Proposals for New Antimalaria Drugs - Part 2
(continued from part 1)
Candidate Compounds with N-O Bonds
Moyna pointed out that N-O and O-O bonds are of a comparable strength :
We note that while the standard bond dissociation energy (BDE) of the peroxo linkage (-O-O-) is 33.2 kcal/mol , the corresponding BDE of the -N-O- linkage is 35.8 kcal/mol . Although the BDEs for these bonds are likely to change when forming part of larger molecules , both moieties will remain highly labile and prone to undergo homolytic cleavage . Therefore , oxazines could follow a reaction pattern (i.e. , -N-O- bond opening and radical generation) , and thus biological activity , related to that of endoperoxides (p. 1851) .
There are many classes of compounds with reactive N-O bonds that could be investigated as antimalaria drug candidates :
Emmons studied the oxaziridines [oxaziranes] in the late 1950s , preparing them by the peroxidation of imines . In a preliminary communication he commented ,
They are active oxygen compounds comparable in many respects to organic peroxides.......
In a second more comprehensive paper , he described their oxidizing nature in greater detail :
The treatment of oxaziranes with ferrous salts causes a one-electron transfer reaction and results in either reductive dealkylation or else isomerizations via free radical chain reactions (p. 5739) .
That sounds very much like the chemistry of the organic peroxides interacting with the iron(II) atom in heme , as mentioned above .
Minisci and Galli reported a new type of free radical addition of amino and chloro to olefinic bonds , involving the reduction of the N-O bond of hydroxylamine-O-sulfonic acid by iron(II) :
H3N+OSO3- + Fe2+ ----------> H3N+ + SO42- + Fe3+
The highly reactive amino free radicals then added across the unsaturated bond . When FeCl2 was used as the Fe(II) source , chlorine free radicals were also be generated , and added with the amino to the olefin .
These two examples suggest that the N-O compounds shown in the sketch above will react , more or less , with heme's iron(II) atoms in Plasmodia's cellular fluid .
Hydroxamic acids (N-hydroxyamides) have been primarily used as chelating agents for Transition metal cations , including those of iron . Hydroxamates even function as essential growth factors for several varieties of microbes , being used as "cellular transport vehicles" [siderophores] for the iron required by the microorganisms for their enzymes . They are being studied as ligands for platinum group metal complexes , proposed as anticancer drugs . Hydroxamates are physiologically active in many life processes in humans , insects , and microorganisms (Fazary et al.) .The simple hydroxamates like acetohydroxamide , benzohydroxamide , and hydroxyurea are weakly effective at inhibiting the growth of Plasmodia (Holland et al.) , with typical IC50 values in the micromolar range , as were a series of aromatic-based hydroxamic acids synthesized by the Holland group . Fosmidomycin , first developed as an antibiotic , was found to be a reasonably effective antimalaria drug (Lell et al.) . Its anti-Plasmodia properties are undoubtedly due to its hydroxamic acid group :
A reassuring aspect of the hydroxamates for the researcher is that they are stable , safe compounds to prepare and manipulate . They can be readily synthesized from carboxylic esters :
The carbitol tail might be installed on a small hydroxamic acid molecule by methylolation with ethyl carbitol and formaldehyde (in this case , derived from the anhydrous soluble trimer s-trioxane) :
The methylolation should occur preferentially at the hydroxamate's oxygen , rather than nitrogen atom . While both atoms would have an enhanced nucleophilicity due to the alpha effect , the nitrogen lone pair of electrons will be partially diverted by resonance with the carbonyl group , as is typical of amides .
The following scheme outlines the possible syntheses of three more interesting hydroxamic acid drug candidates :
N-Alkoxyamines , the N-O equivalent of the hydroperoxides , are a somewhat obscure class of organic compounds . Methoxyamine is probably the best known example of them . They have been prepared by the nucleophilic attack on the sodium salt of hydroxylamine-O-sulfonic acid (HOSA) by alkoxide anions , with displacement of sodium sulfate . The following scheme outlines a possible route to new alkoxyamines , starting from the tertiary alcohols recommended for use in the preparation of the hydroperoxides described earlier :
Insolubility of the sodium-HOSA salt in the reaction solvent might be a problem . Bargigia suspended the pure , dry salt in an excess of the anhydrous alcohol , then added sodium metal to the mix , which became warm and began to boil . The alkali metal alkoxides of tertiary alcohols are well-known in organic synthesis (eg. potassium tert-butoxide , a strong base) , so that part of the scheme should be feasible . The next part would be to get the sodium-HOSA salt to react with the alkoxide . Phenoxyamine [O-phenylhydroxylamine] has been prepared in a simple procedure (but in a low yield of 15%) .While phenoxyamine free base is somewhat unstable , its hydrochloride salt seems to be unaffected by storage at room temperature for several months .
Dissolving metal systems of various sorts are well known to organic chemists in functional group reductions . One such metal-acid combination , iron filings and hydrochloric acid , has been used historically to reduce the nitro group to primary amino . While reviewing one such experimental procedure I noted what at first glance appeared to be an anomaly . In 1939 Johnson and Degering made a detailed study of the reduction of aliphatic nitroalkanes by iron and zinc metal powders with hydrochloric and acetic acids , respectively . The Fe/HCl combination yielded the corresponding amines , while the Zn/HOAc couple provided oximes .
I always thought that the nitro reduction occurred on the surface of the dissolving metal , where presumably the highly reactive nascent hydrogen was being generated . But maybe this wasn't the case in the Johnson-Degering procedure :
To 35g of 40-mesh iron filings are added 75 ml of water and 10 ml of concd. hydrochloric acid . After the evolution of hydrogen has stopped , one-sixth of a mole of nitroparaffin is added and , with rapid stirring , the mass is heated at 100 ºC for thirteen to fifteen hours (p. 3194 ; my emphasis in red) .
2-Nitro-2-methylpropane and other nitroalkanes were reduced to the corresponding aliphatic amines in 8294% yields . The yields were 9397% in the presence of ferric chloride.
