Mutual Prodrug Research Articles

Dual inhibition strategy addressing hyperuricemia and oxidative stress: design, biological evaluation and stability studies of febuxostat-probenecid mutual prodrug

Dual inhibition strategy addressing hyperuricemia and oxidative stress: design, biological evaluation and stability studies of febuxostat-probenecid mutual prodrug

Hybrid/Chimera Drugs - Part 1 - Drug Hybrids Affecting Diseases of the Central Nervous System.

This review, focused on hybrid drugs, is the third in a series of reviews, where the first two reviews dealt with a) dimeric drugs, b) mutual prodrugs - codrugs. The compounds designated as hybrids are comprised of two (and sometimes three) biologically active entities, linked by metabolically stable bridges. In some cases, one of the two components of the hybrids serves as a carrier for the second component, and most frequently, the components elicit their individual biological properties, which are commonly synergistic or complementary. Due to the very large number of publications dealing with hybrid drugs, the present review is restricted to hybrids acting in the central nervous system. Future reviews will cover fields such as antimicrobial, anticancer, and antiviral hybrids, and cardiovascular active hybrids. The selected articles reviewed herein were published between the years 2000-2022 with partial coverage of the year 2023.

Synthesis, characterization, and molecular docking studies of novel hippuric acid anhydrides as potential antiurolithic, analgesic and free radical scavenging agents

Hippuric acid is a biotransformation product in humans which is excreted through urine. Dietary intake of phenolic compounds increases its concentration in urine. Hippuric acid is experimentally proved to be a regulator of calcium oxalate super saturation in human urine and has a solvent effect on oxalate salts. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used analgesics employed in associated renal colic of urolithiasis. The objective of the present study was to chemically link hippuric acid through anhydride linkage to various NSAIDs to synthesize potential mutual prodrugs for urolithiasis and associated renal colic. Hippuric acid was linked to eleven NSAIDs through anhydride linkage by first synthesizing hippuryl chloride using oxalyl chloride as chlorinating agent, followed by its reaction with sodium salts of NSAIDs. Structures of obtained compounds were elucidated using UV spectrophotometry, FTIR spectrometry, 1H NMR and C-13 NMR spectrometry. Synthesized compounds were evaluated for in vitro antioxidant, in vivo acute toxicity, in vivo antiurolithic, in vivo analgesic and in vitro hydrolysis studies. Molecular docking analysis on TNF-α and COX-2 was carried out to determine target protein binding. Synthesized compounds showed stability at acidic pH which indicate potential gastro protective effect of synthesized conjugates. Compounds P9 an P6 showed maximum in vivo antiurolithic activity while P9 and P8 showed maximum in vitro antioxidant activity. All the conjugates except P2 showed significant analgesic activity which show that conjugation of hippuric acid to NSAIDs did not result in loss of analgesic potential. Docking studies showed better affinity with TNF-α as compared to COX-2.

Exploring acetaminophen prodrugs and hybrids: a review.

This critical review highlights the advances in developing new molecules for treating pain syndrome, an important issue for human health. Acetaminophen (APAP, known as paracetamol) and nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in clinical practice despite their adverse effects. Research is being conducted to develop innovative drugs with improved pharmaceutical properties to mitigate these effects. A more practical way to achieve that is to study well-known and time-tested drugs in their molecular combinations. Accordingly, the present work explores APAP and their combined chemical entities, i.e., prodrugs (soft drugs), codrugs (mutual prodrugs), and hybrids. Due to their molecular structure, APAP prodrugs or codrugs could be considered merged or conjugated hybrids; all these names are very fluid terms. This article proposed a structural classification of these entities to better analyze their advances. So, the following: carrier-linked O-modified APAP, -linked N-modified APAP derivatives (prodrugs), and direct- and spacer-N,O-linked APAP hybrids (codrugs) are the central parts of this review and are examined, especially ester and amide NSAID-APAP molecules. The C-linked APAP and nitric oxide (NO)-releasing APAP hybrids were also briefly discussed. Prime examples of APAP-based drugs such as propacetamol, benorylate, acetaminosalol, nitroparacetamol, and agent JNJ-10450232 weave well into this classification. The proposed classification is the first and original, giving a better understanding of the SAR studies for new pain relievers research and the design development for the analgesic APAP-(or NSAID)-based compounds.

