Synthesis of novel polar derivatives of the antimalarial endoperoxides ascaridole and dihydroascaridole

Novel polar derivatives of the antimalarial endoperoxide ascaridole were synthesized based on the zeolite NaY-promoted isomerization of perillyl and nopol derivatives to conjugated cyclohexadienes, followed by reaction with singlet oxygen. Several unsaturated endoperoxides were in situ reduced with diimide to form dihydroascaridole derivatives, as well


Introduction
Terpene peroxides constitute an interesting category of organic compounds that have received a considerable attention for the past two decades because of their often remarkable antimalarial activity at the nanomolar concentration scale. 1 Generally, it is believed that scission of the peroxide O-O bond by Fe(II) species in the erythrocytes generates radicals which are fatal to the parasite.Among them, artemisinin (1), 2 a naturally occurring antimalarial sesquiterpene peroxide possessing an 1,2,4-trioxane group, is considered as one of the most important antimalarial drugs currently in use, with high activity against multidrug-resistant forms of parasite Plasmodium falciparum. 3The enhanced pharmacological activity of 1 has triggered the interest of the organic chemists towards the synthesis of novel artemisinin-based derivatives, 4 such as dihydroartemisinin, artemisone, artemether and artesunate, with even better pharmacological properties.In addition, novel methodologies were developed 5 for the synthesis of peroxo compounds bearing the antimalarial active 1,2,4-trioxane carbon skeleton, and this topic continues to be highly active, from the synthetic point of view.
Meunier and co-workers have synthesized 6 a novel class of compounds termed trioxaquines (such as compound 2) with high antimalarial activity, by combining the 1,2,4-trioxane pharmacophore, covalently bound with another well known antimalarial pharmacophore moiety, the aminoquinolines.Apart of trioxanes, the 1,2,4,5-tetraoxanes, 7 such as the stereoisomers of compound 3, have emerged recently as a promising category of active antimalarial peroxo compounds.The mechanism of antimalarial action for tetraoxanes 8 seems to be different compared to the 1,2,4-trioxanes, involving O-centred radicals as the probable active species being fatal to the parasites.

NHR
Bicyclic endoperoxides, such as the monoterpenes ascaridole (4) and dihydroascaridole (5), have been reported to exhibit moderate antimalarial properties. 9However, the assay may underestimate the potency of these volatile compounds.In addition, the highly lipophilic character of 4 and 5 might be a drawback for an enhanced antimalarial activity.Posner and coworkers have synthesized 9 a series of diaryl substituted ascaridole-type endoperoxides (for example, compound 6), which showed higher activity compared to ascaridole or dihydroascaridole.Apart of the ascaridole-type compounds 4-6, a naturally occurring bicyclic endoperoxide, namely yingzhaosu A (7), 10 was found to be a potent antimalarial.A Japanese group reported recently that bicyclic peroxide 8 11 which possesses the peroxy core skeleton of yingzhaosu A exhibits antimalarial activity comparable to artemisinin (1).On the other hand, monocyclic peroxides (1,2-dioxanes) bearing an epoxide functionality, such as compounds possessing the motif of 9, 12  Generally, however, bicyclic endoperoxides have received a limited attention as possible potent antimalarial compounds.It is possible that the low activity of the volatiles 4 and 5 might be misleading and even discouraging for further examination of compounds bearing an endoperoxide moiety.For this purpose, we attempted the synthesis of less volatile, and polar as well, derivatives of ascaridole and dihydroascaridole with the aim of studying their antimalarial properties.

