A Practical Toolkit for the Detection, Isolation, Quantification, and Characterization of Siderophores and Metallophores in Microorganisms.

Siderophores are well-recognized low-molecular-weight compounds produced by numerous microorganisms to acquire iron from the surrounding environments. These secondary metabolites can form complexes with other metals besides iron, forming soluble metallophores; because of that, they are widely investigated in either the medicinal or environmental field. One of the bottlenecks of siderophore research is related to the identification of new siderophores from microbial sources. Herein we have compiled a comprehensive range of standard and updated methodologies that have been developed over the past few years to provide a comprehensive toolbox in this area to current researchers.

Siderophores and metallophores: Metal complexation weapons to fight environmental pollution.

Siderophores are small molecules of organic nature, released by bacteria to chelate iron from the surrounding environment and subsequently incorporate it into the cytoplasm. In addition to iron, these secondary metabolites can complex with a wide variety of metals, which is why they are commonly studied in the environment. Heavy metals can be very toxic when present in large amounts on the planet, affecting public health and all living organisms. The pollution caused by these toxic metals is increasing, and therefore it is urgent to find practical, sustainable, and economical solutions for remediation. One of the strategies is siderophore-assisted bioremediation, an innovative and advantageous alternative for various environmental applications. This research highlights the various uses of siderophores and metallophores in the environment, underscoring their significance to ecosystems. The study delves into the utilization of siderophores and metallophores in both marine and terrestrial settings (e.g. bioremediation, biocontrol of pathogens, and plant growth promotion), such as bioremediation, biocontrol of pathogens, and plant growth promotion, providing context for the different instances outlined in the existing literature and highlighting their relevance in each field. The study delves into the structures and types of siderophores focusing on their singular characteristics for each application and methodologies used. Focusing on recent developments over the last two decades, the opportunities and challenges associated with siderophores and metallophores applications in the environment were mapped to arm researchers in the fight against environmental pollution.

Preliminary hazard assessment of a new nature-inspired antifouling (NIAF) agent.

A recently synthesized aminated 3,4-dioxygenated xanthone (Xantifoul2) was found to have promising antifouling (AF) effects against the settlement of the macrofouler Mytilus galloprovincialis larvae. Preliminary assessment indicated that Xantifoul2 has reduced ecotoxicological impacts: e.g., being non-toxic to the marine crustacea Artemia salina (<10 % mortality at 50 µM) and showing low bioconcentration factor in marine organisms. In order to meet the EU Biocidal Product Regulation, a preliminary hazard assessment of this new nature-inspired antifouling (NIAF) agent was conducted in this work. Xantifoul2 did not affect the swimming ability of the planktonic crustacean Daphnia magna, the growth of the diatom Phaeodactylum tricornutum, and the cellular respiration of luminescent Gram-negative bacteria Vibrio fischeri, supporting the low toxicity towards several non-target marine species. Regarding human cytotoxicity, Xantifoul2 did not affect the cell viability of retinal human cells (hTERT-RPE-1) and lipidomic studies revealed depletion of lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress only at a high concentration (10 µM). Accelerated degradation studies in water were conducted under simulated sunlight to allow the understanding of putative transformation products (TPs) that could be generated in the aquatic ecosystems. Both Xantifoul2 and photolytic-treated Xantifoul2 in the aqueous matrix were therefore evaluated on several nuclear receptors (NRs). The results of this preliminary hazard assessment of Xantifoul2, combined with the high degradation rates in water, provide strong evidence of the safety of this AF agent under the evaluated conditions, and provide the support for future validation studies before this compound can be introduced in the market.

Antibacterial Potential of Symmetrical Twin-Drug 3,6-Diaminoxanthones.