I wondered if the real reducing agent in their experiments was iron(II) :
RNO2 + 6 Fe2+ + 7 H+ ------------> RNH3+ + 6 Fe3+ + 2 H2O
It may well be ; the ancient Béchamp reduction (1854) is the industrial reduction of aromatic nitro compounds to the corresponding amines by iron , ferrous salts , or iron catalysts in aqueous acid, according to the Merck Index .
The perceptive reader will see where I'm headed here . The cellular fluid inside Plasmodia's food vacuole is known to be mildly acidic [pH = 5.2 , as noted by Vennerstrom(2)] , so perhaps the nitroalkane group will oxidize heme's Fe(II) to Fe(III) in that environment . That might prevent the heme from crystallizing into hemozoin . A properly designed nitroalkane might therefore be worthwhile investigating as an antimalaria drug candidate compound :
The methylolation of simple nitroalkanes with formaldehyde is well known (for example , in the preparation of 2-nitroethanol) , as is their methylolation with formaldehyde and sec-amines (noted immediately above in the sketch) . It would be interesting to try to extend the scope of this nitroalkane reaction using an alcohol such as ethyl carbitol . If successful , it would provide a straightforward and economical route to the drug candidate molecules . The lower nitroalkanes are all relatively inexpensive industrial chemicals .
A word of caution about nitroalkanes , however : while they are generally safe and relatively innocuous compounds at room temperature , they can become dangerously explosive when hot . Distillation of nitroalkanes should be carried out with the greatest of care , excluding all air from the hot vapours , including the pot residue . See Note 4 in the Organic Syntheses procedure in the 2-nitroethanol reference , which discusses the danger of flash explosion in the nitroethanol distillation and the use of an inert "chaser" (in that case , diphenyl ether) to dilute the nitro compound .
The cyclic 1,2-oxazines are the N-O equivalent of the corresponding peroxides , and as such should be reactive with heme's iron(II) . Moyna and his students prepared a series of oxazines , several of which were weakly effective (micromolar range) against Plasmodia :
The key step in their synthesis route was the hetero Diels-Alder reaction of a cyclic diene intermediate with the N=O group of the nitrosobenzene dienophile . This same sort of Diels-Alder reaction is featured in the following scheme , in which vanillin , the main ingredient in vanilla flavouring , is incorporated into a six-atom heterocyclic 1,2-oxazine . I'm assuming the nitrogen atom of the oxazine product will be bonded to the benzylic carbon , as in Moyna's antimalaria drug compound sketched above :
In the following scheme , cinnamaldehyde the main ingredient in cinnamon is combined with a Wittig reagent to provide an enol ether intermediate , which is then cyclized with nitrosobenzene in another hetero Diels-Alder reaction :
The required Wittig salt (precursor to the actual Wittig reagent) might be prepared by the following methylolation reaction :
Ethyl carbitolOH + H2C=O + HP+Ph3 Cl- -----------> Ethyl CarbitolOCH2P+Ph3 Cl- + H2O
Hydroxymethyltriphenylphosphonium chloride , Ph3P+CH2OH Cl- , is a known compound and might be an intermediate in the subsequent condensation with ethyl carbitol :
The nitroxides are stable (under ambient conditions) N-O neutral free radicals . The best known nitroxide is undoubtedly TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) , which is often used as a "spin trap" in ESR (electron spin resonance) studies . The nitroxides could be thought of as an R2NOONR2 type of peroxide that , because of its very bulky R groups , has split in two and is unable to reform . The oxygen free radical is very reactive , and should be able to bond to heme's Fe(II) as the more conventional peroxides , such as artemisinin , are thought to . In the following scheme the nitro candidate compound shown in the sketch above is converted into the corresponding nitroxy molecule (with two carbitol tails) by treatment with sodium metal in DME :
The stoichiometry and mechanism of the condensation are apparently unresolved , and indeed the nitroxy product would be one of several in this somewhat complex reaction .
As mentioned above , Emmons found that oxaziridines (oxaziranes) were able to oxidize iron(II) to iron(III) with the production of dealkylated products or free radical isomerizations . He also observed that his oxaziridine products were all readily hydrolyzed by aqueous acid at room temperature , regenerating the original carbonyl compound (from the imine substrate) . However ,
The oxazirane ring itself does not appear to be very reactive toward basic reagents (p. 5744) .
The oxaziridines are thus somewhat like the a-aminoethers discussed above in part 1 . Like them , their acid lability might be reduced , at least in physiological conditions , by acylation of the nitrogen ring atom . If the N-acyloxaziridines were found to be effective antimalarial drugs , they could be protected against the strongly acidic gastric juices by an enteric coating on the drug dosages (pill , tablet , or capsule) , which would permit them to pass safely into the mildly basic intestinal tract for solution and absorption into the blood stream . N-Acylation should help to preserve the three atom ring intact until the drug molecules are "swallowed" into Plasmodia's food vacuole . Impact of the ring oxygen atom on heme's iron(II) will unzip the oxaziridine with its attendant free radical and/or alkylation reactions .
N-Acyloxaziridines have been prepared by several routes . A practical scheme might involve an adaptation of the oxaziridine synthesis described by Schulz , Becker , and Rieche . They added a primary amine to an olefin undergoing ozonolysis ; a b-aminohydroperoxide , as an unstable intermediate , was produced . Dehydration of this intermediate closes the three atom ring to yield the oxaziridine . In the two schemes below , I've proposed a modification of this reaction in which hydrogen peroxide and a primary amide add to a ketone carbonyl group , with a subsequent dehydration of the hydroperoxide-amide intermediate to the N-acyloxaziridine :
I've suggested an interesting variation of the Mannich reaction , in which a ketone (in this case , acetone) and an alcohol (ethyl carbitol) are methylolated with formaldehyde (from the soluble trimer , s-trioxane) . This reaction would be analogous to the Organic Syntheses preparation of 1-diethylamino-3-butanone , in which diethylamine takes the place of the carbitol .
