Key words
artemisinin - endoperoxide - cerebral malaria - sesquiterpene - central nervous system (CNS) - 1,2,4,5-tetroxane - 1,2,4,5-tetraoxane
1
Introduction
Malaria poses a serious threat to human life and is prevalent in tropical and subtropical areas across the globe.[1] Drugs such as quinine,[2] chloroquine (a synthetic version of quinine), artemisinin, and its derivative compounds have been used to treat malaria. Developing highly effective chemical scaffolds with minimal toxicity is necessary because malarial parasites have become resistant to existing drugs. In this context, 1,2,4,5-tetraoxanes have emerged as a crucial framework with notable antimalarial properties.[3] To improve effectiveness and combat resistance to various antimalarial drugs, 1,2,4,5-tetraoxanes have been combined with a variety of alicyclic, aryl, heteroaryl, and spiro groups including steroid-based, aminoquinoline-based, dispiro-based, triazine-based,[4] diaryl-based, and piperidine-based 1,2,4,5-tetraoxanes. The objective of this review is to provide pharmacists and organic/medical chemists with a current comprehension of the science behind 1,2,4,5-tetraoxane compounds.
Malaria, a lethal infectious disease, has been around for thousands of years. In humans, this disease is transmitted by female Anopheles mosquitoes infected with the Plasmodium parasite. Rarely, it can also be passed through congenital transmission and contact with infected blood products (transfusion malaria).[5] According to the World Malaria Report 2023 released by WHO, there were around 608,000 deaths and 249 million new cases of malaria documented globally in 2022.[6] In humans, malaria is caused by five well-recognized species of Plasmodium: Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium knowlesi, and Plasmodium ovale. Of these, P. falciparum is the most prevalent and lethal species because it infects all types of red blood cells (RBCs) found at different stages of development (from immature young to old RBCs), causing severe types of malaria-like cerebral malaria. Pregnant women and children under the age of five are the main victims of malaria fatalities, it causes the majority of global malaria deaths, resulting in a mortality rate of 20% to 50% when left untreated.[6]
[7]
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[9]
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P. vivax which is less fatal and found frequently in Central America, India, and some parts of the Eastern Mediterranean, while P. falciparum species are found in South and East Asia, South America, the Caribbean, the Middle East, and Africa.[8]
[9]
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P. ovale and P. malariae are the two less prevalent and nonlethal species commonly found in Africa and Papua New Guinea.[6]
[7]
[8]
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[13] However, P. knowlesi has been proven to cause a type of malaria similar to monkey malaria in specific regions of Southeast Asia.[14] Quinine (QN)[15]
[16] and chloroquine (CQ)[17]
[18] both quinoline α-acids from the cinchona plant, were first used to treat illnesses caused by P. falciparum infections.[2]
,
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28] However, their use was stopped due to the development of resistance towards them.[29]
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[31] At the beginning of 1969, Youyou Tu was designated by the Chinese government as the lead scientist for a drug development initiative called Project 523. In her role as the head scientist of Project 523, she traveled extensively throughout China, researching historical documents and 380 plant extracts that were being used to treat mice and monkeys. She discovered that Artemisia annua significantly decreased the P. falciparum parasite in these animals. Based on her findings, Artemisia annua successfully inhibited P. falciparum parasite in these animals. In 2015, the Nobel Prize in Physiology or Medicine was awarded for her discovery of artemisinin and dihydroartemisinin.[32]
[33]
[34] Artemisinin (ART) is a compound with a sesquiterpene lactone and a 1,2,4-trioxane ring system where the peroxide group is placed within the ring structure of the molecule. It has been used to treat drug-resistant P. falciparum malaria. The study disclosed how artemisinin and its semi-synthetic analogues showed that their biological activity is due to the presence of the 1,2,4-trioxane ring.[35]
[36]
[37] The rich heme content of malarial parasites and its catalytic effect on breaking down the endoperoxide bridge may explain why antimalarial drugs like 1,2,4-trioxane are more harmful to the parasites compared to natural antimalarial terpenoids such as artemisinin (1) and its derivatives. Derivatives, like artemistene (2), dihydroartemisinin (3), artemether (4), arteether (5), artesunic acid (6), sodium artesunate (7), and artelinic acid (8), are specifically deadly to malaria-causing parasites[38]
[39] (Figure [1]).