Global health faces a significant issue with the rise of infectious diseases caused by bacteria, fungi, viruses, and parasites. The increasing number of multi-drug resistant microbial pathogens severely threatens public health worldwide. Antibiotic-resistant pathogenic bacteria, in particular, present a significant challenge. Therefore, there is an urgent need to identify new potential antimicrobial targets and discover new chemical entities that can potentially reverse bacterial resistance. The main goal of this research work was to create and develop a library of 3,6-disubstituted xanthones based on twin drugs and molecular extension approaches to inhibit the activity of efflux pumps. The process involved synthesizing 3,6-diaminoxanthones through the reaction of 9-oxo-9H-xanthene-3,6-diyl bis(trifluoromethanesulfonate) with various primary and secondary amines. The resulting 3,6-disubstituted xanthone derivatives were then tested for their in vitro antimicrobial properties against a range of pathogenic strains and their efficacy in inhibiting the activity of efflux pumps, biofilm formation, and quorum-sensing. Several compounds have exhibited effective antibacterial properties against the Gram-positive bacterial species tested. Xanthone 16, in particular, has demonstrated exceptional efficacy with a remarkable MIC of 11 µM (4 µg/mL) against reference strains Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212, and 25 µM (9 µg/mL) against methicillin-resistant S. aureus 272123. Furthermore, some derivatives have shown potential as antibiofilm agents in a crystal violet assay. The ethidium bromide accumulation assay pinpointed certain compounds inhibiting bacterial efflux pumps. The cytotoxic effect of the most promising compounds was examined in mouse fibroblast cell line NIH/3T3, and two monoamine substituted xanthone derivatives with a hydroxyl substituent did not exhibit any cytotoxicity. Overall, the nature of the substituent was critical in determining the antimicrobial spectra of aminated xanthones.

Evaluation of the Cytotoxic and Antiviral Effects of Small Molecules Selected by In Silico Studies as Inhibitors of SARS-CoV-2 Cell Entry.

The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on host cell surface glycans to facilitate interaction with the angiotensin-converting enzyme 2 (ACE-2) receptor. This interaction between ACE2 and the spike protein is a gateway for the virus to enter host cells and may be targeted by antiviral drugs to inhibit viral infection. Therefore, targeting the interaction between these two proteins is an interesting strategy to prevent SARS-CoV-2 infection. A library of glycan mimetics and derivatives was selected for a virtual screening performed against both ACE2 and spike proteins. Subsequently, in vitro assays were performed on eleven of the most promising in silico compounds to evaluate: (i) their efficacy in inhibiting cell infection by SARS-CoV-2 (using the Vero CCL-81 cell line as a model), (ii) their impact on ACE2 expression (in the Vero CCL-81 and MDA-MB-231 cell lines), and (iii) their cytotoxicity in a human lung cell line (A549). We identified five synthetic compounds with the potential to block SARS-CoV-2 infection, three of them without relevant toxicity in human lung cells. Xanthene 1 stood out as the most promising anti-SARS-CoV-2 agent, inhibiting viral infection and viral replication in Vero CCL-81 cells, without causing cytotoxicity to human lung cells.

3,4-Dioxygenated xanthones as antifouling additives for marine coatings: in silico studies, seawater solubility, degradability, leaching, and antifouling performance.

Marine biofouling pollution is a process that impacts ecosystems and the global economy. On the other hand, traditional antifouling (AF) marine coatings release persistent and toxic biocides that accumulate in sediments and aquatic organisms. To understand the putative impact on marine ecosystems of recently described and patented AF xanthones (xanthones 1 and 2), able to inhibit mussel settlement without acting as biocides, several in silico environmental fate predictions (bioaccumulation, biodegradation, and soil absorption) were calculated in this work. Subsequently, a degradation assay using treated seawater at different temperatures and light exposures was conducted for a period of 2 months to calculate their half-life (DT50). Xanthone 2 was found to be non-persistent (DT50 < 60 days) at 50 µM, contrary to xanthone 1 (DT50 > 60 days). To evaluate the efficacy of both xanthones as AF agents, they were blended into four polymeric-based coating systems: polyurethane- and polydimethylsiloxane (PDMS)-based marine paints, as well as room-temperature-vulcanizing PDMS- and acrylic-based coatings. Despite their low water solubility, xanthones 1 and 2 demonstrated suitable leaching behaviors after 45 days. Overall, the generated xanthone-based coatings were able to decrease the attachment of the Mytilus galloprovincialis larvae after 40 h. This proof-of-concept and environmental impact evaluation will contribute to the search for truly environmental-friendly AF alternatives.

Effect of Indole-Containing Pyrazino[2,1-b]quinazoline-3,6-diones in the Virulence of Resistant Bacteria.