N-Acyloxaziridines have also been synthesized by Schmitz , Ohme , and Schramm (addition of aqueous alkaline hydroxylamine-O-sulfonic acid [HOSA] to a ketone to produce an oxaziridine , then its N-acylation) , and by Zong , Shin , and Ryu (tandem a,b-addition of the N and O atoms of hydroxamic acids to methyl propiolate) . N-Acyloxaziridines seem to be chemically quite reactive . In the patent literature they have been described as bleaching agents !
Oximes are another class of familiar , "safe" N-O compounds . They are well-known as crystalline identification derivatives of aldehydes and ketones . Oximes in general can be prepared by a simple reaction of the carbonyl compound with hydroxylamine (free base , from the hydrochloride) :
Amine oxides might oxidize heme's iron(II) in manner similar to the nitroalkanes discussed above :
R3N+O- + 2 Fe2+ + 3 H+ ------------> R3N+H + 2 Fe3+ + H2O
The following scheme takes advantage of the fact that N,N-dialkylanilines can be methylolated at the para position by formaldehyde :
Amine oxides are prepared by the peroxidation of a tertiary amine's nitrogen atom ; hydrogen peroxide is often used for this purpose . Intermediate amine oxide peroxides and hydrates are formed , which must be decomposed and dehydrated to obtain the pure amine oxide .
Many fatty amine oxides are commercially available as amphoteric surfactants (cf. the antiprotozoal drug miltefosine , above) . For example , dimethyl lauryl amine oxide is offered as the product "Ammonyx LO" by the Stepan Chemical Company , Northfield (IL) . A preparation of the corresponding pure compound , N,N-dimethyldodecylamine oxide , has been described in Organic Syntheses . It's also commercially available from the Aldrich Chemical Company (93% pure) . Cocoamidopropylamine oxide , "Macat Ultra CDO" , is manufactured by the Mason Chemical Company , Arlington Heights (IL) . These and other such fatty amine oxide surfactants might be very interesting to examine as potential antimalarial and antiprotozoal compounds .
The pyridine-N-oxides are similarly prepared by the peroxidation (for example , with peracetic acid) of pyridine precursors . In the following scheme , the methyl hydrogens of 2- and 4-picoline are slightly acidic , and can be removed by a strong base such as sodium amide to form the corresponding carbanions , which can then be alkylated by electrophilic halides :
It might be possible to synthesize an entirely new class of peroxides , a-cyanoperoxides , which could potentially be highly toxic to Plasmodia . When the peroxide reacts with heme's iron(II) , it would split in two , and the "free" part in solution would undergo a retro-cyanohydrin reaction to release the parent ketone and a cyanide free radical . The CN free radicals could bond to more iron(II) , or they could attack Plasmodia's enzymes .
There are several possible synthesis routes to the a-cyanoperoxides . A ketone could be converted to its corresponding cyanohydrin by known methods (the bisulfite adduct procedure is probably the best one) ; then its hydroxyl group would be displaced by the surprisingly nucleophilic hydrogen peroxide or hydroperoxide [the alpha effect ; see also the Tiemann modification of the Strecker amino acid synthesis] . Or , the ketone could be reacted first with hydrogen peroxide or hydroperoxide , with a subsequent displacement of the tertiary hydroxyl by nucleophilic cyanide :
And , with the ketone (2 equiv.) and hydrogen peroxide (1 equiv.) :
Even if these a-cyanoperoxides were highly effective against Plasmodia , as cyanide-releasing compounds they could be administered to malaria patients only in a clinical setting , under strict medical supervision . The Merck Index says that the "average fatal dose" for hydrogen cyanide is 50-60 mg ; the actual amount of HCN equivalent in , say , a 100 mg dose of one of the a-cyanoperoxides sketched above would be far less , likely only a few milligrams (I haven't calculated the stoichiometry) . Such a hypothetical 100 mg daily drug dose could probably be tolerated by adults , although maybe not by children .
In this section I'll briefly return to the oxygenated peroxides , specifically those with the aperoxyketal function , the pharmacophore in artemisinin and its relatives . Adam and Rios were among the first researchers to prepare aperoxyketals (1,2,4-trioxanes) :
The use of isobutylene oxide as the starting material for the cyclizing hydroperoxide is important , since no asymmetry will be introduced by it into the resulting trioxane . Unfortunately , when the two unsymmetrical ketones shown in the scheme below are used in the synthesis , their carbonyl carbons will become asymmetric in the 1,2,4-trioxane product :
The heme target molecule has a flat porphyrin structure which would probably be insensitive to asymmetry in the two enantiomers . Plasmodia's enzymes , such as its SERCA enzyme , almost certainly would have enantioselective bonding sites for the peroxides , and therefore would interact differently with the two enantiomers of the 1,2,4-trioxanes shown above .
The remarkably versatile methylolation reaction might be used again to prepare , in a simple and economical manner , a series of linear aperoxyacetals (using formaldehyde) and aperoxyketals :
The above reactions would take advantage of the nucleophilic outer oxygen atom (alpha effect) of the hydroperoxide , which should add readily to the electrophilic carbonyls of formaldehyde and the symmetrical ketones , even when mixed together cold . On the other hand , the carbitol's alcohol oxygen isn't activated , and is only weakly nuclophilic . An acid catalyst , with continuous removal of the water by-product , would be required to combine the aperoxymethylol with the carbitol to form the desired acetal or ketal peroxide . The suggested azeotroping solvent , dichloromethane , isn't as effective as the BTX solvents (benzene , toluene , and xylene) for the azeotropic removal of water from a reaction mix . However , its boiling point is 40 ºC , which should provide a safer reaction medium than the higher-boiling BTX solvents .
