USES THEREOF FOR CONTROL OF PERKINSIOSIS IN BIVALVES"

TECHNICAL DOMAIN OF THE INVENTION

The present invention relates to new endoperoxide compounds and compositions, and to a process for producing them for prophylaxis and control of perkinsiosis in bivalves.

Endoperoxide compounds with biological activity against Perkinsus olseni herein described include 13 trioxolanes and 9 tetraoxanes . Protozoan parasites of the genus Perkinsus are known to infect several species of marine molluscs worldwide, like oysters, abalones, clams, scallops, pearl oysters, cockles or mussels.

Thus, the present invention also describes the synthesis of these compounds, in particular of new endoperoxide compounds of the tetraoxane family.

Compositions comprising endoperoxide compounds are useful for prophylaxis and control of perkinsiosis in bivalves.

Therefore, the present invention also relates to a method of controlling perkinsiosis in bivalves.

The present invention is in the domain of aquaculture, medicine, pharmaceuticals and biochemistry. BACKGROUND OF THE INVENTION

Aquaculture is one of the most fast-growing food production industries, with an average annual growth rate of 5.8% between 2001 and 2016 (FAO, 2018) and a predicted growth of over 50% until 2030, supplying over 60% of marine and freshwater organisms for direct human consumption. However, there are several constraints that could compromise the growth rate of this industry, amongst which are diseases that may affect production due to mortality, or require treatments, impacting the producers with severe annual costs. Parasite-borne infections caused by obligate or opportunistic organisms may affect finfish and shellfish aquaculture, representing a key constraint to the production and economic viability of aquaculture facilities worldwide.

In shellfish aquaculture, perkinsiosis is one of the most feared diseases. It is caused by protozoan parasites of the genus Perkinsus, which are known to infect several species of marine molluscs worldwide, like oysters, abalones, clams, scallops, pearl oysters, cockles or mussels. In Portugal there are reported cases of perkinsiosis in grooved carpet shell clam ( Ruditapes decussatus) culture beds in Ria Formosa since 1983, with 70-80% mortalities in this mollusc species (Azevedo, 1989) attributed to Perkinsus olseni (= Perkinsus atlanticus) , a notifiable protozoan infective agent in molluscs, according to the Office International des Epizooties (OIE, the World Animal Health Organization) (OIE, 2018) .

The development of an in vitro culture of P. olseni created the opportunity to perform in vitro and in vivo studies under controlled conditions using this parasite, boosting further studies of parasite's biology and physiology and allowing for drug testing and screening in this parasite.

Several drugs were already tested in P. olseni, like the herbicides 2 , 4-dichlorophenoxyacetic acid, Linuron, Metolachlor, Pendimethalin, Glyphosate, and the folate pathway inhibitor Pyrimethamine. However, the use of the herbicides in aquaculture is questionable due their toxicity. Besides this, Pyrimethamine is an antimetabolite compound that does not present parasiticidal activity and the control of perkinsiosis requires "fast parasite killers". Selection for resistance to antifolates is also an issue.

The role of Fe (II) intracellular concentration in proliferation, virulence and function of important metabolic pathways in P. olseni was also investigated, unravelling the susceptibility of P. olseni to Fe (II) availability and leading to the proposal of iron chelators for the control of perkinsosis. Cyclohexamine, deferoxamine (DFO) and 2,2- bipyridyl (BIP) exhibited antiproliferative activity on P. olseni. However, these chelators do not have a parasiticidal effect on P. olseni and problems related to lack of selectivity may arise.

Araiijo et al. [Araujo N.C.P, Afonso R., Bringela A., Cancela M.L., Cristiano M.L.S., 2013. Peroxides with antiplasmodial activity inhibit proliferation of Perkinsus olseni , the causative agent of Perkinsosis in bivalves. Parasitol Int. 62, 575-582] describe in a preliminary study the efficacy of some peroxide compounds, namely dihidroartemisinin (DHA) , artesunate (ARS) and 3 trioxolanes as antiparasitic agents against P. olseni. EP2233479 discloses tetraoxane compounds for use in the treatment of malaria and/or cancer, and methods for producing such compounds .

Given the above, it is of the upmost importance to develop compositions that are effective against the diseases caused by P. olseni in bivalve aquaculture, which affect its production impacting the producers with severe annual costs.

The present invention proposes the development of compositions based on peroxide compounds and implementation of a suitable therapeutic strategy for the prophylaxis and control of Perkinsosis in bivalves that is able to reduce the prevalence of P. olseni and thus mortality and morbidity in bivalves.

SUMMARY OF THE INVENTION

The present invention relates to new endoperoxide compounds, in particular to tetraoxane compounds and endoperoxide compositions, to a process for producing them, and to a method for prophylaxis and control of perkinsiosis in bivalves.

In a first aspect, the present invention relates to novel tetraoxane compounds of formula I according to claim 1.

These compounds present high in vitro bioactivity against P. olseni and thus it is particularly useful to prepare pharmaceutical compositions for treatment diseases caused by this parasite and others related to it. Therefore, in a second aspect, the present invention relates to pharmaceutical compositions comprising tetraoxane compounds according to claim 3.

These compositions are very effective in reduction of morbidity and mortality of bivalves infected by P. olseni.

Other known peroxide compounds are also bioactive against P. olseni , such as selected trioxolanes and tetraoxanes and for this reason, in a third aspect, the present invention relates to pharmaceutical compositions comprising one or more of said compounds for treatment of diseases having P. olseni as etiological agent according to claim 6.

Said compounds present IC50 values in a range from 8.1 to 268.3 mM. Among the trioxolanes tested, compound LC131 was shown to be more active, with IC50 value of 38.6 mM. For the tetraoxanes, compounds LC137 and LC139 demonstrated an IC50 value of 11.7 and 8.1 mM respectively. Further, some of these compounds demonstrated activity, in vitro and in vivo, against Plasmodium spp. that cause malaria in humans and their safety profiles were scrutinized, both for human treatment and environmental (aquatic habitats, conferring more safety to their use in new applications .

The present invention also relates to a process for producing a peroxide compound in only two synthetic steps according to claim 7.

This process can be performed with materials that are commercially available at low prices. Finally, the present invention relates to a method of controlling pathologies caused by P. olseni in bivalves by administrating a composition comprising at least a trioxolane and/or tetraoxane compound according to claim 8.