Chemical Characterization and Pharmacological Evaluation of Phytophenols-Etodolac Mutual Prodrugs

Etodolac is choice of drug for pain and inflammation but has major side effects of gastric ulcers that are due to free carboxylic group. Etodolac belongs to the chemical class of non-selective COX-inhibitor but preferentially COX-2 inhibitor. Here the ester linked mutual prodrugs of etodolac with phytophenols like vanillin, carvacrol, umbelliferone, guaiacol, sesamol and syringaldehyde were synthesized. All the prodrugs were characterized by IR-spectroscopy, 1H-NMR, 13C-NMR and mass spectrometry. Among the synthesized prodrugs, the Eto-van, Eto-umbe, Eto-sesa and Eto-syr showed improved analgesic and anti-inflammatory activity compared to etodolac. All the synthesized prodrugs showed less ulcerogenic side effects compared to etodolac.

Simultaneous estimation of propyphenazone, flurbiprofen, and their mutual prodrug by high‐performance liquid chromatography method

AbstractThe current work comprises of development of a simple, rapid, and precise reverse phase‐high‐performance liquid chromatography method for simultaneous estimation of propyphenazone, flurbiprofen, and their mutual prodrug. Column C18 (Shimadzu Shim‐pack Gist, 250 × 4.6 mm, 5 μm) was employed as a stationary phase, and the ratio of acetonitrile:methanol:water (40:40:20 %v/v) was used as the mobile phase. The flow rate was held at 1 mL/min and detection was carried out at 245 nm using an ultraviolet detector. The retention time of propyphenazone, flurbiprofen, and their mutual prodrug was found to be 4.0, 2.5, and 10.0 min, respectively. The proposed method was validated according to the International Council on Harmonization Q2(R1) guideline in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, and solution stability. Calibration plots were linear over the concentration ranges of 2–10 μg/mL (R2 = 0.9998, R2 = 0.9992 and R2 = 0.9994 for propyphenazone, flurbiprofen, and their mutual prodrug, respectively). Results of mean percentage recoveries were in the range of 99.6%–100.1% for flurbiprofen, 99.3%–100.2% for propyphenazone, and 99.9%–100.7% for prodrug, respectively. The developed method can be used for the assay of mutual prodrug and drug release study of the developed mutual prodrug also routine quality control evaluation of mutual prodrug in bulk form.

Synthesis and evaluation of mutual prodrugs of some NSAIDs

The therapeutic efficacy of NSAIDs can be improved by reducing their gastrointestinal side effects by modifying their carboxyl group. Anti-inflammatory drugs Ibuprofen, Ketoprofen were chosen for the preparation of mutual prodrugs with paracetamol. The compounds were synthesized with the aim of achieving a synergistic effect and reducing the gastric irritation of anti-inflammatory drugs. Compounds were confirmed by characterization. The synthesis involved an esterification reaction between the carboxyl group of selected NSAIDs and the hydroxyl group of paracetamol. Mutual prodrugs were evaluated for hydrolysis study in acidic medium and phosphate buffer. Compounds were found to hydrolyze at a controlled rate in the environment, and thus this study found that a mutual prodrug approach can be effectively used to achieve the goals of enhancing the effectiveness of NSAIDs in two ways. First, by masking the carboxyl group of NSAIDs to reduce GI effects and achieve synergistic effects using paracetamol as a pro moiety. Both IP and KP prodrugs were retaining anti-inflammatory activity intact and exhibited much-reduced gastric irritant activity. Prodrug IP however, showed better activity and negligible ulcerogenic tendency than KP.