The 1,2-dioxetanes are also interesting compounds for investigation as potential antimalaria drugs . Most 1,2-dioxetanes are too unstable for any sort of work on them at room temperature , as they usually decompose well below 20 ºC . They have been prepared and studied primarily as transient intermediates in various oxygenation reactions of olefins . However , if electron-rich olefins are used as the substrates in their preparation , the resulting 1,2-dioxetanes can be isolated and are stable at room temperature .
For example , the electron-rich olefin tetrakis(dimethylamino)ethylene [TDAE] reacts exoergically with atmospheric oxygen at 20 ºC , emitting the reaction energy as chemiluminescence . The 1,2-dioxetane product is a fairly stable white solid . TDAE is notable as a two-electron reducer , with a standard oxidation potential roughly comparable to that of zinc metal (0.762 V) .
All four substituents to the olefin substrate must be electron-donating in order to obtain a 1,2-dioxetane that is stable under ambient conditions . Bartlett and Schaap prepared 1,2-dioxetanes from 1,2-diethoxyethylenes (cis and trans) , but they were very unstable , the pure "cis" dioxetane exploding at room temperature . Both the cis and trans isomers decomposed smoothly and quantitatively in solution to ethyl formate between 5060 ºC . Mazur and Foote prepared a 1,2-dioxetane , isolable and stable at room temperature , by the photochemical 1,2-cycloaddition of singlet oxygen to tetramethoxyethylene . Their 3,3,4,4-tetramethoxy-1,2-dioxetane a clear , pale-yellow liquid obtained in 94% yield "was found to be remarkably stable" (p. 3225) , decomposing smoothly at 56 ºC to provide a quantitative yield of dimethyl carbonate :
TDAE was prepared in an unstated yield by Pruett and co-workers by combining a large excess of dimethylamine with fluorotrichloroethylene in an autoclave . Normally , unactivated vinylic halogen atoms are difficult to substitute by nucleophiles under ambient conditions , but apparently this TDAE formation was actually exothermic , and required cooling of the autoclave with a water spray to around 32 ºC . The common , inexpensive solvent tetrachloroethylene (also known as perchloroethylene , or simply "perc") , used extensively in the dry-cleaning industry , could be reacted with four equivalents of a sodium alkoxide to provide the necessary tetraalkoxyethylene substrate . This latter intermediate would then be combined with singlet oxygen in a 1,2-cycloaddition reaction to produce the corresponding 1,2-dioxetane . Singlet oxygen is generated from "ordinary" pure oxygen gas by photolysis in the presence of a suitable sensitizer , usually a porphyrin derivative . A series of dioxetanes could be prepared from the four alcohols : methyl , ethyl , isopropyl , and ethyl carbitol ; first preparing the tetraalkoxyethylene intermediate , then the 1,2-dioxetane for each alcohol . These dioxetanes should be stable at room temperature , making them available for investigation as very reactive peroxides against Plasmodia strains in vitro :
It would be really neat if a "super-duper bug killer" dioxetane could be synthesized from two common , cheap industrial solvents and oxygen gas !
Methylene Blue as an Antimalarial Drug
Over a century ago (in 1891) the German medicinal chemist Paul Ehrlich (1854-1915) discovered that the common laboratory stain Methylene Blue was effective at curing malaria . In the following decades it was sporadically tried as an antimalarial drug , but apparently was unpopular with patients because the dye stained their eyes [the white of the eyeballs] blue , as it did their skin and urine . The effective antimalaria drug quinacrine (mepacrine , atabrine) was similarly disliked by the Allied soldiers in the South Pacific during World War II , as it dyed their skin yellow .
Methylene Blue is thought to inhibit one of Plasmodia's enzymes , glutathione reductase . In 1995 Vennerstrom(3) and co-workers re-examined Methylene Blue and twenty other related laboratory dyes and stains , comparing their effectiveness in vitro against two strains of Plasmodia falciparum . Methylene Blue was remarkably successful in their tests , with the highest IC50 values (3.58 nM , 3.99 nM , versus 24.0 nM for chloroquine) of all the compounds studied .
Methylene Blue is a redox indicator ; it can be used in potentiometric titrations , changing colour abruptly at a certain specific electrode potential . The dye accepts hydrogen ions and electrons in effect , hydrogen atoms and is reduced to its colourless counterpart , Leucomethylene Blue . In turn , the leuco form can be oxidized back to the blue dye by mixing atmospheric oxygen into it . That is , Methylene Blue (MB) is an oxidizer , and Leuco-MB behaves as a reducing agent . MBs oxidizing nature is shown in the laboratory demonstration , " the blue bottle experiment ". An alkaline solution of a reducing sugar such as glucose is mixed with a solution of MB in a flask , which is stoppered . The intial blue colour of the solution fades after a minute or so , the solution becoming colourless as the MB is reduced by the sugar to Leuco-MB . The stopper is removed to let air oxygen into the flask , which is closed again , and shaken . The air oxidizes the Leuco-MB back to MB , and the solution turns blue again . Then , after a short time , the solution decolorizes as before . And so on , until all the sugar has been oxidized , catalytically by the MB , but in essence permanently by oxygen from the air . For a video demonstration (Quicktime format) of the blue bottle experiment , go to this web page . The chemical reactions involved are as follows :
Methylene Blue+ Cl- + 2 H+ + 2 e- (ex glucose) ----------> Leucomethylene Blue + HCl
Then : Leucomethylene Blue + HCl + ½ O2 (air) ------------> Methylene Blue+ Cl- + H2O
Net reaction : 2 H+ + 2 e- (ex glucose) + ½ O2 (air) -------------> H2O
I was wondering : could Methylene Blue catalytically oxidize glucose in mildly acidic conditions , say around pH 5.2 or so , as in Plasmodia's food vacuole ?
It would be interesting to see if the blue bottle experiment could be reproduced in such a mildly acidic environment . If so , we might have another mode of action of MB in Plasmodia : depletion of its oxygen and glucose via catalytic oxidation by the blue dye . The parasite is an animal organism , and so would require oxygen for its energy production processes ; and as noted earlier , it consumes glucose from the human host as the fuel for such an energy-generating metabolism .