Figure 1 Artemisinin and its derivatives
In 2006, the World Health Organization (WHO) advised the utilization of a dual-drug combination in ART-based treatment to prevent the emergence of drug resistance in malaria parasites.[40]
[41]
[42]
[43] However, the first case of antimalarial resistance to monotherapy with ART was documented in Cambodia in 2008 because the treatment reached its maximum effectiveness.[44,45] This resistance is due to single nucleotide polymorphisms that can reduce the efficacy of artemisinin.[46] Artemisinin and its derivatives encounter obstacles such as resistance, as well as issues like low solubility in oil/water and sluggish parasite elimination in malaria patients. Dihydroartemisinin is a derivative of artemisinin which led the foundation for the development of related compounds such as artemether (4), arteether (5), sodium artesunate (7), and artelinic acid (8).[47] These substances are often known as the first-generation derivatives of ART and based upon solubility they can be classified into oil-soluble C(10) β-alkyl ethers (artemether and arteether) and water-soluble C(10) β-(substituted) esters (sodium artesunate and sodium artelinate). These drugs are more soluble in oil/water and more effective against malaria when compared to the ART drug. Yet, the early modifications of ART have downsides in aspects such as oil/water improvement and pharmacokinetics, observed in artemether and arteether, with decreased biological half-lives and potentially harmful effects on the blood system, heart, and central nervous system in an animal model.[48]
[49] Artesunate injection, given once daily for a week, shows a success rate of around 92% in treating malaria patients. However, to prevent resistance from spreading, artesunate is commonly administered in combination with other medications. Sodium artelinate has increased water stability and a longer biological half-life (1.5–3.0 h) than artemisinin, however, it showed decreased efficacy in both in vivo and in vitro experiments and induced nephrotoxicity in healthy rats.[50] Other significant issues related to ART-based antimalarials include higher treatment costs (compared to CQ or QN), inadequate physicochemical/pharmacokinetic properties (like inadequate lipid-/water-partitioning behavior, poor bioavailability, short plasma half-life, etc.), toxicities, and limited availability (scarcity in natural sources).[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58] Due to these obstacles, the treatment of malaria has become more complicated, prompting the search for new drugs to combat resistant strains of the disease. Recently, a variety of 1,2,4-trioxanes, 1,2,4-trioxolanes, and 1,2,4,5-tetraoxanes (Figure [1]) have been developed and tested for their effectiveness in treating malaria.[59] 1,2,4,5-Tetraoxanes, IUPAC and CAS name these as 1,2,4,5-tetroxanes but both names are found in the literature, are entirely synthetic and can be synthesized from inexpensive, easily available materials. Various factors like substrate type, solvent, addition mode, temperature, concentration, and pH play a role in the synthesis of tetraoxanes, which can be accomplished using different techniques.[60] Tetraoxanes show enhanced efficacy against malaria and significantly greater stability.[61] Unsymmetrical dispiro-tetraoxanes, as well as other synthetic tetraoxane compounds, were discovered to have comparable effectiveness to, or greater effectiveness than, artemisinin.[62] Since the 1980s, researchers have developed different basic tetraoxanes and investigated their efficacy in combatting malaria. A series of research papers have assessed the efficacy of more than 250 tetraoxanes for combatting malaria. Many substances have exhibited promising in vivo antimalarial activity.[63]
[64] The objective of this review is to provide pharmacists and organic/medical chemists with a current comprehension of the science behind 1,2,4,5-tetraoxane compounds.
Synthetic Methods for Tetraoxanes
2
Synthetic Methods for Tetraoxanes
Several synthetic approaches have been made to the synthesis of 1,2,4,5-tetraoxanes.[56]
[65] Some commonly used methods are acid-catalyzed hydrogen peroxide cyclocondensation with ketones or aldehydes,[65–68] ozonolysis of alkenes,[69] enol-ethers,[70]
O-ether oxime,[71] and cyclocondensation of bis(trimethylsilyl) peroxide with carbonyl compounds catalyzed by trimethylsilyl trifluoromethanesulfonate (TMSOTf).[72]
[73] Asymmetric tetraoxanes can be synthesized by using different catalysts such as SSA (SiO2-H2SO4),[74] aliphatic and alicyclic gem-hydroperoxides (MeReO3-HBF4 catalyst),[64] steroidal gem-bis-hydroperoxides (H2SO4 catalyst),[75] and gem-bis(trimethylsilylperoxy)alkanes (TMSOTf catalyst).[76] Various other catalysts such as Bi(OTf)3,[77] ClSO3H,[78] I2,[79] Re2O7,[59] PMA,[80] HPA/NaY,[81] ADA-MNPs,[82] and H3+xPMo12–X
+6Mox+5O40
[83] are employed to improve the peroxy acetalization of aldehydes and ketones and promote the specific cyclocondensation of these substances (intermediates) with ketones/aldehydes to generate 1,2,4,5-tetraoxanes. Figure [2] depicts various techniques used in the synthesis of tetraoxanes.[56]
[59]
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[64]
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[78]
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[80]
[81]
[82]
[83] The yields of tetraoxanes obtained from these methods are influenced by a variety of factors, including the structure of the carbonyl compound, temperature, concentration, pH, addition mode, solvent, and the equilibrium between the ketone and precursors of cyclic peroxides.[84] The primary approach involves incorporating hydrogen peroxide into carbonyl compounds, aided by an acid catalyst, and subsequently initiating the cyclization of the hydroperoxide intermediate formed (Scheme [1]).