This method enables to increase shellfish aquaculture of marine molluscs such as oysters, abalones, clams, scallops, pearl oysters, cockles or mussels by reduction of morbidity and mortality of said bivalves infected by Perkinsus spp . It also can be useful to control parasitic infection of bivalves transferred between different areas and even countries.

DESCRIPTION OF THE FIGURES

The figures presented herein show structural representations of the endoperoxides of the invention, new compounds and known compounds for new uses, such as trioxolanes, tetraoxanes, derivatives of artemisinin and fluorescent probes. In particular,

Figure 1 presents the synthetic routes to the trioxolanes LC28, LC32 , LC50 , LC67 , LC68, LC92, LC93, LC135 and LC136.

Figure 2 presents the synthetic routes to the trioxolanes LC94, LC95 , LC129, LC130, LC131 and LC132.

Figure 3 presents the synthetic routes to tetraoxanes LC137, LC138, LC139 , LC140, LC157 and LC163.

Figure 4 presents the synthetic routes to the tetraoxanes LC146, LC153, LC159, LC165, LC179.

Figure 5 depicts the strategy used for desoxygenation of derivatives of artemisinin.

Figure 6 presents the synthetic routes to the 7-nitro-l , 2 , 3- benzoxadiazole (NBD) -tagged peroxides. GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to new endoperoxide compounds, in particular to tetraoxane compounds and endoperoxide compositions, to a process for producing them, and to a method for prophylaxis and control of perkinsiosis in bivalves.

P. olseni and all the other Perkinsus species are phylogenetically close to Dinoflagellates but also to the genus Plasmodium and Toxoplasma, with organelles like plastids in common in the three genera. Several metabolic pathways are also conserved, like the shikimate, the methylerythritol phosphate (MEP) pathway and the folate metabolism.

The role of Fe (II) intracellular concentration in proliferation, virulence and function of important metabolic pathways in P. olseni was also investigated by some groups of researchers such as Wright et al. [Wright, A. C., Ahmed, H., Gauthier, J. D., Silva, A. M., & Vasta, G. R. (2002) . "cDNA cloning and characterization of two iron superoxide dismutases from the oyster parasite Perkinsus marinus". Molecular & Biochemical Parasitology, 1(123), 73-77.] and Leite et al . [Leite, R. B., Brito, A. B., & Cancela, M. L. (2008) "An oxygen molecular sensor, the HIF prolyl 4-hydroxylase, in the marine protist Perkinsus olseni. Protist, 159(3), 355-368.], unravelling the susceptibility of P. olseni to Fe (II) availability and leading to the proposal of iron chelators for the control of perkinsiosis. Having these fundamentals in mind, several endoperoxide-based compounds were selected and tested for their bioactivity against P. olseni. 1. Tetraoxane compounds

Tetraoxane or acetone peroxide define a family of compounds having a six-membered saturated heterocycle ring with two carbon atoms and four oxygen atoms. It is commonly produced by the reaction of acetone and hydrogen peroxide to yield a mixture of linear monomer and cyclic dimer, trimer, and tetramer forms.

Dimer Trimer

Table 1 presents the novel tetraoxane compounds of general formula I, and their identification, namely their chemical formulae, IUPAC names and chemical representation.

Formula I

Wherein R is :

- CHCOOCH2CH3 (LC138);

- NH (LC139 ) ;

- CHCH2OH (LC146);

- CHNH (CH ₂ ) 4NHB0C (LC157);

- CHCH2NH2 (LC165);

- CHCH2 ( 2-methyl-lH-tretrazole-5-amine ) (LC179);

- CHCH2NHNBD (LC200) . Table 1. Compounds of formulae I

2. Endoperoxide compounds

Endoperoxide compounds according to the present invention are compounds that include besides the tetraoxane compounds of the previous section, other known tetraoxane and trioxolane compounds, which present bioactivity against P. olseni, in particular against perkinsiosis in bivalves. Trioxolanes or diperoxides define a family of compounds having a five-membered saturated heterocycle ring with two carbon atoms and three oxygen atoms.

Herein it is disclosed the synthesis and activity of an extended library of synthetic peroxides in Perkinsus olseni, comprising 13 ozonides (trioxolanes) and 9 tetraoxanes, with the objective of developing a suitable therapeutic strategy for the prophylaxis and control of perkinsiosis in bivalves.

The endoperoxide compounds, according to the present invention are shown in Table 2, along with their identification, namely their chemical formulae, IUPAC names and chemical representation .

Table 2. Endoperoxide compounds

3 . Endoperoxide compounds bioactivity

For the purpose of assessing the activity of selected compounds against Perkinsus olseni, in vitro experiments were set out as described previously by Ellandalloussi et al . [Laurence M. Elandalloussi , Ricardo B. Leite, Pedro M. Rodrigues, Ricardo Afonso, Patricia A. Nunes, Cancela M.L. (2005) "Effect of antiprotozoal drugs on the proliferation of the bivalve parasite Perkinsus olseni." Aquaculture, Volume 243, Issues 1- 4] and Leite et al . [Leite RB, Afonso R, and Cancela ML. (2011) "Herbicides and protozoan parasite growth control: implications for new drug development. In Herbicides, Theory and Applications . IntechOpen], and Larramendy et al . [M. Larramendy, S. Soloneski (Eds.), Herbicides, Theory and Applications, InTech, Rijeka, Croatia (2011), pp . 567-580].

Essentially, the screening for candidate drugs is done by exposing a known amount of Perkinsus olseni cells to a predefined range of concentrations of the compound to be tested. This method is based on a commercial assay to determine cell viability. After 72 hours of exposure the cells are tested for viability and statistical analysis is performed to determine IC50 and determined the potential of the candidate inhibitor. Table 3 presents bioactivity values of some peroxide compounds on Perkinsus olseni.

Table 3. Bioactivity of some endoperoxidase compounds against Perkinsus olseni

All the tested compounds, with the exception of LC157, demonstrate a concentration dependent inhibition of Perkinsus olseni in the micromolar range, and some of them, notably compounds LC137, LC138, LC139 and LC140 manifest inhibitory activity at concentrations with one order of magnitude lower, affecting in vitro proliferation, at concentrations as low as 8 mM. This is a clear indicator that the formulation of these compounds is more efficient in the prophylaxis and control of perkinsiosis .

3 . Endoperoxide compositions

The endoperoxide compounds as described in the previous sections can be delivered directly to the bivalve population in powder formulations, in microcapsules, dissolved in DMSO, where the final dilution of DMSO should be below 0.05% v/v to avoid host toxicity, or in other acceptable formulations.