The familiar deep blue dye indigo (blue jeans) is similar to Methylene Blue in its redox activity . Indigo is reduced by 2 H+ + 2e- to leucoindigo , which is yellow . Indigo is highly insoluble in water , unlike the cationic molecule MB , which is quite water-soluble (40 g/L) .
At first glance , Methylene Blue seems to be radically different from the other antimalaria drug candidates discussed in these web pages . However , the partly hydrophilic tails in the typical peroxide compounds , or indeed in the idealized model compound structure (sketched in part 1) , were necessary in most cases to solubilize or stabilize in suspension the drug molecules with their hydrophobic heads. Such a tail is unnecessary for the water-soluble Methylene Blue . The entire MB molecule functions as the "poison package" , and protective hydrophobic groups would be superfluous because of MBs remarkable chemical inertness in blood and other body fluids .
Noting the high affinity of Plasmodia-infected blood cells for Methylene Blue , researchers have attached dye structures (Azure A and proflavine) to modified quinine substrates , hoping to improve the uptake of the latter drug molecules into the blood cells . While improvements in IC50 values were noted for their dye-quinine composites for a D6 falciparum strain , perhaps a more fruitful approach would be to design , synthesize , and assay brand-new redox-active compounds that , like MB , will act in a fundamental manner against Plasmodia by catalyzing the depletion of their glucose and oxygen , in effect "starving them to death". Here are several suggestions in that direction .
The aromatic compound thioxanthene resembles Leucomethylene Blue , with CH2 replacing NH . Its derivatives are used as antipsychotic drugs . Thioxanthene can undergo hydride abstraction , being converted into a thioxanthylium salt , the CH equivalent (without the dimethylamino groups) of Methylene Blue . Thioxanthylium perchlorate has been described as a red crystalline compound . It might have the same sort of activity against Plasmodia as Methylene Blue :
I haven't researched the syntheses of thioxanthene in the literature , but instead I've suggested a possible preparation of it based on the analogous reaction of diphenyl ether with sulfur , catalyzed by anhydrous aluminum chloride , to produce phenoxthin . The following is a scheme to synthesize a thioxanthylium analogue of Methylene Blue via methylolation :
The Merck Index states that potassium perchlorate has been used in the treatment of hyperthyroidism (0.20.8 g/day) , so the perchlorate anion doesn't seem to be too toxic for use as the spectator anion in these compounds . I have proposed the possible use of sodium chlorate , NaClO3 , as an antimalaria drug in Part 3 .
Xanthene (m.p. 101 ºC) , the oxygen analogue of thioxanthene , can also undergo hydride abstraction to provide xanthylium perchlorate , described as "bronze plates", m.p. 225-226 ºC (Bonthrone and Reid , 1959 ; see the reference in hydride abstraction below) . This cationic salt might have the same sort of redox properties as Methylene Blue .
All these novel redox-active candidate compounds should act catalytically to oxidize glucose and in turn be restored to their original cationic form by oxygen . That is , they should give a positive "blue bottle experiment" (or "red bottle" , or whatever) , as Methylene Blue does , so they will be able to catalyze the glucose and oxygen starvation of Plasmodia .
The discovery of artemisinin and its analogues , and the development of a broad range of novel peroxide-containing drug candidate compounds to combat Plasmodia , have been highly educational to medicinal chemists in particular , and to health scientists in general . If we step back for a moment to survey the panorama of the development of these compounds , we see that a multi-disciplinary effort was required and made to achieve the successes realized so far in the production of new antimalaria drugs . Beginning with the humble village herbalists in southern rural China , and proceeding with preliminary extractions and analyses by natural products chemists , the artemisinin saga continued with further sophisticated syntheses and testing in advanced chemical and biological laboratories worldwide . As pointed out in Part 1 , the development of new peroxide-containing compounds seemed to progress in several stages , with the final realization that the artemisinin family of drugs , although highly effective against Plasmodia in their own right , were limited by their natural source of supply and could be readily supplanted by purely synthetic compounds .
While medicinal chemists were busy synthesizing new peroxide-containing molecules , biologists were studying Plasmodia , in effect "taking them apart" and examining their internal structure and functioning . Biochemists found out why the organic peroxides were so successful in killing Plasmodia in the human body , without significantly affecting the host . More importantly , the biochemical mechanisms of the organic peroxides inside Plasmodia were found to be so basic in their nature as to prevent the parasites from developing any significant degree of resistance to them . The peroxides were fundamentally destructive to the parasites' life processes , and they are too primitive a microorganism to have any defense or repair mechanisms to cope with , and to survive , the peroxides' chemical actions on them .
We can and should learn from this experience . Let's start from the "end" of the artemisinin story and work our way backwards . For example , suppose there is a microorganism that causes a certain disease . The biologists and biochemists should "dissect" the invader , finding out exactly how it functions in the host body , and how it harms that body . The microorganism's internal structure and biochemistry should be studied and elaborated . Then , with that information in hand , the medicinal chemists can develop strategies for new drug designs that take advantage of any "Achilles heel" the microorganism might reveal . In particular , chemists can look for vulnerable biochemical processes in the parasite that can be specifically targeted by novel drug candidates , in the search for a "magic bullet" that will efficiently terminate one or more of those life processes . The drugprocess interaction should be of such a fundamental nature that the microorganism would be unable to avoid elimination by the host body by developing any resistance to the drug , nor would it have any repair mechanisms to recover from the chemical attack .
Although herbal medicines have been of immense value in the past for example , quinine and artemisinin the methodology just outlined should permit us in the future to proceed directly with new , rational drug design when we have the biological and biochemical information in hand , without resorting to traditional natural medicinal sources .