Figure 2 An overview of synthetic methods for tetraoxanes
It has been found that the yield of the tetraoxane can be affected by the reaction conditions, and it has been observed that hexaoxonanes might also result form as byproducts.[85] It is well known that tetraoxanes are thermodynamically stable while hexaoxonanes are kinetically controlled products, but recent studies revealed that whether tetraoxanes or hexaoxonanes are kinetically preferred depends on the relative rates of several steps involved in their formation. A hypothesized mechanism for the generation of tetraoxane is illustrated in Scheme [1].[85]
Scheme 1 Mechanism for tetraoxane formation in the presence of H2O2
Various reaction intermediates[85] depicted in Scheme [1] have been isolated under varying reaction conditions. It has always been difficult to purify tetraoxanes: hydroperoxide impurities can be eliminated by using potassium iodide or dimethyl sulfide and hexaoxonanes can be removed by recrystallization or washing the reaction mixture with cold methanol. If the conversion fails, hexaoxonanes can be transformed into tetraoxanes by heating the reaction mixture with perchloric acid in acetic acid.[86]
[87] Spectroscopic techniques can validate the formation of hexaoxonanes.[88]
2.1
Safety Concerns in the Synthesis of Cyclic Organic Peroxides
During the synthesis of cyclic organic peroxides, which are dangerous compounds when present in their solid form especially in quantities above 1 gram, explosions caused by peroxides may occur spontaneously and unpredictably when handled roughly or when the solid is subjected to impact, friction, static charge, or temperature changes. Solid peroxide samples should be handled very carefully and dissolved in a suitable solvent to minimize the risk of explosion. The synthesis of these substances may only be carried out by highly qualified and experienced personnel, under the use of appropriate safety precautions, and only in small quantities (ca. 100 mg).[89]
Antimalarial Activities of Tetraoxane Derivatives
3
Antimalarial Activities of Tetraoxane Derivatives
3.1
Cycloalkanone-Based Tetraoxanes
In 2000, Vennerstrom and co-workers investigated a range of alkyl-substituted dispiro-1,2,4,5-tetraoxanes.[90] Of all the compounds investigated, five (11a–e) showed activity with IC50 values between 10 and 30 nM against the CQ-sensitive D6 and CQ-resistant W2 clones of P. falciparum, in contrast, the IC50 values for artemisinin (1) are 55 and 32 nM and for compound WR 14899929 (12) they are 8.4 and 7.3 nM (Figure [3]).[58] Furthermore, certain compounds within this category have displayed moderate in vivo efficacy (achieving a 40–60% cure rate when given orally at 128 mg/kg/day) against mice infected with P. berghei on days 3, 4, and 5 after infection.
Figure 3 Antimalarial activity of spiro-1,2,4,5-tetraoxanes
Vennerstrom and co-workers also studied the steric effect of the methyl group at C-1 and C-10 on dispiro-1,2,4,5-tetraoxanes. The study revealed that tetramethyl-substituted dispiro-1,2,4,5-tetraoxanes 13a,b had significantly reduced antimalaria effectiveness (IC50 = >1000 nM) against D6 and W2 strains when compared to WR 14899929 (12), whereas tetraoxane 13c, without steric hindrance, was found to be active (Figure [3]).[91]
3.2
Steroid-Based Tetraoxanes
In 1996, Šolaja and co-workers synthesized steroidal 1,2,4,5-tetraoxanes and tested their effectiveness against P. falciparum D6 and W2 strains for antimalarial activity.[75] They found that cis-isomer 14a had no antimalarial effects on tested strains while trans-isomer 14b exhibited some antimalarial activity (IC50 = 155 nM) against the D6 strain (Figure [4]). The lack of effectiveness against malaria was identified as being due to a significant steric effect caused by obstructed ring structures and poor water solubility. In 2000, Šolaja and co-workers prepared tetraoxanes based on cholic acid with varying diester, acid, and diamide connections and they were tested against P. falciparum strains D6 and W2.[73] The cis compounds were more potent than the trans compounds. Two tetraoxanes, particularly 15a,b, with an amide linker, showed strong effectiveness against the D6 strain with IC50 values between 9.29 and 20.08 nM and a significant SI value (Figure [4]). The higher level of activity could be attributed to the existence of hydrophilic linker or a stereochemical structure distinct from 14a,b.