Endoperoxide compositions comprising compounds or mixtures of compounds herein described can be prepared according to the known methods in the art with addition of one or more pharmaceutically acceptable vehicles, carriers, co-adjuvants or alike.

Suitable pharmaceutically acceptable vehicles that can be used in the compositions of the invention are: acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG- 400), propylene glycol, and solketal, and carriers include albumin (BSA) and cyclodextrin ( 2-hydroxypropyl-beta- cyclodextrin, or HPBCD, or similar. Further, compositions according to the invention can be presented in the form of a powder, dissolved powder in a solvent or carrier, or in oil form.

Said compositions may comprise compounds or mixtures of compounds herein described in a concentration of 8 mM to 700 mM, preferably of 10 mM to 500 mM, more preferably of 12 mM to 250 mM, even more preferably of 15 mM to 100 mM, or more advantageously of 20 mM to 50 mM.

4 . Process of synthesis of tetraoxane compounds

Novel tetraoxane compounds of formula I according to the present invention can be easily synthesized from relatively cheap starting materials. 1 , 2 , 4 , 5-tetraoxanes are achiral, and are known to be stable, compared to their 1,2,4- trioxolane counterparts .

The various tetraoxanes are synthesized using a one pot methodology. Ciclohexanone and derivatives are treated with hydrogen peroxide and formic acid to form the respective dihidroperoxides . Adamantanone is subsequently added, to produce tetraoxanes. Further chemical modifications of the substituents on the cyclohexyl ring can then be carried out, to increase chemical diversity or adjust pharmacologic properties .

Thus, tetraoxane compounds of formula I are produced by a process comprising the following steps:

(a) Ciclohexanone and derivatives are treated with hydrogen peroxide and formic acid to form the respective dihidroperoxides; and (b) Adamantanone is subsequently added, to produce tetraoxanes;

(c) Optionally, further chemical modifications of the substituents on the cyclohexyl ring are carried out, to increase chemical diversity or adjust pharmacological properties .

Other endoperoxide compounds can be obtained by known processes. Namely, 1 , 2 , 4 , 5-tetraoxanes , 1 , 2 , 4-trioxolanes and artemisinin derivatives are synthesized by adapting procedures described in the literature (Marti et al . , 2011; Araujo et al., 2013; Lobo et al . , 2018; Fugi et al . , 2010) .

Trioxolanes LC50, LC28, LC23 and LC67 are obtained as described by Griesbaum coozonolysis (Griesbaum et al . , 1997; Griesbaum et al . , 1997) by reactions between freshly prepared 2- adamantanyl-O-methyl oxime (LC29) and the appropriate 4- substituted cyclohexanones. Reduction of trioxolane ester LC67 results in the corresponding trioxolane alcohol LC93, and hydrazinolysis of the alcohol produced the trioxolane phtalimide LC94. Reaction of LC93 with hydrazine hydrate leads to the trioxolane amine LC95. This compound is then coupled to 3-chloro-l , 2-benzisothiazole 1,1-dioxide to produce the saccharyl-trioxolane conjugate LC130. The trioxolane carboxylic acid LC68 is prepared by hydrolysis of the trioxolane ester LC67. Trioxolane amide LC92, is accessed from LC68 and butylamine, through a peptide-like coupling methodology using EDC, HOBt and N-methylmorpholine . Compounds LC135 and LC136 are obtained from LC50, under reductive amination conditions.

The various tetraoxanes are synthesized using a one pot methodology. Ciclohexanone and derivatives are treated with hydrogen peroxide and formic acid to form the respective dihidroperoxides . Adamantanone is subsequently added, to produce tetraoxanes LC137 and LC139. Tetraoxanes LC138 and LC140 are obtained using a similar methodology, but in this case the synthesis begins by formation of adamantanyl dihidroperoxide . Compounds LC163 and LC157 are obtained by reductive amination, with excellent yield. The synthetic approaches used to obtain tetraoxane alcohol LC146, tetraoxane phthalimide LC159, tetraoxane amine LC165 and tetraoxane carboxylic acid LC153 are similar to those used for the chemical modification of trioxolane-based compounds. To assess the cellular accumulation of the drugs using confocal microscopy, a trioxolane and a tetraoxane are coupled to a 7- nitro-1 , 2 , 3-benzoxadiazole moiety (NBD) (Hartwig et al . , 2011) . The fluorescent peroxide probes are prepared using 4- chloro-7-nitro-l , 2 , 3-benzoxadiazole (NBD-C1) as tagging compound. This fluorophore is coupled to trioxolane LC95 and to tetraoxane LC165 to form trioxolane-NBD LC201 and tetraoxane-NBD LC200.

5. Method of controlling perkinsiosis in bivalves

Taking into account the impracticability of treating adult clams in the natural environment, this application is directed to maternity bivalves but also collected in the natural habitat for purposes of repopulation and seeding with sizes between 2 to 10mm. This ensures a repopulation with bivalves free of Perkinsus spp . It is also possible to treat selected bivalves for breeding. In the present invention, the expression "controlling" has the meaning of: treating, preventing or reducing the prevalence of Perkinsus spp . in bivalves.

Compositions according to the present invention, produced from powdered compounds dissolved in 0.05% DMSO are provided to the bivalve population to be controlled along with the conventional microalgae diet.

The exposure is performed in closed-circuit tanks for a period of 24h to 48h, preferably for a period of 48h hours with daily renewal of 10% of the water.

EXAMPLES

Example 1 . Preparation of 3-Chloro-l , 2-benzisothiazole-l , 1- dioxide

The experimental procedure used has been reported previously as described by Brigas et al . [Brigas AF, o ₂

^LC60 Fonseca CSC, Johnstone RAW. 2002. Preparation of 3- chloro-1 , 2-benzisothiazole 1,1-dioxide (pseudo-saccharyl chloride) . J. Chem. Research (S) . 299-300].