Learning how to successfully combat one microorganism may also provide benefits , a sort of "cross-fertilization", in the treatment of other parasitic diseases , and even in research on human illnesses not caused by external organisms , such as cancer . For example , Klayman has pointed out that the artemisinins have been used to treat other parasite infections in humans , such as chlonorchis sinensis , from raw fish . They are also useful anthelmintics (anti-worm drugs) , for example against schistosomes . The artemisinins have been studied as a treatment for influenza , and for an autoimmune disease , systemic lupus erythematosis . Observing that cancerous tumors seem to have a higher than normal concentration of cellular iron , researchers have successfully used artemisinins to control the proliferation of such abnormal growths . Potential use of the new peroxide and N-O compounds in diverse fields such as cancer , autoimmune diseases , and antiviral chemotherapy (and possibly as antibiotics , against drug-resistant bacteria) will make them of considerable interest to pharmaceutical firms which otherwise would ignore them as unprofitable antimalaria drugs . They might even become the "Wonder Drugs of the Twenty-first Century" !
The original antimalaria scope of the artemisinins (and by association , of the synthetic organic peroxides and N-O compounds) has thus dramatically expanded to include many new applications which were barely imagined in the early years of the development of these medications . By keeping an open and educated mind , we can similarly approach new challenges in medicinal chemistry with confidence and determination .
Plasmodia protozoa will always exist ; as their primary hosts , female Anopheles mosquitos will always carry them ; and humans will always be bitten by those mosquitos and become infected with Plasmodia . We can never be free of malaria . But with powerful new peroxide and N-O compounds available , treating malaria and reducing its high mortality will become more of a routine public health management problem . Hopefully some day malaria will be of no greater concern to its victims than a cold or a headache .
Update : several more types of chemical compounds that might have antimalarial activity are discussed in a third web page in Part 3 .
References and Notes
Moyna : H. Ren et al. , "Design , Synthesis , and Biological Evaluation of a Series of Simple and Novel Potential Antimalarial Compounds", Biorg. Med. Chem. Lett. 11 (14) , pp. 1851-1854 (2001) . The same quotation occurs in a related paper by D. Gamenara , H. Heinzen , and P. Moyna , "Design , Synthesis and Biological Evaluation of New Oxazines with Potential Antiparasitic Activity", Tetrahedron Lett. 48 (14) , pp. 2505-2507 (2007) .
preliminary communication : W.D. Emmons , "The Synthesis of Oxaziranes", J. Amer. Chem. Soc. 78 (23) , pp. 6208-6209 (1956) .
comprehensive paper : W.D. Emmons , "The Preparation and Properties of Oxaziranes", J. Amer. Chem. Soc. 79 (21) , pp. 5739-5754 (1957) .
Minisci and Galli : F. Minisci and R. Galli , "New Types of Amination of Olefinic , Acetylenic and Aromatic Compounds by Hydroxylamine-O-Sulfonic Acid and Hydroxylamines / Metal Salts Redox Systems", Tetrahedron Lett. 1965 (22) , pp. 1679-1684 .
primarily used : H.L. Yale , "The Hydroxamic Acids", Chem. Rev. 33 , pp. 209-256 (1943) ; J.B. Bapat , D. St. C. Black , and R.F.C. Brown , "Cyclic Hydroxamic Acids", Adv. Heterocyclic Chem. 10 , pp. 199-240 , H.R. Katritzky and A.J. Boulton (eds.) , Academic Press , New York , 1969 .
Fazary et al. : A.E. Fazary et al. , "The Role of Hydroxamic Acids in Biochemical Processes", Med. J. Islamic Acad. Sci. 14 (3) , pp. 109-116 (2001) [available as a free download (PDF , 46 KB) , from http://www.medicaljournal-ias.org/14_3/Fazary.pdf ] .
Holland et al. : K.P. Holland et al. , "Antimalarial Activities of Polyhydroxyphenyl and Hydroxamic Acid Derivatives", Antimicrobial Agents Chemotherapy 42 (9) , pp. 2456-2458 (1998) [available as a free download (PDF , 100 KB) , from http://aac.asm.org/cgi/reprint/42/9/2456.pdf ] .
Lell et al. : B. Lell et al. , "Fosmidomycin , A Novel Chemotherapeutic Agent for Malaria", Antimicrobial Agents Chemotherapy 47 (2) , pp. 735-738 (2003) [available as a free download (PDF , 58 KB) , from http://aac.asm.org/cgi/reprint/47/2/735.pdf ] .
Methoxyamine : Also referred to as methoxylamine in the mini-review by L.F. Fieser and M. Fieser , Reagents for Organic Syntheses , vol. 1 , pp. 670-671 , John Wiley , New York , 1967 ; idem. , vol. 11 , pp. 322-323 (1984) , where it is said to be "highly poisonous". Preparation : R.A. Goldfarb , "N-Alkoxysulfanilimide Derivatives", J. Amer. Chem. Soc. 67 (10) , pp. 1852-1853 (1945) .
potassium tert-butoxide : Reviewed by L.F. Fieser and M. Fieser , Reagents for Organic Syntheses , vol. 1 , pp. 911-927 , John Wiley , New York , 1967 . A safe preparation of potassium t-butoxide is described by W.S. Johnson and W.P. Schneider , "b-Carbethoxy-g,g-Diphenylvinylacetic Acid", Org. Synth. Coll. Vol. 4 , pp. 132-135 (1963) [available as a free download (PDF , 190 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV4P0132.pdf ] .
Phenoxyamine : C.L. Bumgardner and R.L. Lilly , "O-Phenylhydroxylamine", Chem. Ind. 1962 (12) , pp. 559-560 .
hydrochloride salt : J.S. Nicholson and D.A. Peak , "O-Phenylhydroxylamine Hydrochloride", Chem. Ind. 1962 (27) , p. 1244 . The authors noted , The hydrochloride is relatively stable in comparison with the free base with only a slight discoloration over several months.
Johnson and Degering : K. Johnson and E.F. Degering , "The Utilization of Aliphatic Nitro Compounds . (I) The Production of Amines and (II) The Production of Oximes", J. Amer. Chem. Soc. 61 (11) , pp. 3194-3195 (1939) .