Figure 4 Antimalarial activity (in vitro and in vivo) of steroidal 1,2,4,5-tetraoxanes
Again in 2016, Šolaja and co-workers reported the in vivo antimalarial activity of steroidal tetraoxane 16 that inhibited liver-stage P. berghei infection (IC50 = 0.33 μM) (Figure [4]).[92] Additionally, at a quantity of 100 mg/kg, it demonstrated a 91% drop in the parasite liver burden in mice. Moreover, the compound displayed a certain degree of efficacy with an IC50 of 1.16 μM against IV-V P. falciparum gametocytes from the 3D7elo1-pfs16-CBG99 transgenic strain, compared to DHART with an IC50 of 0.44 μM in the identical model.[93] In 2018, Kazakova and co-workers investigated the effectiveness of 1,2,4,5-tetraoxanes based on deoxycholic acids against P. falciparum isolates that are CQ-resistant K1 and CQ-sensitive (T96) (Figure [4]).[63] In contrast to CQ (IC50 = 29 nM), compounds 17a,b showed greater potency with IC50 values between 3.0–7.6 nM against the K1 strain. However, when it came to the T96 strain, 17a,b exhibited moderate activity.
3.3
Adamantane-Based Tetraoxanes
In 2006, the O’Neill group developed unsymmetrical dispiro- and spiro-1,2,4,5-tetraoxanes and evaluated their efficacy against malaria under in vitro and in vivo studies.[94] Out of all the substances examined, compounds 18a,b displayed notable in vivo efficacy (100% inhibition) when administered orally at a dose of 30 mg/kg (Figure [5]).
Figure 5 Antimalarial activity of novel adamantane-based 1,2,4,5-tetraoxanes
Compounds 18a,b were tested in the 4-day Peter’s test to determine their oral in vivo effectiveness against P. berghei (ANKA) in mice, comparing their ED50 and ED90 values to artemether (4) having ED50 = 5.88 and ED90 = 10.57 mg/kg. Compound RKA216 (18b) demonstrated exceptional efficacy when given orally with an ED50 of 3.18 mg/kg and an ED90 of 3.88 mg/kg.
In 2008, the O’Neill group investigated achiral dispiro-1,2,4,5-tetraoxanes and evaluated their efficacy in combatting malaria.[95] Compound 19a,b showed strong efficacy with IC50 values of 5.55 and 3.52 nM, respectively, against the P. falciparum 3D7 strain (Figure [5]). When compared to RKA216 (18b) with an ED50 of 3.18 mg/kg, compounds 19a,b showed considerable in vivo efficacy with ED50 values of 6.61 and 7.93 mg/kg. It was also found that compounds 19a,b showed no toxicity.