Drug resistance is rising to alarming levels, constituting one of the major threats to global health. The overexpression of efflux pumps and the formation of biofilms constitute two of the most common resistance mechanisms, favoring the virulence of bacteria. Therefore, the research and development of effective antimicrobial agents that can also counteract resistance mechanisms are extremely important. Pyrazino[2,1-b]quinazoline-3,6-diones, from marine and terrestrial organisms and simpler synthetic analogues, were recently disclosed by us as having relevant antimicrobial properties. In this study, using a multi-step approach, it was possible to synthesize new pyrazino[2,1-b]quinazoline-3,6-diones focusing on compounds with fluorine substituents since, to the best of our knowledge, the synthesis of fluorinated fumiquinazoline derivatives had not been attempted before. The new synthesized derivatives were screened for antibacterial activity and, along with previously synthetized pyrazino[2,1-b]quinazoline-3,6-diones, were characterized for their antibiofilm and efflux-pump-inhibiting effects against representative bacterial species and relevant resistant clinical strains. Several compounds showed relevant antibacterial activity against the tested Gram-positive bacterial species with MIC values in the range of 12.5-77 µM. Furthermore, some derivatives showed promising results as antibiofilm agents in a crystal violet assay. The results of the ethidium bromide accumulation assay suggested that some compounds could potentially inhibit bacterial efflux pumps.

Emerging Target-Directed Approaches for the Treatment and Diagnosis of Microbial Infections.

With the rising levels of drug resistance, developing efficient antimicrobial therapies has become a priority. A promising strategy is the conjugation of antibiotics with relevant moieties that can potentiate their activity by target-directing. The conjugation of siderophores with antibiotics allows them to act as Trojan horses by hijacking the microorganisms' highly developed iron transport systems and using them to carry the antibiotic into the cell. Through the analysis of relevant examples of the past decade, this Perspective aims to reveal the potential of siderophore-antibiotic Trojan horses for the treatment of infections and the role of siderophores in diagnostic techniques. Other conjugated molecules will be the subject of discussion, namely those involving vitamin B12, carbohydrates, and amino acids, as well as conjugated compounds targeting protein degradation and ß-lactamase activated prodrugs.

Natural and Synthetic Xanthone Derivatives Counteract Oxidative Stress via Nrf2 Modulation in Inflamed Human Macrophages.

Natural products have attracted attention due to their safety and potential effectiveness as anti-inflammatory drugs. Particularly, xanthones owning a unique 9H-xanthen-9-one scaffold, are endowed with a large diversity of medical applications, including antioxidant and anti-inflammatory activities, because their core accommodates a vast variety of substituents at different positions. Among others, α- and γ-mangostin are the major known xanthones purified from Garcinia mangostana with demonstrated anti-inflammatory and antioxidant effects by in vitro and in vivo modulation of the Nrf2 (nuclear factor erythroid-derived 2-like 2) pathway. However, the main mechanism of action of xanthones and their derivatives is still only partially disclosed, and further investigations are needed to improve their potential clinical outcomes. In this light, a library of xanthone derivatives was synthesized and biologically evaluated in vitro on human macrophages under pro-inflammatory conditions. Furthermore, structure-activity relationship (SAR) studies were performed by means of matched molecular pairs (MMPs). The data obtained revealed that the most promising compounds in terms of biocompatibility and counteraction of cytotoxicity are the ones that enhance the Nrf2 translocation, confirming a tight relationship between the xanthone scaffold and the Nrf2 activation as a sign of intracellular cell response towards oxidative stress and inflammation.

New diarylpentanoids and chalcones as potential antimicrobial adjuvants.

Antimicrobial resistance arises due to several adaptation mechanisms, being the overexpression of efflux pumps (EPs) one of the most worrisome. In bacteria, EPs can also play important roles in virulence, quorum-sensing (QS) and biofilm formation. To identify new potential antimicrobial adjuvants, a library of diarylpentanoids and chalcones was synthesized and tested. These compounds presented encouraging results in potentiating the activity of antimicrobials, being diarylpentanoid 13 the most promising. Compounds 9, 13, 16, 19, 22, and 23 displayed EP inhibitory effect, mainly in Staphylococcus aureus 272123. Compounds 13, 19, 22, and 23 exhibited inhibitory effect on biofilm formation in S. aureus 272,123 while 13 and 22 inhibited QS in the pair Sphingomonas paucimobilis Ezf 10-17 and Chromobacterium violaceum CV026. The overall results, demonstrated that diarylpentanoid 13 and chalcone 22 were active against all the resistance mechanisms tested, suggesting their potential as antimicrobial adjuvants.