Merck Index : Merck Index , 8th edition , P.G. Stecher et al. (eds.) , Organic Name Reactions section , p. 1144 (Merck and Co. , Rahway , NJ , 1968) .
Vennerstrom(2) : J. L. Vennerstrom , "Amine Peroxides as Potential Antimalarials", J. Med. Chem. 32 (1) , pp. 64-67 (1989) .
2-nitroethanol : W.E. Noland , "2-Nitroethanol", Org. Synth. Coll. Vol. 5 , pp. 833-838 (1973) [available as a free download (PDF , 137 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV5P0833.pdf ] .
Wittig reagent : A. Maercker , "The Wittig Reaction", Organic Reactions , Vol. 14 , Ch. 3 , pp. 270-490 , A.C. Cope (ed.) , John Wiley , New York , 1965 ; in Wikipedia , the online encyclopedia : "Wittig reaction", at http://en.wikipedia.org/wiki/Wittig_reaction .
known compound : G. Wittig and M. Schlosser , "Phosphinealkylenes as Olefin-forming Reagents . IV . Preparation of Vinyl Ethers , Vinyl Thioethers , and Vinyl Halides from Ylids", Chem. Ber. 1961 (94) , pp. 1373-1383 ; CAN 55:124531 ; AN 1961:124531 .
oxaziridines : J.K. Mishra , "Oxaziridines", Synlett Spotlight 112 (3) , pp. 543-544 (2005) [available as a free download (PDF , 103 KB) , from http://www.thieme-connect.com/ejournals/pdf/synlett/doi/10.1055/s-2005-862374.pdf ] ; N. Magomedov , "Chemistry of Oxaziridines", seminar , December 1 , 2000 [available as a free download (PDF , 222 KB) , from http://daecr1.harvard.edu/pdf/smnr_2000-2001_Magomedov_Nabi.pdf ] ; M.B. Soellner , "The Chemistry of Oxaziridines", document dated January 30 , 2003 [available as a free download (PDF , 133 KB) , from http://www.chem.wisc.edu/areas/organic/studsemin/soellner/soellner-abs.pdf ] .
Schulz , Becker , and Rieche : M. Schulz , D. Becker , and A. Rieche , "Preparation of Oxaziridines from Olefins", Angew. Chem. Internat. Ed. Engl. 4 (6) , pp. 525-526 (1965) . The authors advise , Owing to the danger of explosions , no more than 0.5g of olefin should be used , and all necessary precautions taken (p. 526) . Only low yields (2336%) of oxaziridines were obtained .
1-diethylamino-3-butanone : A.L. Wilds , R.M. Nowak , and K.E. McCaleb , "1-Diethylamino-3-Butanone", Org. Synth. Coll. Vol. 4 , pp. 281-282 (1963) [available as a free download (PDF, 121 KB) from : http://www.orgsyn.org/orgsyn/pdfs/CV4P0281.pdf ] .
Schmitz , Ohme , and Schramm : E. Schmitz , R. Ohme , and S. Schramm , "Synthesis and Reactions of 2-Acyl-Oxaziridines", Tetrahedron Lett. 1965 (23) , pp. 1857-1862 .
Zong , Shin , and Ryu : K. Zong , S.I. Shin , and E.K. Ryu , "A New Method for Preparing N-Acyloxaziridines via Tandem O,N-Addition of Hydroxamic Acids to Methyl Propiolate", Tetrahedron Lett. 39 (34) , pp. 6227-6228 (1998) .
derivatives : R.L. Shriner et al. , The Systematic Identification of Organic Compounds , seventh ed. , John Wiley , New York , 1998 ; p. 323 .
amine oxide peroxides : H.J. Shine and L. Hughes , "Organic Nitrogen Compounds . I . Peroxide Intermediates of Tertiary Alkylamine Oxidation by Hydrogen Peroxide", J. Org. Chem. 31 (10) , pp. 1558-1562 (1967) . A highly efficient preparation of amine oxides from tert-amines , using a tungstate catalyst deposited on an Mg-Al double hydroxide layer carrier combined with 30% hydrogen peroxide solution , permits a rapid , nearly quantitative yield of product to be obtained at room temperature : B.M. Choudary et al. , "The First Example of Catalytic N-Oxidation of Tertiary Amines by Tungstate-exchanged Mg-Al Layered Double Hydroxide in Water : A Green Protocol", Chem. Commun. 2001 (18) , pp. 1736-1737 . The tungstate , which is a mild oxidizer , might have aided in the decomposition of the intermediate amine oxideperoxide complex .
N,N-dimethyldodecylamine oxide : M.N. Sheng and J.G. Zajacek , "N,N-Dimethyldodecylamine Oxide", Org. Synth. Coll. Vol. 6 , pp. 501-503 (1988) [available as a free download (PDF , 154 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV6P0501.pdf ] . Note that t-butyl hydroperoxide is the peroxidizing agent in this preparation , not hydrogen peroxide .
can be removed : Several more examples of the alkylation (or acylation) of the methyl group of picoline and lutidine : R.B. Woodward and E.C. Kornfeld , "Ethyl-2-Pyridyl Acetate", Org. Synth. Coll. Vol. 3 , pp. 413-415 (1955) [available as a free download (PDF , 142 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV3P0413.pdf ] ; L.A. Walter , "1-(a-Pyridyl)-2-Propanol", ibid. , vol. 3 , pp. 757-759 [available as a free download (PDF , 156 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV3P0757.pdf ] ; W.G. Kofron and L.M. Baclawski , "Metalation of 2-Methylpyridine Derivatives : Ethyl 6-Methylpyridine-2-Acetate", ibid. , vol. 6 , pp. 611-612 (1988) [available as a free download (PDF , 141 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV6P0611.pdf ] .
cyanohydrin : R.B. Wagner and H.D. Zook , Synthetic Organic Chemistry , John Wiley , New York , 1953 ; pp. 604-605 . I like to call this valuable reference the "Zook Cook Book" ! For a typical example of a cyanohydrin preparation , see R.F.B. Cox and R.T. Stormont , "Acetone Cyanohydrin", Org. Synth. Coll. Vol. 2 , pp. 7-8 (1943) [available as a free download (PDF , 148 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV2P0007.pdf ] .
bisulfite adduct : E.C. Wagner and M. Baizer , "5,5-Dimethylhydantoin", Org. Synth. Coll. Vol. 3 , pp. 323-325 (1955) [available as a free download (PDF , 145 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV3P0323.pdf ] . The method is described in Note 1 , p. 324 .