A New Avenue for Enhanced Treatment of Hyperuricemia and Oxidative Stress: Design, Synthesis and Biological Evaluation of Some Novel Mutual Prodrugs Involving Febuxostat Conjugated with Different Antioxidants

Febuxostat (FEB) is the first non-purine xanthine oxidase inhibitor (XOI) used for the treatment of hyperuricemia and gout. The oxidative stress induced by reactive oxygen species (ROS) which accompany purine metabolism by XO, could contribute to cellular damage and several pathological conditions. In this view, the present work addresses the evaluation of combining the hypouricemic effect of FEB and the free radical scavenging potential of various natural antioxidants in a single chemical entity by implementing the “mutual prodrug” strategy. Accordingly, a series of five ester prodrugs containing FEB together with different naturally occurring antioxidants namely, thioctic acid (4), thymol (5), menthol (6), vanillin (7), and guaiacol (8) was synthesized. Prominently, all the chemically conjugated prodrugs (4 – 8) revealed an obvious increase in the hypouricemic and antioxidant potentials when compared with their corresponding promoieties and physical mixtures. Moreover, they showed a potential protective effect against CCl4-induced hepatotoxicity and oxidative stress, together with no cytotoxicity on normal breast cells (MCF10A). Furthermore, the in vitro chemical and enzymatic stability studies of the prodrugs (4 – 8) using a developed HPLC method, verified their stability in different pHs, and rapid hydrolysis in rabbit plasma and liver homogenate to their parent metabolites. Moreover, the prodrugs (4 – 8) showed higher lipophilicity and lower aqueous solubility when compared to the parent drugs. Finally, the obtained merits from the implementation of the mutual prodrug strategy would encourage further application in the development of promising candidates with high therapeutic efficacy and improved safety profiles.

Development of 5-fluorouracil-dichloroacetate mutual prodrugs as anticancer agents

5-Fluorouracil (5-FU) is one of the most widely applied chemotherapeutic agents with a broad spectrum of activity. However, despite this versatile activity, its use poses many limitations. Herein, novel derivatives of 5-FU and dichloroacetic acid have been designed and synthesized as a new type of codrugs, also known as mutual prodrugs, to overcome the drawbacks of 5-FU and enhance its therapeutic efficiency. The stability of the obtained compounds has been tested at various pH values using different analytical techniques, namely HPLC and potentiometry. The antiproliferative activity of the new 5-FU derivatives was assessed in vitro on SK-MEL-28 and WM793 human melanoma cell lines in 2D culture as well as on A549 human lung carcinoma, MDA-MB-231 breast adenocarcinoma, LL24 normal lung tissue, and HMF normal breast tissue as a multicellular 3D spheroid model cultured in standard (static) conditions and with the use of microfluidic systems, which to a great extent resembles the in vivo environment. In all cases, new mutual prodrugs showed a higher cytotoxic activity toward cancer models and lower to normal cell models than the parent 5-FU itself.

Synthesis and in vitro kinetic study of new mutual prodrug for colon cancer associated with constipation

Objective: a new mutual prodrug was synthesized for colon targeting in the treatment of colon cancer associated with constipation. Methods: a new mutual prodrug was synthesized through several steps included amide hydrolysis in a strong acidic medium, amide synthesis, diazotization and coupling reactions. The stability of this prodrug in HCl buffer, in phosphate buffer and in rat fecal matter was monitored. The chemical structure of mutual prodrug was characterized by physical and spectroscopic techniques as FTIR, UV-Visible and 13C NMR spectra. In colon, the mutual prodrug was proposed to split by the action of bacterial azoreductase into two N-substituted benzamides, metoclopramide and declopramide, that constituted two apoptotic agents, also the local application of metoclopramide on colonic smooth muscles was proposed to enhance their contractions affords relief of constipation. In vitro kinetic studies in a hydrochloric acid buffer showed an insignificant release of metoclopramide and declopramide while in a phosphate buffer, only (9.12٪) release was observed over six hours. In order to confirm the hydrolysis of mutual prodrug in colon, the release study in a rat fecal matter was monitored over six hours and showed that the hydrolysis was almost complete (90.88٪) with a half-life of (166.19 min) followed first order kinetics. The prodrug approach that is based on enzymes specification may offer a new method to improve drug efficacy and reduce side effects.