Up-to-Date Overview of the Use of Natural Ingredients in Sunscreens.

The photoprotective skincare segment is in high demand to meet consumer concerns on UV-induced skin damage, with a recent trend towards sunscreen alternatives with a natural origin. In this study, the use of natural ingredients, either from terrestrial or marine origin, in a panel of 444 sunscreen commercial formulations (2021) was analyzed. Ingredients from terrestrial organisms represent the large majority found in the analyzed sunscreen formulations (48%), whereas marine ingredients are present only in 13% of the analyzed products. A deeper analysis regarding the most prevalent families of ingredients from terrestrial and marine organisms used as top ingredients is also presented, as well as their mechanisms of action. This study provides an up-to-date overview of the sunscreen market regarding the use of natural ingredients, which is of relevance for scientists involved in the development of new sunscreens to identify opportunities for innovation.

Indole-Containing Pyrazino[2,1-b]quinazoline-3,6-diones Active against Plasmodium and Trypanosomatids.

Malaria, leishmaniasis, and sleeping sickness are potentially fatal diseases that represent a real health risk for more than 3,5 billion people. New antiparasitic compounds are urgent leading to a constant search for novel scaffolds. Herein, pyrazino[2,1-b]quinazoline-3,6-diones containing indole alkaloids were explored for their antiparasitic potential against Plasmodium falciparum, Trypanosoma brucei, and Leishmania infantum. The synthetic libraries furnished promising hit compounds that are species specific (7, 12) or with broad antiparasitic activity (8). Structure-activity relationships were more evident for Plasmodium with anti-isomers (1S,4R) possessing excellent antimalarial activity, while the presence of a substituent on the anthranilic acid moiety had a negative effect on the activity. Hit compounds against malaria did not inhibit ß-hematin, and in silico studies predicted these molecules as possible inhibitors for prolyl-tRNA synthetase both from Plasmodium and Leishmania. These results disclosed a potential new chemotype for further optimization toward novel and affordable antiparasitic drugs.

From Natural Xanthones to Synthetic C-1 Aminated 3,4-Dioxygenated Xanthones as Optimized Antifouling Agents.

Biofouling, which occurs when certain marine species attach and accumulate in artificial submerged structures, represents a serious economic and environmental issue worldwide. The discovery of new non-toxic and eco-friendly antifouling systems to control or prevent biofouling is, therefore, a practical and urgent need. In this work, the antifouling activity of a series of 24 xanthones, with chemical similarities to natural products, was exploited. Nine (1, 2, 4, 6, 8, 16, 19, 21, and 23) of the tested xanthones presented highly significant anti-settlement responses at 50 µM against the settlement of mussel Mytilus galloprovincialis larvae and low toxicity to this macrofouling species. Xanthones 21 and 23 emerged as the most effective larval settlement inhibitors (EC50 = 7.28 and 3.57 µM, respectively). Additionally, xanthone 23 exhibited a therapeutic ratio (LC50/EC50) > 15, as required by the US Navy program attesting its suitability as natural antifouling agents. From the nine tested xanthones, none of the compounds were found to significantly inhibit the growth of the marine biofilm-forming bacterial strains tested. Xanthones 4, 6, 8, 16, 19, 21, and 23 were found to be non-toxic to the marine non-target species Artemia salina (<10% mortality at 50 µM). Insights on the antifouling mode of action of the hit xanthones 21 and 23 suggest that these two compounds affected similar molecular targets and cellular processes in mussel larvae, including that related to mussel adhesion capacity. This work exposes for the first time the relevance of C-1 aminated xanthones with a 3,4-dioxygenated pattern of substitution as new non-toxic products to prevent marine biofouling.

Usage of Synthetic Peptides in Cosmetics for Sensitive Skin.