Adam and Rios : W. Adam and A. Rios , "Perhydrolysis of Epoxides", J.C.S. Chem. Commun. 1971 (15) , pp. 822-823 .
azeotroping solvent : Handbook of Chemistry and Physics , various eds. , azeotropes Tables .
transient intermediates : E.W.H. Asvold and R.M. Kellogg , "Formation of 1,2-Dioxetanes and Probable Trapping of an Intermediate in the Reactions of Some Enol Ethers with Singlet Oxygen", J. Amer. Chem. Soc. 102 (10) , pp. 3644-3646 (1980) .
electron-rich olefins : R.W. Hoffmann , "Reactions of Electron-Rich Olefins", Angew. Chem. Internat. Ed. Engl. 7 (10) , pp. 754-765 (1968) .
tetrakis(dimethylamino)ethylene : R.L. Pruett et al. , "Reactions of Polyfluoro Olefins . II . Reactions with Primary and Secondary Amines", J. Amer. Chem. Soc. 72 (8) , pp. 3646-3650 (1950) .
two-electron reducer : N. Wiberg , "Tetraaminoethylenes as Strong Electron Donors", Angew. Chem. Internat. Ed. Engl. 7 (10) , pp. 766-779 (1968) .
Bartlett and Schaap : P.D. Bartlett and A.P. Schaap , "Stereospecific Formation of 1,2-Dioxetanes from cis- and trans-Diethoxyethylenes by Singlet Oxygen", J. Amer. Chem. Soc. 92 (10) , pp. 3223-3225 (1970) .
Mazur and Foote : S. Mazur and C.S. Foote , "Chemistry of Singlet Oxygen . IX . A Stable Dioxetane from Photoxygenation of Tetramethoxyethylene", J. Amer. Chem. Soc. 92 (10) , pp. 3225-3226 (1970) .
Pruett : see the reference for tetrakis(dimethylamino)ethylene above .
glutathione reductase : P.E. Meissner et al. , "Methylene Blue for Malaria in Africa : Results from a Dose-Finding Study in Combination with Chloroquine", Malaria Journal 5 (84) 2006 [available as a free download (PDF , 242 KB) , from http://www.malariajournal.com/content/pdf/1475-2875-5-84.pdf ] . The authors admitted , ...... the combination of CQ-MB [chloroquine-methylene blue] is clearly not effective in the treatment of malaria in Africa.
Vennerstrom(3) : J.L. Vennerstrom et al. , "Antimalarial Dyes Revisited : Xanthenes , Azines , Oxazines , and Thiazines", Antimicrobial Agents Chemotherapy 39 (12) , pp. 2671-2677 (1995) [available as a free download (PDF , 252 KB) , from http://aac.asm.org/cgi/reprint/39/12/2671.pdf ] .
mildly acidic environment : A buffer solution with a pH of around 5.2 could be used as the solvent for the glucose , with the addition of a small quantity of Methylene Blue solution . Buffers with various pH values are commercially available , eg. "Hydrion Buffer", pH = 5.00 , from the Aldrich Chemical Company . Chemistry handbooks also provide formulas for buffer solutions . For example , the Handbook of Chemistry and Physics gives the following recipe for a buffer solution whose pH is 5.20 : potassium hydrogen phthalate (0.1 molar , 50 ml) + NaOH (0.1 molar , 28.8 ml) . This same solution (pH = 5.00) is commercially available from the Fisher Scientific Company .
dye structures : J. Howarth and D.G. Lloyd , "Redox Systems as Conduits for Antimalarial Compounds", J. Antimicrob. Chemother. 47 (1) , pp. 122-124 (2001) [available as a free download (PDF , 70 KB) , from http://jac.oxfordjournals.org/cgi/reprint/47/1/122.pdf ] .
hydride abstraction : W. Bonthrone and D.H. Reid , "Hydride Ions in Organic Reactions . Part I . Dehydrogenation by Triphenylmethyl Perchlorate", J. Chem. Soc. 1959 , pp. 2773-2779 ; idem. , "Dehydrogenation of Heterocyclic Hydroaromatic Compounds by Triphenylmethyl Perchlorate", Chem. Ind. 1960 (38) , pp. 1192-1193 ; G.A. Olah et al. , "Preparative Carbocation Chemistry . 13 . Preparation of Carbocations from Hydrocarbons via Hydrogen Abstraction with Nitrosonium Hexafluorophosphate and Sodium NitriteTrifluoromethanesulfonic Acid", J. Org. Chem. 43 (1) , pp. 173-175 (1978) ; J. Holmes and R. Pettit , "Hydride Ion Abstraction with Antimony Pentachloride", J. Org. Chem. 28 (6) , pp. 1695-1696 (1963) .
phenoxthin : C.M. Suter and C.E. Maxwell , "Phenoxthin", Org. Synth. Coll. Vol. 2 , pp. 485-486 (1943) [available as a free download (PDF , 111 KB) , from http://www.orgsyn.org/orgsyn/pdfs/CV2P0485.pdf ] .
Klayman : D.L. Klayman , "Qinghaosu (Artemisinin) : An Antimalarial Drug from China", Science 228 (4703) , pp. 1049-1055 (1985) ; additional artemisinin applications are discussed on p. 1054 .
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