An Essay on Isoxazoline Derivatives

The Pharmacologic Ability of Isoxazoline Derivatives

What are Isoxazolines and the Uses there of

From agriculture to vital components in pharmaceutical syntheses, heterocycles reign king. These unsuspecting, but mighty rings are ubiquitous in many realms of industry. Rings in drugs and other compounds in general are important not only because they offer scaffold rigidity, three dimensionality and electronic distribution, but because they themselves, play key roles in a whole molecule’s reactivity, lipophilicity, polarity, metabolic stability and toxicity.5 More often than not, they are integrated with electronegative elements. Replacing one or more of the carbons in a cyclic system with electronegative heteroatoms (e.g. oxygen, nitrogen, or sulfur) offers powerful bonding opportunities and stability on a molecular level. As a result, heterocycles find their way into the most mundane – caffeine in coffee to the most complex – the nitrogenous bases in DNA, along with nearly every name-brand pharmaceutical.4

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Isoxazolines, a class of five-membered heterocycles are composed of one of both nitrogen and oxygen heteroatoms. They showcase promising insecticidal, anti-microbial, anti-cancer and anti-inflammatory uses1-3, mainly attributed by the strongly electronegative elements they possess, which results in a great deal of reactivity by way of hydrogen bonding. This is essential in the general mechanism for many pharmaceutical agents, where these electronegative elements bind to hydrogens from targeted proteins’ functional groups. These proteins are inhibited by the binding action and are effectively turned off, ultimately switching on or off responses in the body.4 This process is therefore facilitated by isoxazolines, whereby both oxygen and nitrogen heteroatoms may hydrogen bond. Because of this, isoxazolines provide frameworks that are extremely well-suited to biological settings and are vastly conducive to pharmacological applications.2

Prevention of Vector-Borne Diseases

Of the many biological interests of isoxazolines, it is their widely appropriated use in veterinary settings that are most pertinent in controlling pathogenic vectors. Malaria, leishmaniasis and Zika continue to ravage subtropical areas where they are the major cause of death alongside West Nile Virus in temperate locales.3 Isoxazolines offer a powerful solution to the numerous vector-borne illnesses that befall humans, having been used extensively and successfully in reducing pathogen transmission via blood-feeding insect (e.g. mosquitos, fleas and ticks) in companion animals. Afoxolaner is one such isoxazoline that provides low, to non-existent neurotoxicity in GABA receptors and very limited brain penetration level in mammals.3 Once ingested, the compounds enter into the organism’s bloodstream where they bindto the plasma proteins.3-4 After a blood feeding, the isoxazoline binds GABA receptors of the target vector, actively blocking the glutamate-gated chloride channels within the receptor,3 inducing convulsions and death of the insect.

Using canine pharmacokinetics, a median human dosage of 260 mg of Afoxolaner provides an impressive 74 days of drug circulation above IC99, effective for species of Culex and 50 days of equivalent inhibitory concentration for Phlebotomus.3 Compared to ivermectin, which has a half-life of only 18 hours, a single human dose of less than 500 mg of Afoxolaner provides sufficient plasma exposure above the insecticidal threshold for over 2 months, effective for a wide range of vector species.3 Models were constructed with this data and used to visualize drug impact in a population that suffers from malaria and Zika fever. With only a once-yearly 30% coverage scenario, reduction was upwards of 97% in those years of administration, with a 3% increase in rebound infection compared to a non-intervention scenario.3 This overshoot of a treated population is the result of Zika’s immunizing ability, which is described by reduction in herd immunity by any death of those immune and births of the susceptible.3 While malaria is not advantageous in immunizing like Zika, a significant decrease would be observed despite  rebound. Under 17% malaria parasite occurrence within a 6-month transmission season, a 30% coverage saw a reduction of up to 66% for an intervention year and a 28% reduction over a period of 4 years with the first two being intervention years.3 Because of the severity of vector-borne maladies in specific regions of the world, it is crucial to develop simple solutions for the elimination of these pathogenic carriers. This not only benefits those in sub-tropical populations, but the world over.

Elimination of Multiple Drug Resistant Bacteria

β-Lactams are one of the three most prevalent classes of antibiotic agents and have been heavily reviewed for nearly 100 years, beginning with the discovery of penicillin.8 Due to their extensive documentation, their general mechanism is very well understood and consequently, they constitute nearly 65% of the world’s antibiotic market (as of 2004).8 Although powerful agents against a whole host of bacterial species, they are not impenetrable and have a weakest link in their molecular structure. Certain bacteria, once evolved to produce the enzyme β-Lactamase, can become multiple drug resistant (MDR). This enzyme allows the bacteria to chemically alter the β-Lactam ring found in β-Lactam antibiotics, which destroys the drug’s structure and ultimate function in killing the bacteria.8 Because many MDR strains of bacteria such as S. aureus are urgent concerns, new classes of antibiotics are necessary in the fight against them.5