Sensitive skin is characterized by symptoms of discomfort when exposed to environmental factors. Peptides are used in cosmetics for sensitive skin and stand out as active ingredients for their ability to interact with skin cells by multiple mechanisms, high potency at low dosage and the ability to penetrate the stratum corneum. This study aimed to analyze the composition of 88 facial cosmetics for sensitive skin from multinational brands regarding usage of peptides, reviewing their synthetic pathways and the scientific evidence that supports their efficacy. Peptides were found in 17% of the products analyzed, namely: acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-8, acetyl tetrapeptide-15, palmitoyl tripeptide-5, acetyl hexapeptide-49, palmitoyl tetrapeptide-7 and palmitoyl oligopeptide. Three out of seven peptides have a neurotransmitter-inhibiting mechanism of action, while another three are signal peptides. Only five peptides present evidence supporting their use in sensitive skin, with only one clinical study including volunteers having this condition. Noteworthy, the available data is mostly found in patents and supplier brochures, and not in randomized placebo-controlled studies. Peptides are useful active ingredients in cosmetics for sensitive skin. Knowing their efficacy and synthetic pathways provides meaningful insight for the development of new and more effective ingredients.

Marine Ingredients for Sensitive Skin: Market Overview.

Marine ingredients are a source of new chemical entities with biological action, which is the reason why they have gained relevance in the cosmetic industry. The facial care category is the most relevant in this industry, and within it, the sensitive skin segment occupies a prominent position. This work analyzed the use of marine ingredients in 88 facial cosmetics for sensitive skin from multinational brands, as well as their composition and the scientific evidence that supports their efficacy. Marine ingredients were used in 27% of the cosmetic products for sensitive skin and included the species Laminaria ochroleuca, Ascophyllum nodosum (brown macroalgae), Asparagopsis armata (red macroalgae), and Chlorella vulgaris (microalgae). Carotenoids, polysaccharides, and lipids are the chemical classes highlighted in these preparations. Two ingredients, namely the Ascophyllum nodosum extract and Asparagopsis armata extracts, present clinical evidence supporting their use for sensitive skin. Overall, marine ingredients used in cosmetics for sensitive skin are proposed to reduce skin inflammation and improve the barrier function. Marine-derived preparations constitute promising active ingredients for sensitive skin cosmetic products. Their in-depth study, focusing on the extracted metabolites, randomized placebo-controlled studies including volunteers with sensitive skin, and the use of extraction methods that are more profitable may provide a great opportunity for the cosmetic industry.

Determination of the Absolute Configuration of Bioactive Indole-Containing Pyrazino[2,1-b]quinazoline-3,6-diones and Study of Their In Vitro Metabolic Profile.

In recent decades, fungi-derived naturally occurring quinazolines have emerged as potential drug candidates. Nevertheless, most studies are conducted for bioactivity assays, and little is known about their absorption, distribution, metabolism, and elimination (ADME) properties. To perform metabolic studies, the synthesis of the naturally occurring quinazolinone, fiscalin B (1), and its chloro derivative, 4-((1H-indol-3-yl)methyl)-8,10-dichloro-1-isobutyl-1,2-dihydro-6H-pyrazino[2,1-b]quinazoline-3,6(4H)-dione (2), disclosed as an antibacterial agent, was performed in a gram scale using a microwave-assisted polycondensation reaction with 22% and 17% yields, respectively. The structure of the non-natural (+)-fiscalin B was established, for the first time, by X-ray crystallography as (1R,4S)-1, and the absolute configuration of the naturally occurring fiscalin B (-)-1 was confirmed by comparison of its calculated and experimental electronic circular dichroism (ECD) spectra as (1S,4R)-1. in vitro metabolic studies were monitored for this class of natural products for the first time by ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). The metabolic characteristics of 1 and 2 in human liver microsomes indicated hydration and hydroxylation mass changes introduced to the parent drugs.

Microsequential injection analysis/lab-on-valve system for the automatic evaluation of acetylcholinesterase inhibitors.

A miniaturized microsequential injection/lab-on-valve (µSIA-LOV) system was developed and shown to be a useful alternative to perform inhibitory studies on acetylcholinesterase. These studies are essential for the evaluation of the potential therapeutic effect of drugs commonly used in the treatment of Alzheimer's disease. Donepezil, galantamine, and rivastigmine were tested, in addition to compounds based on the xanthone scaffold. Four of these xanthone derivatives were identified as having EC50 values between 676 and 4466 µmol/l, showing a potential inhibitory effect higher than the clinical agent rivastigmine. The developed automatic system added advantages of reduction of reagents and sample consumption (around 55 µl per analysis), lower cost per analysis, and the generation of less waste (around 1.2 ml per analysis). The µSIA-LOV system is also a robust, rapid, reliable, and simple system to use. Docking studies suggested a possible mode of interaction with the target acetylcholinesterase protein.