In 2010, the O’Neill group identified a powerful antimalarial medication named RKA182 (20) that was effective against the 3D7 and K1 strains of P. falciparum, with IC50 values of 0.87 and 1.1 nM, respectively.[96] RKA182 (20) demonstrated noticeable in vivo efficacy in studies, with ED50 and ED90 doses of 1.33 and 4.18 mg/kg, outperforming the therapeutic medications artemether (4) and artesunate (7). Additionally, RKA182 (20) showed higher water solubility, reduced toxicity, and improved absorption, distribution, metabolism, and excretion (ADME) characteristics.[97] The O’Neill group developed a novel series of second-generation RKA182 (20) analogues by eliminating the amide bond to enhance metabolic stability.[98] They developed various polar dispiro-1,2,4,5-tetraoxanes and evaluated their in vitro efficacy against P. falciparum strains 3D7 and K1. Each compound exhibited strong antimalarial properties at very low nanomolar concentrations, outperforming both CQ and artesunate. Compounds 21a,b displayed superior effectiveness against the CQ-resistant K1 strain, which is 25 times more potent than artesunate and 5 times stronger than RKA182 (20) (Figure [5]). Compound 21c (HCl salt) and 21d (ditosylate salt) showed superior effectiveness compared to artesunate in treating P. berghei ANKA infected mice with ED90 values of 11 and 10 mg/kg, respectively. These results show that this group of molecules has a high level of activity and better metabolic stability than RKA182 (20). Further investigation by the O’Neill group revealed that two mannoxane structure compounds 22a,b containing a Mannich base displayed strong in vivo antimalarial properties (>99.7% decrease in parasitemia after 4 days in P. berghei infected mice) and in vitro tests (IC50s = 4.8–5.7 nM against NF54 and K1 strains, respectively) (Figure [5]).[99] Furthermore, mice that were given 22a,b lived an average of 27 and 25 days, with 22a curing 66% of them successfully. Compound 22a exhibits significantly higher efficacy than aterolane (OZ277) (achieving a 60% cure rate in 25 days) and RKA182 (20) (requiring 22 days for cure). Unlike OZ277 and RKA182, compounds 22a,b exhibit a level of in vitro inhibition of hematin dimerization that is comparable to CQ and amodiaquine. This research also demonstrated the significance of the Mannich base pharmacophore and the adamantyl ring in the effectiveness against malaria.
3.4
Dispiro-Based Tetraoxanes
In 2010, Vennerstrom and co-workers synthesized unsaturated dispiro-1,2,4,5-tetraoxane (+)-dihydrocarvone and reported its antimalarial efficacy.[100] It was found that enhancement in the polarity of the compound leads to a decrease in the efficacy of antimalarial drugs. Compound 23a showed the most effectiveness with an IC50 of 2.1 nM against the K1 and NF54 strains (Figure [6]).
Figure 6 Antimalarial activity of novel unsaturated 1,2,4,5-tetraoxanes
Compound 23b shows average in vitro antimalarial effectiveness with IC50 of 6.9 and 6.6 nM against K1 and NF54 strains, respectively. In addition, it shows better in vivo effectiveness (99.9% cure) in mice infected with P. berghei when given orally, outperforming ART treatment (98% cure in the same study).[101]
3.5
Diaryl-Based Tetraoxanes
In 2009, Rawat and co-workers developed and assessed the in vitro efficacy of substituted 3,6-diaryl-1,2,4,5-tetraoxanes molecules against malaria.[102]
Figure 7 Antimalarial activity of 3,6-diaryl-1,2,4,5-tetraoxanes
The results indicate that symmetrical tetraoxanes 24a–c, with ethyl, propyl, and isopropyl groups on the phenyl ring, have notable antimalarial activity against the D6 and W2 strains of P. falciparum. The values of IC50 range from 0.61 to 0.99 μM and 0.76 to 1.03 μM, respectively (Figure [7]). A few compounds 25a–f in the asymmetric tetraoxanes displayed strong effectiveness against the D6 and W2 strains, showing IC50 values ranging from 0.35 to 0.79 M. Compounds 25a–f are not as potent against the W2 strain as ART, but they have a similar effectiveness to CQ (0.41 μM). In 2011, Rawat and co-workers investigated the in vitro antimalarial efficacy of tetraoxane-imine/amine/amide conjugates against P. falciparum strains D6 and W2. When compared to therapeutic drugs like CQ and ART, four compounds 26a–d, imine and amine derivatives, showed considerable effectiveness, with IC50 values ranging from 0.38 to 2.64 μM (D6 strain) and 0.57–1.62 μM (W2 strain) (Figure [7]).[103]
3.6
Di-adamantane-Based Tetraoxanes
In 2013, the O’Neill group synthesized novel tetraoxane dimer compounds and evaluated their efficacy in treating malaria.[104] Among them compounds 27a–c exhibited the highest in vitro antimalarial efficacy against the 3D7 strain with IC50 values of 4.0, 3.5, and 4.7 nM, respectively (Figure [8]).
Figure 8 Antimalarial activity of dimer tetraoxanes
Moreover, compounds 27a and 27d showed the best efficacy against the K1 strain with IC50 values of 2.6 and 2.7 nM, respectively. Additionally, some compounds displayed moderate oral antimalarial effectiveness against P. berghei ANKA mice but were not as active as artesunate.