Towards the Development of Dual Hypouricemic and Anti-inflammatory Candidates: Design, Synthesis, Stability Studies and Biological Evaluation of Some Mutual Ester Prodrugs of Febuxostat-NSAIDs

Treatment of gout involves two basic approaches: reducing the serum uric acid mainly by xanthine oxidase inhibitors (XOIs) and alleviating the intensity of the accompanying acute arthritic inflammation using non-steroidal anti-inflammatory drugs (NSAIDs). Febuxostat (FEB) is the first non-purine XOI approved for the treatment of hyperuricemia and gout. The present study aims at combining the hypouricemic effect of FEB and the anti‐inflammatory (AI) properties of NSAIDs in a single entity by adopting the “mutual prodrug” approach. Accordingly, a series of seven ester prodrugs comprising basically FEB together with different NSAIDs namely, diclofenac (4), ibuprofen (5), ketoprofen (6), indomethacin (7), naproxen (8), ketorolac (9) and etodolac (10) was synthesized. All the investigated seven prodrugs (4–10) were equipotent or even superior to their corresponding parent drugs in the hypouricemic and AI activities, together with a gastrointestinal (GI) safety profile. Among this series, the prodrug FEB-DIC (4) showed excellent dual in vivo hypouricemic and anti-inflammatory activity (43.60 % and 15.96 %, respectively) when compared to the parent drugs FEB and diclofenac (36.82 % and 12.10 %, respectively) and its physical mixture (37.28 % and 12.41 %, respectively). Investigation of the in vitro chemical stability and hydrolysis of the prodrug (4) in aqueous and biological samples using a developed HPLC method confirmed its stability in various pHs, whereas rapid hydrolysis to the parent drugs in liver homogenate and human plasma was proven. Finally, it is concluded that the mutual prodrug approach could be successfully used in drug design and development for overcoming undesirable difficulties without losing the desired activities of the parent drugs.

Discovery of SI 1/20 and SI 1/22 as Mutual Prodrugs of 5-Fluorouracil and Imidazole-Based Heme Oxygenase 1 Inhibitor with Improved Cytotoxicity in DU145 Prostate Cancer Cells.

In this work, we extend the concept of 5-fluorouracil/heme oxygenase 1 (5-FU/HO-1) inhibitor hybrid as an effective strategy for enhancing 5-FU-based anticancer therapies. For this purpose, we designed and synthesized new mutual prodrugs, named SI 1/20 and SI 1/22, in which the two active parent drugs (i. e., 5-FU and an imidazole-based HO-1 inhibitor) were connected through an easily cleavable succinic linker. Experimental hydrolysis rate, and in silico ADMET predictions were indicative of good drug-likeness and pharmacokinetic properties. Novel hybrids significantly reduced the viability of prostate DU145 cancer cells compared to the parent compounds 5-FU and HO-1 inhibitor administered alone or in combination. Interestingly, both compounds showed statistically significant lower toxicity, than 5-FU at the same dose, against non-tumorigenic human benign prostatic hyperplasia (BPH-1) cell line. Moreover, the newly synthesized mutual prodrugs inhibited the HO-1 activity both in a cell-free model and in vitro, as well as downregulated the HO-1 expression and increased the reactive oxygen species (ROS) levels.

A Short Review on Biological Activities of Paracetamol Derivatives

Paracetamol reduces body temperature with multiple mechanisms. Paracetamol is chemically 4-hydroxy acetanilide and has a good safety profile. Following its successful use as an over-the-counter antipyretic and analgesic medication, several attempts were made to increase the potency, mask the bitter taste, and decrease the toxicity of this drug by modifications at the phenyl ring, acetamido group, and hydroxyl group. The free hydroxyl group of paracetamols was masked to obtain prodrugs (carbonate prodrugs, ester prodrugs like alanine-prodrug, proline-prodrug, galactosylated prodrug, and mutual prodrugs with other drugs and NSAIDs). Propacetamol is a commercially available prodrug derived from paracetamol that is effective in parenteral form. Paracetamol ester prodrugs with sulfur-containing amino acids such as N-acetyl cysteine, cysteine, and methionine showed low hepatotoxicity compared to the parent drug. In addition, paracetamol derivatives including metal complexes, chalcones, Mannich bases, nucleoside analogs, hybrids with the aryl-imidazolidinyl ring, thymol, and triazole ring displayed diverse activities like antioxidant, anticancer, and antimicrobial activities.