Starting from saccharin (56 mmol) and phosphorus pentachloride (66 mmol), heated at 200 °C. Colourless needles from ethanol (63% yield); m.p. 143-145 °C. IR v _{max (} cm ¹ ): 1724, 1654, 1603 (C=C), 1346 (S0 ₂₎ , 775 (Ar-H) and 692 (C-Cl); ^! H-NMR (400 MHz, CDCI ₃₎ : d 7.85 (4H, m, Ar-H) ppm. Found: C, 41.5%; H, 2.0%; N, 6.9%; calcd for C ₇ H _4N O ₂ SCI: C, 41.7%; H, 2.0%; N, 7.0%. MS (El), m/z 201 [M] ⁺ . Example 2 . Preparation of l-Phenyl-lH-tetrazole-5-one, LC133

O 5-Chloro-l-phenyl-tetrazole (1 eq) was added to a

HN ^/ N" ^Ph solution of sodium hydroxide (5M, 10 mL) . The

N=N

reaction mixture was stirred at room temperature for 24 h. The resulting solution was cooled to room temperature and acidified by addition of HC1 (aq) (10%; pH«l) . A precipitate was formed, filtered and washed with chloroform and hexane to give the product (90% yield) as a colourless powder; m.p. 97-99°C; ¹ H-NMR (400 MHz, CDCI3): d 7.57 (m, 2H) , 7.66 (s, 1H) , 7.70 (d, J = 6.5 Hz, 1H) ppm; MS (IE), m/ z : 162, 05 [M + H] ⁺ .

Example 3 . Preparation of l-Methyl-lH-tetrazole-5-amine, LC126I

A solution of sodium hydroxide (20%) was added dropwise to a suspension of 5-aminotetrazole monohydrate (120

mmol) in water (30 mL), with a drop of phenolphthalein . The mixture was stirred until complete dissolution of the suspended material. Dimethyl sulphate (110 mmol) was then added in small portions, keeping an alkaline medium through addition of aqueous sodium hydroxide. The final mixture was refluxed for 1 h, then cooled, and finally left in ice bath for 48h. Colourless needles of the desired compound were filtered and dried (51% yield); m.p. 220-221°C. ^! H-NMR (400 MHz, CDCI3) : d 4.15 (3H, s) ppm; MS (El), m/z 99 [M] ⁺ .

Example 4 . Preparation of 2-Methyl-2H-tetrazole-5-amine, LC126II

H N The filtrate from 1-methyl-lH-tetrazole-5-amine ,

Nl N LC126I synthesis was evaporated under reduced pressure

N-N

\ to afford a solid residue. Water (50 mL) was added, and the mixture was then extracted with diethyl ether (3 x 50 mL) . The organic extract was dried over anhydrous sodium sulfate, filtered, and the filtrate evaporated to afford colourless crystals. Recrystallization from diethyl ether gave the desired compound as colourless needles (25% yield); m.p. 104.5- 105.5°C. ^! H-NMR (400 MHz, CDC1 ₃ ) : d 3.32 (3H, s) ppm; MS (El), m/z 99 [M] ⁺ .

Example 5 . Preparation of Tert-butyl ( 4-aminobutyl ) carbamate, LC64

⁰ I A solution of di-tert-butyl dicarbonate (2.50 x

H _2N ^^ _N A ₀ " 10 ² mol) in 1,4-dioxane (100 mL) , under

H

LC64

stirring, was added by cannula, over 3 hours, to a stirring solution of 1 , 4-diaminobutane (1.40 x 10 ^_1 mol) in 1,4-dioxane (100 mL) . The final reaction mixture was stirred at room temperature for 20 h and then concentrated under reduced pressure. Water was added to precipitate the formed conjugate. The aqueous residue was extracted with DCM (2 x 30 mL) . The combined organic extracts were dried over anhydrous MgSCq, filtered, and the filtrate was evaporated to dryness under reduced pressure to give a clear oil (97 % yield) . ¹ H- NMR (400 MHz, CDCI ₃ ) : d 1.42 (s, 9H) , 1.48 (d, J = 7.0 Hz, 4H) , 2.71 (s, 2H) , 3.10 (s, 2H) ppm; MS (MALDI-TOF ) , m/z 189,17 [M + H ] ⁺ .

Example 6 . Preparation of 0-methyl-2-adamantanone oxime, LC29. To a solution of 2-adamantanone (30 mmol) in methanol

(30 mL) were added pyridine (55.6 mmol) and

methoxylamine hydrochloride (45.0 mmol) . The reaction mixture was stirred at room temperature for 48 h. The final mixture was concentrated and then diluted with DCM (50 mL) and water (50 mL) . The organic layer was separated and the aqueous layer was extracted with DCM (30 mL) . The combined organic extracts were washed with aqueous HC1 (1 M; 30 mL x2), then with saturated aqueous NaCl (30 mL) . The final organic extract was dried over MgSCq, filtered and concentrated under reduced pressure to give O-methyl-2-adamantanone oxime (89% yield) as a colourless solid (m.p. 69-70°C) . ¹ H-NMR (400 MHz, CDCI3) : d 1.78-1.97 (12H, m) , 2.53 (1H, s), 3.45 (1H, s), 3.81 (3H, s) ppm; MS (MALDI-TOF ) , m/z 180.02 [M + H] ⁺ .

Example 7 . General procedure for the preparation of Adamantane- 2-spiro-3 ' -8 ' -oxo-1 ' , 2 ' , 4 ' -trioxaspiro [ 4 , 5 ] decane LC50 and Adamantane-2-spiro-3 ' -8 ' -ethoxycarbony1-1 ' , 2 ' , 4 ' - trioxaspiro [ 4 , 5 ] decane LC67.

Trioxolanes were prepared by coupling O-methyl-2-adamantanone oxime, LC29 with a cyclohexanone derivative, through ozonolysis as described by Vennerstrom.

Ozone, produced with an ozone generator Sander Labor- Ozonizator 301.7 (0.5 L/min O2, 140 V), was passed through a solution of dichloromethane at -78°C and flushed into a solution of O-methyl ketone oxime and a ketone, in pentane/dichloromethane (6:4) at 0°C. After completion, the solution was flushed with nitrogen for 5 min and concentrated under reduced pressure at room temperature to give a crude material that was purified by column chromatography.

Example 8 . Preparation of Adamantane-2-spiro-3'-8'-oxo- l',2',4'-trioxaspiro[4,5] decane, LC50.

A solution of O-methyl 2-adamantanone oxime, LC29 (8.4 mmol) and 1 , 4-cyclohexanedione (11 mmol) in pentane (60 mL) and dichloromethane (40 mL) was treated with ozone as described in Example 7. The crude product was purified by column chromatography (silica gel; ethyl acetate/n-hexane 1/9) to give product LC50 (42% yield) as a colourless solid; m.p. 127-128°C. ¹ H-NMR (400 MHz, CDCI3) : d 1.69-2.02 (m, 14H), 2.14 (t, J = 7,1 Hz, 4H) , 2.51 (t, , J = 7,1 Hz, 4H ) ppm; ¹³ C-NMR (100 MHz, CDCI3) : 25.9, 26.31, 31.09, 32.59, 34.25, 35.70, 36.18, 37.35, 106.46, 111.95, 208.90 ppm; MS (El) , m/z 278.9 [M + H] ⁺ .