3.7
Benzylamino- and Aryloxy-Based Tetraoxanes
In 2016, the O’Neill group investigated an effective method for synthesizing aryloxy 1,2,4,5-tetraoxanes and evaluated their efficacy against malaria.[105] Each compound exhibited excellent antimalarial potency (less than 10 nM) against the P. falciparum 3D7 strain. Three compounds 28a–c showed strong effectiveness against the strain being tested, with IC50 values between 0.5–3.7 nM, similar to the IC50 of 2.2 nM of artesunate (Figure [9]).
Figure 9 Antimalarial activity of benzylamino- and aryloxy-based 1,2,4,5-tetraoxanes
When given at a dosage of 30 mg/kg, the lead compound E209 (28d) exhibited the highest level of in vivo effectiveness (prevention rate of 99.65%) in mice from P. berghei infection. Furthermore, they described the most effective approach for synthesizing the lead compound E209.[105] After a thorough assessment, it was concluded that E209 (28d) fulfills all requirements specified in the Medicines for Malaria Venture (MMV) target candidate profile 1 (TCP1), having the pharmacokinetic attributes needed for a single treatment, alone or with other drugs, and a fast efficacy (PRR equal to or better than DHART).[102] E209 (28d) exhibited strong in vivo inhibitory effects in the nanomolar range against various strains of P. falciparum and P. vivax. Additionally, its effectiveness against P. falciparum was validated in trials (rodent models). It demonstrates favorable PK-PD characteristics and reduced levels of parasites similar to DHART.[106]
In 2018, the O’Neill group synthesized some new arylcarboxamide and benzylamino-based dispiro-1,2,4,5-tetraoxanes.[107] Compounds 29a–c exhibited the strongest in vitro effectiveness against P. falciparum strain 3D7 showing IC50 values ranging from 0.84–1.8 nM (Figure [9]). It was also found that N205 (29b) showed greater efficacy against P. falciparum compared to artesunate (ED90 = 10 mg/kg) following four doses administered daily. According to the effectiveness of N205 (29b),[107] a single oral dose of N205 is equally potent as several doses of artesunate.
3.8
Aminoquinoline-Based Tetraoxanes
Several research groups have tried to synthesize hybrids of 1,2,4,5-tetraoxanes with other known pharmacophores to provide potential leads against malaria. In 2008, Šolaja and co-workers studied the development and antimalarial effects of hybrid compounds that merged tetraoxane with 7-chloro-4-aminoquinoline, which they called tetraoxquines.[108] According to activity data compounds, 30a,b exhibited in vitro activity against P. falciparum stains (D6 and W2) with IC50 values between 2.0–2.33 nM and 5.76–9.05 nM, respectively (Figure [10]).
Figure 10 Antimalarial activity of aminoquinoline-based 1,2,4,5-tetraoxane hybrids
Additionally, compounds 30a,b, with a minimal curative dose (MCD) of 80 mg/kg, a minimum active dose (MAD) of 20 mg/kg/day, and a maximum tolerated dose (MTD) of >960 mg/kg, successfully treated mice in a modified Thompson test for antimalarial blood stage activity.
In 2014, Lopes and co-workers investigated a few hybrids based on 1,2,4,5-tetraoxane and 8-aminoquinoline, assessing their effectiveness against malaria (Figure [10]).[109] Intraperitoneal injection of the most effective molecule 31, connected to the other two pharmacophores by an amide linker, effectively treated animals with blood-stage P. berghei infection.
Furthermore, Lopes and co-workers also conducted the development of another series of hybrids based on 1,2,4,5-tetraoxane-8-aminoquinoline, linking aryl/heteroaryl groups to the metabolically labile C-5 position of the 8-aminoquinoline.[110] The majority of the compounds exhibited high effectiveness against P. falciparum W2 and P. berghei in laboratory tests, with EC50 values ranging from low nanomolar to micromolar concentrations. Compounds 32a–d demonstrated the strongest potency and had comparable effects to ART on CaCo-2 cells, without causing toxicity (CC50 = >50 μM) (Figure [10]). Hybrid molecules with C-5 aryl modification on the 8-aminoquinoline were discovered to exhibit improved metabolic stability in microsomes compared to primaquine analogues (C-5 unsubstituted 8-aminoquinolines) while retaining their dual-stage antimalarial efficacy.