Amide Codrug Approach: Synthesis and Biopharmaceutical Evaluation of Nonsteroidal Anti-Inflammatory Drug and Calcium Channel Blocker Conjugates

Background and Aim Geriatric patients often suffer from osteoarthritis and hypertension comorbidity. The codrugs approach was shown to be an effective strategy for targeting diseases synergistically hence improving the quality of life of patients. The present study aimed to synthesize various Nonsteroidal Anti-inflammatory Drugs NSAIDs and Calcium channel blocker CCB Co-drugs and biopharmaceutical study to eliminate the adverse gastrointestinal effects of the NSAIDs to treat comorbid conditions in geriatric patients with significant reduction of polypharmacy.Methods Various conjugates were synthesized by a one-pot amidation reaction of Amlodipine with various NSAIDs. Further characterization by melting point TLC Fourier transform infrared nuclear magnetic resonance and mass spectroscopy followed by solubility partition coefficient and hydrolysis study in SGF and SIF.Results The synthesized codrugs satisfied the structural criteria of the proposed plan. From the biopharmaceutical and hydrolysis study it was observed that the co-drugs underwent significant hydrolysis in SIF pH 7.4 and have showed delayed onset of action with respect to the standard drugs. The delayed onset may be due to the hydrolysis of amide linkage followed by the release of the prodrug which finally releases the active drug.Conclusion The findings illuminated the codrugs pros and cons aiding optimization and development. This research advances amide-based mutual prodrugs and their use in pharmaceutical research. The outcome of this exploration confirmed that the described co-drug can offer desirable safety and therapeutic benefits. Hence these conjugates could be considered for further development.

Fine-tuning the sequential drug release of nano-formulated mutual prodrugs dictates the combination effects.

The maintenance of robust ratiometric loading of dual therapeutic agents and fine-tuning release kinetics for consistent in vitro and in vivo optimization of combination effects is vital for discovering new anticancer drug combinations and remains challenging. Smart nanomedicine strategies have been investigated for this purpose, but most of the reported strategies focus either on ratiometric delivery or on unimodal sequential release of the two different agents, which hampers effective optimization of combination effects. Herein we report a sequential drug release system based on nanoformulated mutual prodrugs constructed by the formation of ketal linkages with different acid sensitivities, thus enabling the acid-triggered release of two anticancer drugs, paclitaxel and gemcitabine, in various sequences. We found that in several cell lines, the sequence of drug release substantially affected the combination effects; specifically, in A549 cells, time-staggered release profiles showed enhanced synergistic effects relative to those of a simultaneous release profile. Moreover, in vivo assessment of the antitumor efficacy of the nanoformulations in A549 xenograft models indicated that the best therapeutic effects were obtained with time-staggered release profiles, which was consistent with the in vitro results. Our strategy for precisely controlled sequential drug release can be expected to facilitate the screening of optimal drug combinations and maximize combination effects both in vitro and in vivo.

Synthesis and preliminary biological evaluation of gabactyzine, a benactyzine-GABA mutual prodrug, as an organophosphate antidote

Organophosphates (OPs) are inhibitors of acetylcholinesterase and have deleterious effects on the central nervous system. Clinical manifestations of OP poisoning include convulsions, which represent an underlying toxic neuro-pathological process, leading to permanent neuronal damage. This neurotoxicity is mediated through the cholinergic, GABAergic and glutamatergic (NMDA) systems. Pharmacological interventions in OP poisoning are designed to mitigate these specific neuro-pathological pathways, using anticholinergic drugs and GABAergic agents. Benactyzine is a combined anticholinergic, anti-NMDA compound. Based on previous development of novel GABA derivatives (such as prodrugs based on perphenazine for the treatment of schizophrenia and nortriptyline against neuropathic pain), we describe the synthesis and preliminary testing of a mutual prodrug ester of benactyzine and GABA. It is assumed that once the ester crosses the blood–brain-barrier it will undergo hydrolysis, releasing benactyzine and GABA, which are expected to act synergistically. The combined release of both compounds in the brain offers several advantages over the current OP poisoning treatment protocol: improved efficacy and safety profile (where the inhibitory properties of GABA are expected to counteract the anticholinergic cognitive adverse effects of benactyzine) and enhanced chemical stability compared to benactyzine alone. We present here preliminary results of animal studies, showing promising results with early gabactyzine administration.