Results and Discussion
In this paper we describe a methodology for the synthesis of polar derivatives of ascaridole (4) and dihydroascaridole (5) using as starting materials commercially available terpenoids such as derivatives of nopol and perillyl alcohol.The retrosynthetic analysis, presented in Scheme 1, was triggered by our observation 13 that several monoterpenes like limonene (10) and αor β-pinene (11) undergo transformation to the aromatic p-cymene (12) via intermediate formation of the conjugated cyclohexadiene α-terpinene (13), in a sequence promoted by zeolite NaY (Scheme 2).Under certain reaction conditions (temperature, time, and substrate/zeolite loading level) the reaction mixture might be enriched for the desired intermediate conjugated cyclohexadiene, which upon reaction with singlet oxygen in a [4+2] fashion generates an endoperoxide (ascaridole, 4, in the case of α-terpinene, 13).We envisioned formation of the appropriately substituted cyclohexadienes, structurally related to α-terpinene (13), by the zeolite NaYpromoted isomerization of nopol a terpenoid with a structure similar to β-pinene, and from derivatives of perillyl alcohol, a terpene structurally similar to limonene.
Retrosynthetic analysis for the synthesis of ascaridole-type endoperoxides.Endoperoxides from nopol ( 14) and methyl nopoate (15) By using 2.5 mmoles of nopol (14) or methyl nopoate (15) per 1 gr of dry NaY suspended in 10 mL of hexane, and heating to 70 o C for 8 hours, an inseparable mixture of products was formed in 75% combined yield (Scheme 3).Analysis by GC and GC-MS revealed that the total ratio of the mainly formed (16+17+18) relative to other isomeric byproducts was ~ 4/1.From the crude 1 H NMR spectrum it was obvious the presence of the desired diene 16 in ~25% relative yield, the isomeric diene 17 in comparable amounts, while 18 was the major one (~30%).Prolonged reaction times increase the relative percentage of the dehydrogenation product 18.Yet, perillyl aldehyde (23) under certain reaction conditions transforms cleanly to the isomeric conjugated dienal 24 (p-mentha-l,3-dien-7-al) in 77% yield, and without formation of the aromatic aldehyde 25 (Scheme 5).We found that the optimum reaction conditions require 1.1 mmoles of perillyl aldehyde per 1 g of dry NaY, and heating to 70 o C for 24 hours.Prolonged reaction time results to the gradual formation of 25.Formation of dienal 24 has been reported 14 to result in excellent yield by heating perillyl aldehyde with excess of concentrated H 2 SO 4 at 120-130 o C. In our hands, this experimental procedure proceeds in 80% combined product yield, however, generates in addition to 24, the aromatic aldehyde 25 in ~15-20% product ratio, and approximately 10% of unidentified byproducts (GC analysis).Having in our hands the dienal 24 we easily accomplished its further functionalization (Scheme 6) to form triene 26, and dienes 27-28, suitable for transformation to endoperoxides by reaction with singlet oxygen.Thus, 24 reacted with the stabilized ylide Ph 3 P=CHCOOMe to form triene 26 in 50% isolated yield.Furthermore, selective carbonyl reduction of 24 with LiAlH 4 /AlCl 3 15 yielded alcohol 27 16

Endoperoxides from methyl perillate (33)
The facile formation of endoperoxides 29-32 using perillyl aldehyde as starting material, urged us to examine the synthesis of analogous ascaridole-type endoperoxides from methyl perillate (33).Surprisingly, the isomerization of methyl perillate to the desired diene 35 was very slow with the intermediate non-conjugated diene 34 to predominate under identical reaction conditions applied to the intrazeolite isomerization of perillyl aldehyde.After 6 hour of zeolite treatment, methyl perillate was consumed and 1 H NMR analysis of the crude reaction mixture revealed that diene 34 was the predominant product (>80% product ratio).The structure determination of 34 was tentative, and was mainly based on several spectral similarities to the known terpene terpinolene 13a (see Scheme 7).On prolonged reaction time, 34 isomerizes slowly to the desired conjugated diene 35, 16   Photooxygenation of the crude reaction mixture followed by careful flash chromatography allowed the isolation of the endoperoxide 37 (Figure 3) in pure form, however, in relatively low yield (~15%) from methyl perillate.

Dihydroascararidole derivatives
The observation that saturated bicyclic endoperoxides are more potent antimalarials relative to their unsaturated counterparts, 9 urged us to reduce the endoperoxides 29-32 with in situ generated diimide (HN=NH). 18The reaction was very clean without affecting the peroxide O-O bond, and the novel saturated endoperoxides 38-41 were isolated in almost quantitative yield.In our hands, approximately 30 equivalents of the diimide precursor (ΚΟΟC-N=N-COOK) per alkene double bond are necessary to achieve complete reduction.