Isoxazoline scaffolds, having previously shown notable activity against M. tuberculosis5 as well displaying activity against resistant microorganisms7 make potential candidates to take on MDR bacteria. The nitrofuran isoxazoline used in the successful elimination of M. tuberculosiswere incorporated with varying pyridyl side chains along with a piperazine spacer between the two.5 Remarkably, yet unknowingly, the positioning of the pyridyl side chain was crucial in the development of a more potent derivative, with the strongest having a minimum inhibitory concentration (MIC) of 4–16 µg/mL against a Staphylococcus panel, which was either improved or comparable to ciprofloxacin.5 In the search for other drugs against resistant microorganisms, isoxazolinyl oxazolidinones were used and bound with various groupings.7 The most powerful compound in the series produced was effective against E. faecalis with a MIC of 0.0866 µM compared to conventional vancomycin at 0.5249 µM; suggesting the meta positioning of the electron-withdrawing nitro group had a great impact.7 Even though drugs like ciprofloxacin and vancomycin show significant activity against strains of MDR bacteria they are not effective for all and can technically be improved in terms of potency. Moreover, each antibiotic is used in different settings with different scenarios, and ciprofloxacin and vancomycin may not be administered for these reasons; requiring new drugs like isoxazolines to come forth as to better treat MDR bacteria.

Treatment of Inflammatory Diseases and Cancer

Among the world’s diseases, cancer is one of the most threatening as a leading cause of mortality with more than 10 million diagnosed per year.2 When developing new drug-based treatments, natural products are reached for first; where over half of the anticancer drugs approved worldwide are either naturally derived compounds or their analogs.2 Coumarins are such naturally-occurring molecules that exhibit cytotoxicity along with immuno-stimulating and antimicrobial effects.1-2 Unprecedentedly, these compounds are amplified in their biological effect when combined with other heterocycles.1 Isoxazolines with their own anticancer and immuno-potentiating properties were thereby coupled with coumarins in hopes of a synergetic effect in a single molecule.1-2

Various novel analogs of isoxazoline coumarins were synthesized and tested for their immunostimulant and cytotoxic effects.1-2 immuno-potentiating derivatives 2, 4 and 8 showcased little to no cell toxicity and such exceptional in vitro lymphocyte proliferation that they were paneled for in vivo studies.1 At dosages of 0.1, 0.01 and 0.001 mg/kg body weight in mice, compounds 2, 4 and 8 enhanced antibody synthesis, cytokine release and splenocyte proliferation significantly higher than the standard, Levamisole did.1 These derivatives in particular owe their effects to their aromatic ring at the 3rd position of the isoxazoline and the ortho/para orientation of their methoxy groupings. In particular, the positioning of these electron-donating groups (EDG) increase the total electron density of the adjacent isoxazoline ring, leading to better interaction with receptor binders.1 The same ideas were applied in vitro against melanoma cancer cell line UACC903 and normal fibroblast cell line FF2441 for analysis of their cytotoxicity.2 This resulted in the emerging of 7c, which acted only on the viability of melanoma cells at IC50 levels of 10.5 µM without inhibiting the normal fibroblast cells.2 By administering the drug to tumor-bearing mice (100 mg/kg body weight), they saw an 85% suppression in tumor volume, reduction of ascites volume, cell number and increased in the life span when compared to the control.2 This was seen as a direct result of the presence of 3,4-dimethoxy groups on the phenyl ring adjacent to the isoxazoline ring;2 promoting electron density by EDGs.1 Because the world around us is inspired by nature, as is mankind, mimicking the many bioactive compounds it offers serve as ideal frameworks for future novel pharmaceuticals.

What’s Next

Of these synthetic isoxazoline pharmaceuticals, none of them were ever tested in vivo in human trials. Although multiple in vivo studies involving mice were conducted, they can only predict what would actually happen within a human.

References

1.   Ismail, T. et al. Synthesis and immunopotentiating activity of novel isoxazoline functionalized coumarins. European Journal of Medicinal Chemistry, 2016, 123, pp. 90-104.

2.   Lingaraju, G., Balaji, K., Jayarama, S., Anil, S., Kiran, K., Sadashiva, M. Synthesis of new coumarin tethered isoxazolines as potential anticancer agents. Bioorganic & Medicinal Chemistry Letters, 2018, 28(23-24), pp. 3606-12.

3.   Miglianico, M. et al. Repurposing Isoxazoline Veterinary Drugs for Control of Vector-Borne Human Diseases. Proceedings of the National Academy of Sciences, 2018, 115(29).

4.   O'Brien, P. The changing face of Heterocyclic Chemistry in the pharmaceutical industry. https://www.openaccessgovernment.org/changing-face-heterocyclic-chemistry-pharmaceutical-industry/41175/ (accessed Nov 22, 2019).

5.   Picconi, P. et al. Novel pyridyl nitrofuranyl isoxazolines show antibacterial activity against multiple drug resistant Staphylococcus species. Bioorganic & Medicinal Chemistry, 2017, 25(15), pp. 3971-79.

6.   Taylor, R. D., Maccoss, M., Lawson, A. D. G. Rings in Drugs. Journal of Medicinal Chemistry, 2014, 57(14), pp. 5845–59.

7.   Varshney, V., Mishra, N. N., Shukla, P. K., Sahu, D. P. Synthesis and Antibacterial Evaluation of Isoxazolinyl Oxazolidinones:               Search for Potent Antibacterial. Bioorganic & Medicinal Chemistry Letters, 2009, 19(13), pp. 3573–76.

8. Worthington, R. J., Melander, C. Overcoming Resistance to β-Lactam Antibiotics. The Journal of Organic Chemistry, 2013, 78, pp. 4207–13.