Probing the effect of microenvironment on the enzyme-like behavior of catalytic peptide assemblies

As bridging species between short peptides and macromolecular proteins, peptide assemblies not only provide a supramolecular approach for the fabrication of controllable molecular machines with enzyme-like functions, but also a simplified model for understanding the catalytic mechanism of natural enzymes. In this study, we focused on probing the effect of microenvironment on the catalytic behavior of peptide assemblies. Upon simply replacing the X residue in Fmoc-FFXAH-CONH2, we realized the modulation of the microenvironment of the amyloid assemblies, which thus appeared esterase-like function with different catalytic abilities. The chemistry, structure and activity were analyzed to explore the principles that how the hydrophobic, charged, polar and chiral microenvironment deciding the catalytic behavior of the esterase mimic. In addition, we also presented the potential of the catalytic assemblies in the encapsulation, delivery and enzymatic metabolization of a mutual prodrug. This work sheds new insights for understanding the structure–function relationship of catalytic peptide assemblies and natural enzymes, and also provides a new avenue for the designing of artificial enzymes with better functions.

Mitigation of doxorubicin-induced cardiotoxicity with an H2O2-Activated, H2S-Donating hybrid prodrug

Doxorubicin (DOX) is one of the most effective anticancer agents in clinical oncology. Its continued use, however, is severely limited by its dose-dependent cardiotoxicity which stems, in part, from its overproduction of reactive oxygen species (ROS) and often manifests itself as full-blown cardiomyopathy in patients, years after the cessation of treatment. Therefore, identifying DOX analogs, or prodrugs, with a diminished cardiotoxic profile is highly desirable. Herein, we describe a novel, H2O2-responsive DOX hybrid codrug (mutual prodrug) that has been rationally designed to concurrently liberate hydrogen sulfide (H2S), a purported cardioprotectant with anticancer activity, in an effort to maintain the antitumor effects of DOX while simultaneously reducing its cardiotoxic side effects. Experiments with cardiomyoblast cells in culture demonstrated a rapid accumulation of prodrug into the cells, but diminished apoptotic effects compared with DOX, dependent upon its release of H2S. Cells treated with the prodrug exhibited significantly higher Nrf2 activation relative to DOX-treated cells. Preliminary indications, using a mouse triple-negative breast cancer cell line sensitive to DOX treatment, are that the prodrug maintains considerable toxicity against the tumor-inducing cell line, suggesting significant promise for this prodrug as a cardioprotective chemotherapeutic to replace DOX.

Synthesis of 5‐Fluorouracil–Naproxen Conjugates as a Mutual Prodrug for Targeting Cancer Tissues

AbstractA new 5‐fluorouracil–naproxen conjugate is synthesized as a mutual prodrug for targeting cancer tissues. The structure of the target compound and their intermediate are characterized by their melting point, IR, 1H NMR, 13C NMR, and elemental microanalysis. The cytotoxic activity is preliminarily evaluated using nonsmall lung cancer CRL‐2049, human breast cancer CAL‐51, and one type of normal cell line; rat embryo fibroblast cell line. The synthesized compound shows a good cytotoxic effect at the cancer cell and no significant effect at rat embryo fibroblast cell line.

Design, synthesis and biological evaluation of hybrid of ubenimex-fluorouracil for hepatocellular carcinoma therapy

In our previous study, we discovered a ubenimex-fluorouracil (5FU) conjugates BC-02, which displays significant in vivo anti-tumor activity, however, the instability of BC-02 in plasma limits its further development as a drug candidate. Herein, we designed and synthesized four novel ubenimex-5FU conjugates by optimizing the linkers between ubenimex and 5FU based on BC-02. Representative compound 20 is more stable than BC-02 in human plasma and displays about 100 times higher CD13 inhibitory activity than the positive control ubenimex. Meanwhile, the antiproliferative activity of 20 was comparable with 5FU in vitro. The preliminary mechanism study indicated that compound 20 exhibited significant anti-invasion and anti-angiogenesis activities in vitro. Furthermore, compound 20 obviously inhibits tumor growth and metastasis in vivo and prolong the survival time of tumor-bearing mice. Our study may have an important implication reference for the design of more druglike mutual prodrug, and compound 20 can be used as a lead compound for further design and development.