 4-((fluorosulfonyl)oxy)benzoic acid giving fluorosulfonylated amides (FSAs) as products. We also demonstrated that FSAs are useful building blocks in drug discovery. The conjugation of FSA with pharmaceutical phenols by SuFEx efficiently introduced amino acid moiety into target molecule, providing a series of prodrugs with diverse property.

Synthesis, bioconversion, pharmacokinetic and pharmacodynamic evaluation of N-isopropyl-oxy-carbonyloxymethyl prodrugs of CZh-226, a potent and selective PAK4 inhibitor

We have previously disclosed compound 3 (CZh-226), a potent and selective PAK4 inhibitor, but its development was delayed due to poor oral pharmacokinetics. In an attempt to improve this issue, we synthesised a series of prodrugs by masking its terminal nitrogen of the piperazine moiety. Most synthesised prodrugs of 3 have low or no inhibition of PAK4 activity. The stability of synthetic prodrugs was evaluated in PBS, SGF, SIF, rat plasma and liver S9 fraction. Of these, prodrug 19 was not only stable under both acidic and neutral conditions but also could be quickly converted to parent drug 3 in rat plasma and liver S9 fraction. Such effective conversion into parent drug 3 was observed in rats, providing higher exposure of 3 compared to its direct administration. When given via oral route at daily doses of 25 and 50 mg/kg, the prodrug 19 was effective and well tolerated in mouse model of HCT-116 and B16F10.

Delivering bioactive cyclic peptides that target Hsp90 as prodrugs

The most challenging issue facing peptide drug development is producing a molecule with optimal physical properties while maintaining target binding affinity. Masking peptides with protecting groups that can be removed inside the cell, produces a cell-permeable peptide, which theoretically can maintain its biological activity. Described are series of prodrugs masked using: (a) O-alkyl, (b) N-alkyl, and (c) acetyl groups, and their binding affinity for Hsp90. Alkyl moieties increased compound permeability, Papp, from 3.3 to 5.6, however alkyls could not be removed by liver microsomes or in-vivo and their presence decreased target binding affinity (IC50 of ≥10 µM). Thus, unlike small molecules, peptide masking groups cannot be predictably removed; their removal is related to the 3-D conformation. O-acetyl groups were cleaved but are labile, increasing challenges during synthesis. Utilising acetyl groups coupled with mono-methylated amines may decrease the polarity of a peptide, while maintaining binding affinity.

Discovery of a para-Acetoxy-benzyl Ester Prodrug of a Hydroxamate-Based Glutamate Carboxypeptidase II Inhibitor as Oral Therapy for Neuropathic Pain.

4-Carboxy-α-[3-(hydroxyamino)-3-oxopropyl]-benzenepropanoic acid 1 is a potent hydroxamate-based inhibitor of glutamate carboxypeptidase II. In an attempt to improve its poor oral pharmacokinetics, we synthesized a series of prodrugs by masking its hydrophilic hydroxamate group. Prodrugs were evaluated for oral availability in mice and showed varying degree of plasma exposure to 1. Of these, para-acetoxybenzyl-based, 4-(5-(((4-acetoxybenzyl)oxy)amino)-2-carboxy-5-oxopentyl)benzoic acid, 12, provided 5-fold higher plasma levels of 1 compared to oral administration of 1 itself. Subsequently, para-acetoxybenzyl-based prodrugs with additional ester promoiety(ies) on carboxylate(s) were examined for their ability to deliver 1 to plasma. Isopropyloxycarbonyloxymethyl (POC) ester 30 was the only prodrug that achieved substantial plasma levels of 1. In vitro metabolite identification studies confirmed stability of the ethyl ester of benzoate while the POC group was rapidly hydrolyzed. At oral daily dose-equivalent of 3 mg/kg, 12 exhibited analgesic efficacy comparable to dose of 10 mg/kg of 1 in the rat chronic constrictive injury model of neuropathic pain.

Design, synthesis, and biological evaluation of NAD(P)H: Quinone oxidoreductase (NQO1)-targeted oridonin prodrugs possessing indolequinone moiety for hypoxia-selective activation

The enzyme NQO1 is a potential target for selective cancer therapy due to its overexpression in certain hypoxic tumors. A series of prodrugs possessing a variety of cytotoxic diterpenoids (oridonin and its analogues) as the leaving groups activated by NQO1 were synthesized by functionalization of 3-(hydroxymethyl)indolequinone, which is a good substrate of NQO1. The target compounds (29a-m) exhibited relatively higher antiproliferative activities against NQO1-rich human colon carcinoma cells (HT-29) and human lung carcinoma (A549) cells (IC50 = 0.263–2.904 μM), while NQO1-defficient lung adenosquamous carcinoma cells (H596) were less sensitive to these compounds, among which, compound 29h exhibited the most potent antiproliferative activity against both A549 and HT-29 cells, with IC50 values of 0.386 and 0.263 μM, respectively. Further HPLC and docking studies demonstrated that 29h is a good substrate of NQO1. Moreover, the investigation of anticancer mechanism showed that the representative compound 29h affected cell cycle and induced NQO1 dependent apoptosis through an oxidative stress triggered mitochondria-related pathway in A549 cells. Besides, the antitumor activity of 29h was also verified in a liver cancer xenograft mouse model. Biological evaluation of these compounds concludes that there is a strong correlation between NQO1 enzyme and induction of cancer cell death. Thus, this suggests that some of the target compounds activated by NQO1 are novel prodrug candidates potential for selective anticancer therapy.

The design, synthesis and biological evaluation of pro-EGCG derivatives as novel anti-vitiligo agents

A series of prodrugs of EGCG derivatives were designed, synthesized, and the protective effect on melanocytes against H2O2-induced cell damage were extensively evaluated, demonstrating the potential application value of them in anti-vitiligo treatment.