Mahmud and co-workers, in 2020, investigated the molecular docking and quantitative structure-activity relationship (QSAR) of various hybrids that linked 1,2,4,5-tetraoxane with 8-aminoquinoline.[111] Lopez and co-workers have already investigated the efficacy of these drugs against the W2 strain of P. falciparum within red blood cells.[110] It was discovered through molecular docking analysis that 1,2,4,5-tetraoxane-8-aminoquinoline hybrids have a stronger binding affinity with P. falciparum lactate dehydrogenase (pfLDH) than chloroquine (CQ). This study indicates that these substances may serve as more effective inhibitors of pfLDH than CQ.
3.9
2-Cyanopyrimidine-Based Tetraoxanes
In 2014, Moreira, O’Neill, and co-workers synthesized hybrids of 2-cyanopyrimidine-based 1,2,4,5-tetraoxanes and examined their efficacy against atovaquone-resistant (FCR3), 3D7, and W2 strains of P. falciparum for their antimalarial effects. Moreover, their ability to block falcipain-2 (FP-2) was also evaluated.[112]
Figure 11 Antimalarial activity of 2-cyanopyrimidine-based 1,2,4,5-tetraoxane hybrids
Out of the tested compounds, three compounds 33a–c exhibited the most favorable balance of activity and safety in the evaluation against HEK-293 cells. Compounds 33a and 33c effectively blocked the W2 and 3D7 strains with IC50 values between 9.8 and 13.1 nM (Figure [11]). The hybrid compound 33b was selected for its in vivo potent FP-2 inhibitory activity in mice infected P. berghei. In comparison to the control, the in vivo findings indicated a reduction in parasitemia levels and enhancements in the survival rate of mice.
Mannich Base Based Tetraoxanes
4
Mannich Base Based Tetraoxanes
In 2016, Rudrapal and co-workers investigated a novel category of unique Mannich bases made from tetraoxane-phenol conjugate and evaluated their in vivo efficacy against P. falciparum strains, which included CQ-sensitive (RKL-2) and CQ-resistant (RKL-9) strains (Figure [12]).[113] Compared to other compounds, two compounds 34a,b with indole and phenol rings, showed higher efficacy against RKL-9 (with IC50 of 8.19 and 5.30 μg/mL, respectively). However, they were not as effective as the standard drug CQ (IC50 = 0.04 μg/mL) when tested against the same strain.
Figure 12 Antimalarial activity of Mannich bases of tetraoxane-phenol hybrid and 3,6-disubstituted 1,2,4,5-tetraoxane derivatives
In 2019, Kumawat and co-workers synthesized modified forms of 3,6-disubstituted 1,2,4,5-tetraoxane derivatives and evaluated their efficacy in combating malaria[114] (Figure [12]). All the prepared compounds were examined for activity against P. falciparum strains 3D7, RKL-2, and RKL-9. Compounds with changes at the 3,6-positions of the 1,2,4,5-tetraoxane structure, like trimethyl 35a (IC50 = 1.953 μg/mL against 3D7), methyl triphenyl 35b (IC50 = 3.906 μg/mL against RKL-9), and dimethyl/diphenyl 35c (IC50 = 3.906 μg/mL against RKL-2), showed enhanced effectiveness.
The structure-activity relationship analysis showed that the antimalarial activity of the tetraoxane moiety was promoted by trimethyl substitution at positions 3 and 6 on the tetraoxane core. It could be because the peroxide bond has the proper steric barrier. The authors postulated that the enhanced effectiveness against malaria was observed with phenyl ring additions at positions 3 and 6 of tetraoxane which receive electrons from tetraoxane.
4.1
N-Sulfonylpiperidine-Based Tetraoxanes
In 2022, Awasthi and co-workers synthesized a range of symmetrical and non-symmetrical N-sulfonylpiperidine dispiro-1,2,4,5-tetraoxanes and assessed their effectiveness against malaria (in vitro and in vivo).[115] They initially tested the in vitro antiplasmodial effectiveness of both symmetrical and non-symmetrical N-sulfonylpiperidine dispiro-1,2,4,5-tetraoxanes using the HRP-2 assay on the erythrocytic phases of P. falciparum strain 3D7, found that the non-symmetrical tetraoxane showed higher antiplasmodial activity compared to the symmetrical tetraoxane (Figure [13]).