Example 9 . Preparation of Adamantane-2-spiro-3 ' -8 ' - ethoxycarbony1-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane , LC67.

A solution of O-methyl 2-adamantanone oxime, LC29 (20 mmol) and ethyl 4- oxocyclohexanecarboxylate (20 mmol), in pentane (60 mL) and DCM (40 mL), was treated with ozone, according to the described in Example 7. The crude product was purified by column chromatography (silica gel, ethyl acetate/n-hexane 1/9) to afford trioxolane LC67 as a colourless oil (46% yield) . ¹ H-NMR (400 MHz, CDCI3 ) : d 1.26 (t, J = 7,2 Hz, 3H) , 1.70-1.76 (m

11H) , 1.92-2.03 (m, 12H, ) , 2.33 (m, 1H) , 4.15 (dd, J = 7.1 Hz, J = 7.0 Hz, 2H) ppm; MS (MALDI-TOF ) , m/z 337.34 [M + H] ⁺ .

Example 10 . Preparation of Adamantane-2-spiro-3 ' - 8 ' - hydroxymethyl-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane , LC93.

A solution of adamantane-2-spiro-3 ' -8 ' - ethoxycarbony1-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane ,

LC67 (11.3 mmol), lithium borohydride (11.3 mmol, 2M in THF) and lithium triethylborohydride (1.13 mmol, 1M in THF) in ether (15 mL) was stirred overnight, at room temperature. The reaction mixture was diluted with ether (5 mL) , washed with aqueous NaOH (3M; 2 x 10 mL), brine and water (2 x 10 mL) . The organic extract was dried over MgSCq, filtered, and concentrated under reduced pressure to give product LC93 (90% yield) as a yellow crystalline solid; m.p. 99-101°C. ¹ H-NMR (400 MHz, CDCI3 ) : d 1.25 (m, 2H) , 1.51-2.08 (m, 21H), 3.46 (t, J = 7.1 Hz, 2H) ppm; MS (MALDI-TOF), m/z 318.30 [M + Na] ⁺ .

Example 11. Preparation of Adamantane-2-spiro-3 ' -8 ' - phtalimidomethyl-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane , LC94.

A solution of adamantane-2-spiro-3 ' -8 ' - hydroxymethyl-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane ,

LC93 (2.8 g, 9.52 mmol) in dry THF (25 mL) was cooled to 0 °C. Ph ₃ P (3.5 g, 1.33 mmol), phthalimide (1.55 g, 10.5 mmol) and DIAD (2.6 mL, 1.33 mmol) were gradually added. The mixture was stirred at room temperature for 24 hours. The solvent was then evaporated to dryness and the crude product was purified by column chromatography (silica gel, ethyl acetate/n-hexane 1/9) to give product LC94 (80% yield) as a white powder (m.p. 149- 151 °C) . ^X H NMR (300 MHz, CDC1 ₃ ) : d 1.30-1.34 (m, 2H) , 1.51- 2.08 (m, 21H), 3.55 (d, J = 6.7 Hz, 2H) , 7.71 (m, 2H) , 7.84 (m, 2H) ppm; MS (MALDI-TOF ) , m/z 462.19 [M + K] ⁺ .

Example 12. Preparation of Adamantane-2-spiro-3 ' -8 ' - ( aminomethyl ) -1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane , LC95.

A solution of adamantane-2-spiro-3 ' -8 ' - phtalimidomethyl-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane , LC94 (3.20 g, 7.56 mmol) and hydrazine monohydrate (1.45 g, 45.4 mmol) in chloroform and methanol (7:3, 50 mL total) was heated at 60 °C for 35 h. The reaction mixture was cooled to room temperature and filtered to remove solid by-products. The filtrate was washed with water (50 mL) and brine (50 mL) , dried over MgSCg, filtered, and concentrated to give product LC95 (77% yield) as light yellow oil. ¹ H NMR (400 MHz, CDCI ₃ ) : d 1.14-1.33 (m, 3H) , 1.68-1.96 (m, 22H) , 2.54 (d, J = 7.0 Hz,

2H) ppm; MS (MALDI-TOF ) , m/z 293.20 [M + H] ⁺ .

Example 13. Preparation of Adamantane-2-spiro-3 ' -8 ' - hydroxymethyl-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane-1 , 2- benzisothiazole-1 , 1-dioxide, LC129, as described by Lobo et al . [Lobo L, Cabral LIL, Sena MI, et al . 2018. New endoperoxides highly active in vivo and in vitro against artemisinin-resistant Plasmodium falciparum. Malaria Journal. 17:1-11. doi :10.1186/sl2936-018-2281-x].

3-Chloro-l, 2-benzisothiazole-l , 1-dioxide, LC60 (4.08 mmol) was added to a solution of

adamantane-2-spiro-3 ' -8 ' -hydroxymethyl-1 ' , 2 ' , 4 ' - trioxaspiro [ 4 , 5 ] decane , LC93 (3.4 mmol) in dry toluene (30 mL) . The solution was stirred at 45°C for 15 minutes, followed by addition of triethylamine (6.8 mmol) until disappearance of all of the starting material. The precipitate of triethylamine hydrochloride was filtered off and the filtrate was evaporated to give a yellow crystalline solid, which was recrystallized from ethanol (50% yield); m.p. 150-151°C. ¹ H-NMR (400 MHz, CDCI3) : d 1.35-1.38 (m, 2H) , 1.62-1.96 (m, 21H), 4.36 (d, J =

7.2 Hz, 2H) , 7.64 (d, J = 7.1 Hz, 1H) , 7.69 (d, J = 7.0 Hz, 2H) , 7.82 (d, J = 7.2 Hz, 1H) ppm; ¹³ C-NMR (100 MHz, CDCI3 ) : 17.60, 26.87, 29.23, 32.36, 35.39, 37.95, 39.14, 66.39, 108.22, 117.62, 123.30, 127.03, 133.44, 134.12, 143.61, 169.23 ppm;

Found: C, 62.85%; H, 6.44%; N, 2.99%; calculated for C24H29 O6S: C, 62.73%; H, 6.36%; N, 3.05%; MS (El), m/z 482.25 [M + Na] ⁺ .