Xanthones Active against Multidrug Resistance and Virulence Mechanisms of Bacteria.

The emergence of multidrug and extensively drug-resistant pathogenic bacteria able to resist to the action of a wide range of antibiotics is becoming a growing problem for public health. The search for new compounds with the potential to help in the reversion of bacterial resistance plays an important role in current medicinal chemistry research. Under this scope, bacterial efflux pumps are responsible for the efflux of antimicrobials, and their inhibition could reverse resistance. In this study, the multidrug resistance reversing activity of a series of xanthones was investigated. Firstly, docking studies were performed in the AcrAB-TolC efflux pump and in a homology model of the NorA pump. Then, the effects of twenty xanthone derivatives on bacterial growth were evaluated in Staphylococcus aureus 272123 and in the acrA gene-inactivated mutant Salmonella enterica serovar Typhimurium SL1344 (SE03). Their efflux pump inhibitory properties were assessed using real-time fluorimetry. Assays concerning the activity of these compounds towards the inhibition of biofilm formation and quorum sensing have also been performed. Results showed that a halogenated phenylmethanamine xanthone derivative displayed an interesting profile, as far as efflux pump inhibition and biofilm formation were concerned. To the best of our knowledge, this is the first report of xanthones as potential efflux pump inhibitors.

From Natural Products to New Synthetic Small Molecules: A Journey through the World of Xanthones.

This work reviews the contributions of the corresponding author (M.M.M.P.) and her research group to Medicinal Chemistry concerning the isolation from plant and marine sources of xanthone derivatives as well as their synthesis, biological/pharmacological activities, formulation and analytical applications. Although her group activity has been spread over several chemical families with relevance in Medicinal Chemistry, the main focus of the investigation and research has been in the xanthone family. Xanthone derivatives have a variety of activities with great potential for therapeutic applications due to their versatile framework. The group has contributed with several libraries of xanthones derivatives, with a variety of activities such as antitumor, anticoagulant, antiplatelet, anti-inflammatory, antimalarial, antimicrobial, hepatoprotective, antioxidant, and multidrug resistance reversal effects. Besides therapeutic applications, our group has also developed xanthone derivatives with analytical applications as chiral selectors for liquid chromatography and for maritime application as antifouling agents for marine paints. Chemically, it has been challenging to afford green chemistry methods and achieve enantiomeric purity of chiral derivatives. In this review, the structures of the most significant compounds will be presented.

Xanthenes in Medicinal Chemistry - Synthetic strategies and biological activities.

BACKGROUND: Xanthenes are a special class of oxygen-incorporating tricyclic compounds. Structurally related to xanthones, the presence of different substituents in position 9 strongly influences their physical and chemical properties, as well as their biological applications. This review explores the synthetic methodologies developed to obtain 9H-xanthene, 9-hydroxyxanthene and xanthene-9-carboxylic acid, as well as respective derivatives, from simple starting materials or through modification of related structures. Azaxanthenes, bioisosteres of xanthenes, are also explored. Efficiency, safety, ecological impact and applicability of the described synthetic methodologies are discussed. Synthesis of multi-functionalized derivatives with drug-likeness properties are also reported and their activities explored. Synthetic methodologies for obtaining (aza)xanthenes from simple building blocks are available, and electrochemical and/or metal free procedures recently developed arise as greener and efficient methodologies. Nonetheless, the synthesis of xanthenes through the modification of the carbonyl in position 9 of xanthones represents the most straightforward procedure to easily obtain a variety of (aza)xanthenes. (Aza)xanthene derivatives displayed biological activity as neuroprotector, antitumor, antimicrobial, among others, proving the versatility of this nucleus for different biological applications. However, in some cases their chemical structures suggest a lack of pharmacokinetic properties being associated with safety concerns, which should be overcome if intended for clinical evaluation.