Conclusions
In conclusion, we have developed a novel methodology for the synthesis of polar derivatives of the endoperoxides ascaridole and dihydroascaridole using as starting materials readily available terpenoids.The key reaction is the isomerization of the terpenoids to conjugated cyclohexadienes promoted by zeolite NaY.While the yield of the endoperoxides arising from nopol and methyl perillate are very low (5-15%), in the case of perillyl aldehyde due to the very clean isomerization to its conjugated dienal, the overall yield of the endoperoxides 29-32 is relatively good (~30-40% from perillyl aldehyde).The antimalarial potency of these compounds is currently under investigation.

Experimental Section
General Procedures.Nuclear magnetic resonance spectra were recorded on a 500 MHz spectrometer in CDCl 3 .Isomeric purities were determined by 1 H NMR spectroscopy, by analytical gas chromatography on an SP-5 capillary column, and by GC-MS.Photooxygenation reaction were carried out using a 300 W Xenon lamp.
Starting materials.Nopol ( 14) and perillyl aldehyde (23) are commercially available.Methyl nopoate (15) was prepared by Jones oxidation of nopol to nopoic acid (in 10 mmoles scale) followed by esterification of the resulting acid with CH 2 N 2 (65% yield over the two steps).In a 50 mL flask were placed 1 g of zeolite NaY, which had been previously dried at 120-130 o C under vacuum (10 -4 torr) for at least 6 hours prior to use, 10 ml of hexane and then 0.4 gr of nopol ( 14) or methyl nopoate (15).The slurry was refluxed to 70 o C for 8 hours.The reaction mixture was cooled to room temperature and then filtered.The filtrate was kept and the solid material was further treated with 2 x 10 mL of methanol for 30 minutes each time, and then filtered again.The combined solvent extracts were evaporated to afford 0.3 gr of a mixture of products (mainly 16, 17 and 18).Characteristic absorptions of the desired cyclohexadienes 16, from the crude reaction mixture: 5.70 (d, 1H, J = 5.5 Hz), 5.60 (d, 1H, J = 5.5 Hz).Photooxygenation of the crude reaction mixture (methylene blue, 10 -4 M, as sensitizer) in dichloromethane, followed by flash column chromatography using hexane/ethyl acetate = 5/1 as eluant afforded 22 mg of endoperoxide 19 and 19 mg of endoperoxide 20 as colourless oils.
display weak to moderate antimalarial properties.

Inc Endoperoxides from perillyl alcohol (21) and its derivatives
As a next step, following the discouraging low yield formation of the ascaridole derivatives 19 and 20 from nopol, we examined the possibility of forming the appropriate conjugated cyclohexadienes via isomerization of perillyl alcohol (21) or its derivatives.Disappointingly, upon treatment with NaY, 21 or its acetate (22) were transformed to p-cymene(12), via dehydration or acetic acid elimination, respectively, under the acidic zeolite conditions (Scheme 4).
(79% yield), while acetylation of 27 with acetic anhydride formed 28 17 in >90% yield.Photooxygenation of 26-28 afforded the corresponding endoperoxides 29-31 in almost quantitative yield.It is notable that in the case of triene 26, the cycloaddition occurs exclusively on the diene system within the 6-membered ring, most probably due to the electron-deficiency of the double bond next to the ester functionality.Furthermore, endoperoxide 29, which was isolated as a white crystalline solid, was hydrolyzed quantitatively by LiOH in THF/H 2 O = 10/1 to the corresponding acid 32, also isolated as white solid.
however, the aromatic ester 36 is gradually formed.The optimum reaction time was 60 hours, after which, a mixture of 34/35/36 appeared in a relative yield of 25/50/25, and in 65% isolated yield (Scheme 7).It is quite surprising the stability of non-conjugated 34 towards isomerization to 35 under NaY treatment, taking into account that, terpinolene (Scheme 7) a non-conjugated monoterpene structrurally similar to 34 is an intermediate product in the isomerization of several monoterpenes (such as limonene or pinenes), however, it does not persist under zeolite treatment13and undergoes fast isomerization.