Example 14. Preparation of Adamantane-2-spiro-3 ' -8 ' - aminomethyl-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane-1 , 2- benz isothiazole-1 , 1-dioxide , LC130. 3-Chloro-l, 2-benzisothiazole-l , 1-dioxide, LC60 (0.71 g, 3.53 mmol) was added to solution of adamantane-2-spiro-3 ' -8 ' - ( aminomethyl ) -1 ' , 2 ' , 4 ' - trioxaspiro [ 4 , 5 ] decane , LC95 (1 g, 3.41 mmol) in dry THF (20 mL) . The solution was stirred at 60 °C until all of the starting material had disappeared. The reaction mixture was cooled to r.t. and evaporated. Recrystallization from ethanol gave the desired compound as yellow crystalline solid (61% yield); m.p. 152-154 °C. ^X H RMN (CDC1 ₃ ): d 1.21-1.30 (m, 2H) , 1.65-2.0 (m,

22H), 3.43 (s, 2H), 7.68 (d, J = 7.1 Hz, 1H) , 7.75 (dd, J =

7.0 Hz, 2H) , 7.89 (d, J = 7.4 Hz, 1H) ppm; MS (MALDI-TOF ) , m/z 481.16 [M + Na] ⁺ .

Example 15. Preparation of Adamantane-2-spiro-3 ' -8 ' - hydroxymethyl-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane-1-phenyl-1H- tetrazole, LC132. To a solution of adamantane-2-spiro-3 ' -8 ' - hydroxymethyl-1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane ,

LC93 (1.83 mmol) in THF (10 mL) was added mesyl chloride (2.0 mmol) and triethylamine (3.65 mmol) . The solution was stirred at room temperature for 3 hours. Then a solution of 5-chloro-l-phenyl-tetrazole (2.75 mmol) in THF (10 mL) was added dropwise to the stirred suspension, over 30 minutes. The mixture was stirred at 65 °C for 24 hours. Excess solvent was then removed. Recrystallization from ethanol gave LC132 as a white solid (32% yield) . m.p. 82-84 °C. ¹ H-NMR (400 MHz, CDCI3) 7.73 (d, J = 7.1 Hz, 2H) , 7.57 (t, J = 7.2 Hz, 2H) , 7.48 (t,

J = 7.1 Hz, 1H) , 4.50 (d, J = 7.4 Hz, 2H) , 2.01 (d, J = 7.1 Hz, 7H) , 1.71-1.86 (m, 13H) , 1.39-1.46 (m, 2H) , 1.27 (s, 1H) ppm; MS (MALDI-TOF), m/z 477.11 [M + K] ⁺ . Example 16. General procedure for the preparation of Adamantane-2-spiro-3 '-8 ' - [ ( ( tert-butyl-4- aminobutyl ) carbamate ) amino ] -1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane , LC135 and Adamantane-2-spiro-3 ' -8 ' [ ( aminotetrazole ) amino ] - 1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane, LC136.

The required amine ( tert-butyl ( 4-aminobutyl ) carbamate , LC64 or 5-aminotetrazole ) (3.4 mmol) was added to a solution of compound adamantane-2-spiro-3 ^, -8 ^, -oxo-l ^, ,2 ^, ,4'- trioxaspiro [ 4 , 5 ] decane , LC50 (3.4 mmol) in anhydrous 1,2- dichloroethane (20 mL) and acetic acid (3.4 mmol) . The mixture was allowed to stir at room temperature for 30 minutes, followed by addition of sodium triacetoxyborohydride (8.5 mmol) . After stirring at room temperature for 16 hours, the final reaction mixture was washed with aqueous NaOH (5M; 2 x

10 mL) and dichloromethane (2 x 20 mL) . The organic extract was dried over MgSCq, filtered, and the solvent evaporated. The crude product was purified by column chromatography (silica gel, ethyl acetate/n-hexane 3/7).

Example 17. Preparation of Adamantane-2-spiro-3 ' -8 '-[(( tert- butyl-4-aminobutyl ) carbamate ) amino ] -1 ' , 2 ' , 4 ' - trioxaspiro [ 4 , 5 ] decane, LC135.

Prepared according to general procedure 2 to give LC135 as a brown oil (83% yield). ^! H-NMR (400 MHz, CDC1 ₃ ) : d 0.83 (m, 3H) , 1.25 (m, 3H) , 1.36 (m, 9H), 1.61 (m, 12H) , 1.91 (m, 8H) , 2.66 (m, 3H) , 3.05 (s, 2H) ppm; MS (MALDI-TOF ) , m/z 451,31 [M + H] ⁺ .

Example 18. Preparation of Adamantane-2-spiro-3 ' - 8 ' [ ( aminotetrazole ) amino ] -1 ' , 2 ' , 4'-trioxaspiro [4, 5] decane , LC136. Prepared according to general procedure 2 to give LC136 as a white solid (80% yield); m.p.

98-100 °C . ^! H-NMR (400 MHz, CDC1 ₃ ) : d 1.24-1.33 (m, 2H) , 1.69- 1.72 (m, 1 OH ) , 1.90-2.05 (m, 10H), 2.66 (m, 1H) ppm; ¹³ C-NMR (100 MHz, CDCI3 ) : 21.05, 27.07, 28.42, 33.67, 36.78, 37.64, 58.57, 117.87, 127.78, 156.15 ppm; MS (MALDI-TOF ) , m/z 347.31 [M + H] ⁺ .

Example 19. General Procedure for the preparation of adamantane-2-spiro-3 '-l',2',4',5' -tetraoxane-6 ' -spiro-1 " - cyclohexane LC137, adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' -spiro-l"-cyclohexane-4"-ethyl carboxylate

LC138 , adamantane-2-spiro-3 ^, -l ^, ,2 ^, ,4 ^, ,5' -tetraoxane-6 ' -spiro- l"-piperidine LC139 and Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 '-spiro-l"-cyclohexanone LC140.