Figure 13 Antimalarial activity of N-sulfonylpiperidine dispiro-1,2,4,5-tetraoxanes
Three compounds 36a–c containing a cycloheptyl ring displayed superior efficacy compared to other analogues, particularly compound 36b (IC50 = 4.7 ± 0.3 nM against 3D7) which demonstrated nearly equivalent antimalarial activity to artemisinin (IC50 = 4.74 ± 0.78 nM against 3D7). Compounds 37, 38, and 39 showed strong antimalarial effects with IC50 values of 10.54 ± 0.81, 8.43 ± 0.71, and 10.9 ± 1.4 nM, respectively, against the 3D7 strain. Compounds 37 and 38 showed the best in vivo antimalarial effectiveness with only 0.25% parasitemia after 72 hours. Also, the SAR study of the synthesized tetraoxane revealed that ring size influenced the antimalarial activity.
4.2
N-Benzoylpiperidine-Based Tetraoxanes
Again in 2024, Awasthi and co-workers synthesized a certain number of mixed N-benzoylpiperidine-based 1,2,4,5-tetraoxane analogues and reported their antimalarial property against chloroquine-sensitive P. falciparum strain 3D7.[116] Some of them showed very good antimalarial activity in the nanomolar range comparable to the artemisinin (IC50 = 5.97 ± 0.61 nM). Compounds 40 and 41 both have the same structural units (N-benzoyl-substituted tetraoxane) except compound 40 with the formyl group present in the para-position showed higher antimalarial activity (IC50 = 6.35 ± 0.55 nM) than 41 (IC50 = 7.28 ± 0.57 nM); compounds 42 and 43 were found to be less active with than 40 and 41.
Figure 14 Antimalarial activity of N-benzoylpiperidine-based dispiro-1,2,4,5-tetraoxanes
In all above reported compounds as shown in Figure [14], it is observed that substituents and their nature have a deciding role in antimalarial activity; formyl-, fluoro-, and chloro-substituted tetraoxanes possessed greater activity. They also performed molecular docking studies by taking falcipain-2 enzyme (receptor macromolecule) on synthesized tetraoxanes. Molecular docking studies revealed that tetraoxanes 40 and 43 bind the active site of the falcipain-2 enzyme through H-hydrogen bonding and hydrophobic interactions which are the foremost interpretation for their activity.
Mechanism of Action of Dispiro-1,2,4,5-tetraoxanes
5
Mechanism of Action of Dispiro-1,2,4,5-tetraoxanes
The specific mechanism of action for these tetraoxanes remains to be identified. The O’Neill group have demonstrated the possible agents responsible for the antimalarial effects of RKA182 (20) in 1,2,4,5-tetraoxane activity,[96] by conducting mechanistic examinations of dispiro-1,2,4,5-tetraoxane 20 in THF with iron(II) bromide and a radical trapping agent TEMPO. The authors hypothesized that regioisomeric alkoxy radicals are formed by the cleavage of the O1–O2 bond. These regioisomeric radicals undergo β-scission, resulting in the formation of primary and secondary carbon-centered radicals. TEMPO captured these radicals to produce the respective aminoxy adducts 44 and 45 (Scheme [2]). The O’Neil group[117] conducted modeling research with heme and RKA182 (20), and found that heme favored binding with 1,2,4,5-tetraoxane through a less hindered oxygen atom. They also conducted docking experiments and discovered that the closest distance was between heme Fe(II) and the oxygen of RKA182, with the lowest energy conformations measuring between 2.4 and 2.8 Å (Scheme [3]). Docking simulations showed that the heme Fe(II) tends to interact more with the less hindered oxygen atom of 1,2,4,5-tetraoxane.
Scheme 2 Proposed mechanisms for the iron-mediated degradation of RKA182 and its TEMPO adducts
Scheme 3 Heme alkylation by RKA182
6
Conclusion
Tetraoxane derivatives/hybrids have been identified as a new category of antimalarial endoperoxides, serving as an important treatment option for malaria with potent antimalarial properties over the last three decades. There is scope for further exploration in the synthesis of new 1,2,4,5-tetraoxane derivatives that have the potential to exhibit druglike effects against different types of Plasmodium species. 1,2,4,5-Tetraoxane compounds linked with a steroid, triazine, amine, aminoquinoline, dispiro, piperidine, or diaryl derivatives were designed and synthesized for their effectiveness against malaria. Some of these compounds exhibit impressive in vitro antimalarial activity. In this current review article, we have outlined the synthesis and effectiveness against malaria of 1,2,4,5-tetraoxane derivatives/hybrids and the impact of structural changes on their antimalarial properties. Eventually, these 1,2,4,5-tetraoxane derivatives coupled with different aryl/heteroaryl/alicyclic/spiro groups will assist medicinal chemists in concentrating on creating powerful new compounds against diverse strains of Plasmodium species.