To a stirring solution of the appropriate ketone (cyclohexanone, A and 4-piperidinone, B) , 2-adamantanone or LC133 (5 mmol) in acetonitrile (5.5 mL) and formic acid (3.7 mL) at 0°C was added 50% aq. hydrogen peroxide (1.9 ml) . The solution was allowed to warm to room temperature and stirred for 45 min. The solution was diluted with dichloromethane (100 ml) and washed with water (100 ml) . The organic phase was dried over Na2SC>4, filtered and concentrated to give the corresponding gem-dihydroperoxide. A solution of this intermediate in dichloromethane (5 mL) was added to a stirring solution of 2-adamantanone or ketone (Ethyl 4- oxocyclohexanecarboxylate , C and 1,4-cyclohexanone, D) (7.5 mmol) and 54% ethereal solution of HBF4 (0.1 mL) in dichloromethane (5 mL) at 0°C. The mixture was allowed to warm to room temperature and stirred for 4h. The organic layer was washed with a saturated solution of NaHCCy and dried over MgSCq and the solvent removed. The resulting residue was purified by flash column chromatography to give the desired dispiro- 1,2,4, 5-tetraoxane .

Example 20. Preparation of Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' -spiro-1 " -cyclohexane , LC137 as described by Ghorai et al . [Ghorai P & Dussault PH. 2009. Broadly Applicable Synthesis of 1 , 2 , 4 , 5-Tetraoxanes . Org. Lett. 11, 213-216]. i/ T'o- ⁰ '— prepared according to the described in example 19 to give LC137 as a white solid (32% yield); m.p. 57-59°C. ¹ H- NMR (400 MHz, CDCI3 ) : d 1.46-1.99 (m, 21H), 2.27 (s, 3H) ppm; ¹³ C-NMR (100 MHz, CDCI3) : 25.42, 27.14, 33.23, 37.04, 108.16, 110.13 ppm; Found: C, 68.49%; H, 8.73%; inferred for C16H24O4:

C, 68.54%; H, 8.63%. MS (MALDI-TOF ) , m/z 318.33 [M + K] ⁺ .

Example 21. Preparation of Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' -spiro-l"-cyclohexane-4"-ethyl carboxylate, LC138.

Prepared according to the described in example 19 to give LC138 as a white solid (58% yield) .

^! H-NMR (400 MHz, CDCI3 ) : d 1.26 (m, 3H) , 1.57- 1.96 (m, 22H) , 2.40 (m, 1H) , 4.14 (m, 2H) ppm; ¹³ C-NMR (100 MHz, CDCI3) : 14.23, 27.07, 33.15, 36.96, 41.69, 60.42, 107.94, 110.52, 174.62 ppm; MS (MALDI-TOF), m/z 393.47 [M + K] ⁺ .

Example 22. Preparation of Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' -spiro-l"-piperidine, LC139. Prepared according to the described in example 19 to give LC139 as a yellow solid (51% yield); m.p. 65-67 ⁰ C . ^! H-NMR (400 MHz, CDCI3) : d 1.26 (s, 1H) , 1.75 (d, J = 7.3 Hz, 6H) , 2.04 (m, 14H), 3.08 (t, J = 7.1 Hz, 2H) ppm; ¹³ C- NMR (100 MHz, CDCI3 ) : 27.80, 29.67, 32.67, 33.81, 36.68, 107.39, 109.46 ppm; MS (MALDI-TOF), m/z 282.29 [M + H] ⁺ .

Example 23. Preparation of Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' -spiro-l"-cyclohexanone, LC140, as described by Marti et al . [Marti F, Chadwick J, Amewu RK, et al . 2011. Second generation analogues of RKA182: synthetic tetraoxanes with outstanding in vitro and in vivo antimalarial activities. Med. Chem. Commun. 2, 661-665. doi: 10.1039/clmd00102g] Prepared according to the described in example 19 to give LC140 as a white solid (51% yield) .

!H-NMR (400 MHz, CDCI3) : d 1.57-1.72 (m, 9H) ,

1.89-2.07 (m, 8H) , 2.47 (m, 5H) , 2.71 (s, 1H) , 3.18 (s, 1H) ppm; ¹³ C-NMR (100 MHz, CDCI3 ) : 27.09, 29.71, 30.20, 31.44,

33.14, 34.26, 35.67, 38.88, 38.75, 106.60, 111.01, 209.30 ppm; MS (MALDI-TOF ) , m/z 318.29 [M + Na] ⁺ .

Example 24. Preparation of Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' -spiro-l"-cyclohexane-4"-hydroxymethyl , LC146.

A solution of adamantane-2-spiro-3 ' - 1 ' , 2 ' , 4 ' , 5 ' -tetraoxane-6 ' -spiro-1 "-cyclohexane-

4"-ethyl carboxylate, LC138 (11.3 mmol), lithium borohydride (11.3 mmol, 2M in THF) and lithium triethylborohydride (1.13 mmol, 1M in THF) in ether (15 mL) was stirred overnight, at rt . The reaction mixture was diluted with ether (5 mL), washed with aqueous NaOH (3M; 2 x 10 mL), brine and water (2 x 10 mL) . The organic extract was dried over MgSCq, filtered, and the solvent removed. The crude product was purified by column chromatography (silica gel, ethyl acetate/n-hexane 2/8) to give product LC146 as a white solid (36% yield) . m.p. 175-177°C. ¹ H-NMR (400 MHz, CDCI ₃ ) : d 1.19 (m, 2H), 1.64 (s, 6H) , 1.69-1.93 (m, 13H) , 2.14 (d, J = 6.7 Hz, 2H) , 3.47 (s, 2H) ppm; ¹³ C-NMR (100 MHz, CDCI3) : 26.57, 26.78, 27.14, 33.64, 34.91, 34.99, 36.59, 36.98, 39.03, 67.72, 108.68, 111.59 ppm; MS (MALDI-TOF ) , m/z 309.35 [M]-.

Example 25. Preparation of Adamantane-2-spiro-3 ^, -l',2',4',5'- tetraoxane-6 ' -spiro-1 "-cyclohexane-4"-aminomethy1 , LC165. solution of adamantane-2-spiro-3 ' - 1 ' , 2 ' , 4 ' , 5 ' -tetraoxane-6 ' -spiro-1

cyclohexane-4"-phtalimidomethyl , LC159 (7.56 mmol) and hydrazine monohydrate (45.4 mmol) in chloroform and methanol (7:3, 50 mL total) was heated at 60°C for 35 h. The reaction mixture was cooled to room temperature and filtered to remove solid by-products. The filtrate was washed with water (50 mL) and brine (50 mL), dried over MgSCg, filtered, and concentrated to give product LC165 (45% yield) as light yellow oil. ^! H-NMR (400 MHz, CDC1 ₃ ) : d 1.15-1.38 (m, 2H), 1.62-1.96

(m, 22H) , 2.66 (d, J = 7.3 Hz, 2H) ppm; ¹³ C-NMR (100 MHz,

CDCI3) : 18.63, 26,65, 28.94, 33.40, 33.93, 36.44, 41.53, 46.76, 108.16, 110.13 ppm; MS (El), m/z 310.36[M + H] ⁺ .

Example 26. Preparation of Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' spiro-l"-cyclohexane-4"-carboxylic acid, LC153. To a solution of adamantane-2-spiro-3 ' -

1 ' , 2 ' , 4 ' , 5 ' -tetraoxane-6 ' -spiro-1

cyclohexane-4"-ethyl carboxylate, LC138 (4 mmol) in methanol

(15 mL) was added a solution of potassium hydroxide (20 mmol) in water (6 mL) . The mixture was refluxed for 6 hours. Then the solution was allowed to cool to room temperature and concentrated under reduced pressure. The crude was taken up in water (50 ml) and washed with dichloromethane (30 ml) . The aqueous layer was acidified to pH 1 with concentrated hydrochloric acid and then extracted with dichloromethane (3 x 40 ml) . The combined organic phases were washed with brine (30 ml), dried over Na2SC>4, filtered and concentrated under reduced pressure to give the pure compound as a white solid (80% yield), m.p. 179-182 °C. ^! H-NMR (400 MHz, CDC1 ₃ ) : d 1.18- 1.85 (m, 2 OH ) , 2.29-2.30 (m, 1H) , 2.90 (brs, 1H) , 3.17 (brs, 1H) ppm; ¹³ C-NMR (100 MHz, CDCI3 ) : 25.05, 26.06, 27.24, 32.97, 34.58, 35.04, 36.57, 36.96, 41.17, 106.95, 111.73, 181.13 ppm; MS (El), m/z 323.29 [M - H]-.

Example 27. General Procedure for the preparation of adamantane-2-spiro-3 ' -1' , 2' , ' , b' -tetraoxane-6 ' spiro-1"- cyclohexane-4"- [ ( ( tert-butyl-4-aminobutyl ) carbamate) amino] LC157 and Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' -tetraoxane- 6 ' spiro-l"-cyclohexane-4"- [( aminotetrazole ) amino ] LC163.

To a solution of adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' -tetraoxane- 6 ' -spiro-l"-cyclohexanone, LC140 (3.4 mmol) in anhydrous 1,2- dichloroethane (20 mL) was added amino compounds (3.74 mmol) and acetic acid (3.4 mmol) . The mixture was allowed to stir at room temperature for 30 minutes followed by addition of sodium triacetoxyborohydride (8.5 mmol) . After stirring at room temperature for 16 hours, the reaction mixture was washed with aqueous NaOH (5M; 2 x 10 mL) and dichloromethane (2 x 20 mL) . The organic extract was dried over MgSCq, filtered, and the solvent removed. The crude product was purified by column chromatography (silica gel, ethyl acetate/n-hexane 3/7) to give product .

Example 28. Preparation of Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' spiro-l"-cyclohexane-4"- [ ( ( tert-butyl-4- aminobutyl ) carbamate ) amino ] , LC157. Prepared according to general procedure of example 27 to give LC157 as yellow oil

(32% yield). ^! H-NMR (400 MHz, CDCI3 ) : d 0.93 (m, 3H), 1.34 (d, J = 6.9 Hz, 3H), 1.47 (s, 9H) , 1.49-1.76 (m, 12H) , 2.06 (m, 8H) , 2.86 (t, J = 7.0 Hz, 3H) , 3.15 (s, 2H) ppm; ¹³ C-NMR (100 MHz, CDCI3) : 22.53, 23.42, 26.55, 27.37, 28.11, 30.01, 32.02, 34.31, 36.05, 38.21, 47.61, 60.53, 79.49, 108.90, 111.01, 156.14 ppm; MS (MALDI-TOF ) , m/z 467,32 [M + H] ⁺ .

Example 29. Preparation of Adamantane-2-spiro-3 ' -1 ' , 2 ' , 4 ' , 5 ' - tetraoxane-6 ' spiro-1 "-cyclohexane-4"- [( aminotetrazole ) amino ] , LC163.

Prepared according to general procedure of example 27 to give LC163 as a white solid (95%

yield); m.p. 142-144°C. ^! H-NMR (400 MHz, CDC1 ₃ ) : d 1.15-1.22 (m, 2H) , 1.60-1.70 (m, 10H), 1.80-2.05 (m, 10H),

2.6 (m, 1H) ppm; ¹³ C-NMR (100 MHz, CDCI3) : 20.05, 26.06, 27.42, 29.67, 32.78, 33.64, 59.57, 106.44, 111.91 ppm; MS (MALDI-

TOF) , m/z 363.43 [M] .

Example 30. Preparation of Adamantane-2-spiro-3 -1 , 2 , 4 ' , 5 ' - tetraoxane-6 ' spiro-l"-cyclohexane-4"- [ ( 2-methyl-2H- tetrazole ) aminomethyl ] , LC179 To a solution of adamantane-2-spiro-3 ' - 1 ' , 2 ' , 4 ' , 5 ' -tetraoxane-6 ' -spiro-1"-

cyclohexane-4"-hydroxymethyl , LC146 (1.83 mmol) in THF (10 mL) was added mesyl chloride (2.0 mmol) and triethylamine (3.65 mmol) . The solution was stirred at room temperature for 3 hours. Then a solution of 2-methyl-2H- tetrazole-5-amine, LC126II (2.75 mmol) in THF (10 mL) was added dropwise to the stirred suspension over 30 minutes. The mixture was stirred at 65°C for 24 hours. Excess solvent was then removed. The crude product was purified by column chromatography (silica gel, ethyl acetate/n-hexane 3/7) to give LC179 as a white solid (25% yield) . m.p. 134-136°C. ¹ H- NMR (400 MHz, CDC1 ₃ ) : d 1.06-1.09 (m, 2H) , 1.17-1.24 (m, 10H), 1.62-1.64 (m, 3H), 1.73-1.85 (m, 6H) , 2.04 (d, J = 7.1 Hz, 2H) , 2.95 (d, J = 7.4 Hz, 2H ) , 3.85 (d, J = 6.5 Hz, 3H ) ppm; ¹³ C-NMR (100 MHz, CDCI3) : 18.93, 26.25, 28.44, 33.00, 33.70, 36.53, 38.56, 41.53, 54.96, 108.16, 110.13 ppm; MS (El), m/z 391.31 [M] ⁺ .