Synthesis and Biological Evaluation of Phosphate Prodrugs of 4-Phospho-d -erythronohydroxamic Acid, an Inhibitor of 6-Phosphogluconate Dehydrogenase

We have previously reported the discovery of potent and selective inhibitors of 6-phosphogluconate dehydrogenase, the third enzyme of the phosphate pentose pathway, from Trypanosoma brucei, the causative organism of human African trypanosomiasis. These inhibitors were charged phosphate derivatives with restricted capacity to enter cells. Herein, we report the synthesis of five different classes of prodrugs: phosphoramidate; bis-S-acyl thioethyl esters (bis-SATE); bis-pivaloxymethyl (bis-POM); CycloSaligenyl; and phenyl, S-acyl thioethyl mixed phosphate esters (mix-SATE). Prodrugs were studied for stability and activity against the intact parasites. Most prodrugs caused inhibition of the growth of the parasites. The activity of the prodrugs against the parasites appeared to be related to their stability in aqueous buffer.


Introduction
Human African trypanosomiasis (HAT), also known as sleeping sickness, is a life threatening disease that affects many people in sub-Saharan Africa. [1] It is caused by the protozoan Trypanosoma brucei, of which two subspecies (T. b. rhodesiense and T. b. gambiense) [2] are pathogenic to humans. These different subspecies give rise to different clinical symptoms. Three of the four drugs used against this disease were developed more than 50 years ago; the fourth one and the most recent, eflornithine (d,l-a-difluoromethylornithine, DFMO) is active only against T. b. gambiense. As a result of increasing resistance and the side effects associated with the available drugs there is an urgent need to develop new treatments to fight this disease. [2] The bloodstream form of T. brucei spp are entirely dependent on glycolysis for production of ATP; thus the parasite is susceptible to inhibition of glycolysis, and some of the enzymes involved in the metabolism of glucose are potential targets for the development of new treatments. [3] Glucose is also metabolised by the pentose phosphate pathway (PPP), the third enzyme of which, 6-phosphogluconate dehydrogenase (6-PGDH), has been shown to be essential for the viability of T. brucei. [4] The enzyme catalyses the conversion of 6-phosphogluconate (6PG) to ribulose 5-phosphate (Ru5P) with concomitant reduction of one mole of NADP + to NADPH. Inhibition of the enzyme will diminish production of NADPH, thus increasing the parasite's vulnerability to oxidative stress. Moreover, the levels of ribose 5-phosphate needed for nucleotide biosynthesis will decline and 6PG will accumulate in the cell. 6PG is known to be an inhibitor of 6-phosphoglucose isomerase, the enzyme which converts glucose 6-phosphate to fructose 6phosphate during glycolysis. Inhibition of this enzyme conse-quently should lead to more glucose 6-phosphate entering the PPP potentially creating a self-feeding loop with lethal consequences for the parasite. [5] Previous work from our group, has identified a series of potent and selective inhibitors of T. brucei 6-PGDH [6,7] ( Figure 1). Unfortunately these compounds were inactive in vitro against the intact parasite probably because of poor uptake into the parasites. The IC 50 values of these compounds against T. b. rhodesiense were 229 mm, > 332 mm, > 389 mm for A, B, and C respectively. Low cellular penetration is found with drugs bearing phosphate or phosphonate groups, because of these groups being deprotonated at physiological pH. An increasing number of phosphate esters of pharmaceutical interest (mainly antiviral agents and signaling regulators) has en-We have previously reported the discovery of potent and selective inhibitors of 6-phosphogluconate dehydrogenase, the third enzyme of the phosphate pentose pathway, from Trypanosoma brucei, the causative organism of human African trypanosomiasis. These inhibitors were charged phosphate derivatives with restricted capacity to enter cells. Herein, we report the synthesis of five different classes of prodrugs: phosphoramidate; bis-S-acyl thioethyl esters (bis-SATE); bis-pivaloxymethyl (bis-POM); CycloSaligenyl; and phenyl, S-acyl thioethyl mixed phosphate esters (mix-SATE). Prodrugs were studied for stability and activity against the intact parasites. Most prodrugs caused inhibition of the growth of the parasites. The activity of the prodrugs against the parasites appeared to be related to their stability in aqueous buffer.
couraged the advancement of the prodrug approach for the delivery of such compounds into the target cells. [8,9] Several kinds of phosphate masking group have been developed. [8,[10][11][12][13] Different mechanisms then operate to release the parent drug inside the cell. These range from simple chemical hydrolysis [12] to a multienzymatic cleavage of the prodrugs by the action of several enzymes, mainly esterases. [10,[13][14][15] In this paper we discuss the conversion of the 6-PGDH inhibitor B into prodrugs to increase its activity against the T. brucei by enhancing uptake by passive permeation across the plasma membrane. Five different phosphate-masking groups (phosphoramidate, bis-S-acyl thioethyl esters, bis-pivaloxymethyl, Cy-cloSaligenyl and phenyl, S-acyl thioethyl mixed phosphate esters) have been produced.
The synthesised prodrugs were then evaluated for activity against the parasite T. brucei brucei. Their stability was also studied in phosphate-buffered saline (PBS) at 37 8C.

Chemistry
The retrosynthetic analysis of the target compounds is shown in Scheme 1. The synthesis of all five types of masked phosphate can be obtained from the same intermediate: the 2,3-Oisopropylidene erythrono hydroxamic acid 4, which can be coupled with different chloro phosphate diesters 5 or with phosphine-like derivatives 6.
The acidity of the hydroxamic group was found to be a problem in the synthesis of prodrugs of the 4-phosphate of the erythrono hydroxamic acid 1. To limit the possible side reactions, a suitable protecting group for the hydroxamic acid was studied. Indeed, attempts at phosphorylating the compound when the hydroxamate was unprotected were unsuccessful and led to complex mixtures. [7] Our first synthesis of the lead B was achieved by opening the 2,3-isopropylidene-d-erythronolactone 7 with O-benzyl hydroxylamine followed by phosphorylation with tribenzylphosphite and final cleavage of the benzyl groups by hydrogenolysis. [7] Unfortunately the benzyl protecting group was too stable to be used in the synthesis of prodrugs, and could not be removed in the presence of the masked phosphate groups. Therefore various other protecting groups for the hydroxamate were investigated. Attempts with tert-butyldimethylsilyl (TBDMS), trityl, THP, and polymer-supported benzyl were not successful; instability, low yields, or difficulties monitoring the reaction (in the case of PS-benzyl) were the main problems encountered.
In our search for an alternative protective groups we found that O-2,4-dimethoxybenzyl and O-4-methoxybenzyl hydroxylamines were successfully used by Barlaam et al. for the synthesis of hydroxamic acids. [16] These protected hydroxylamines can be readily prepared from the 4-methoxybenzyl alcohol 9 a and the 1,4-dimethoxy benzyl alcohol 9 b by Mitsunobu reaction with N-hydroxyl phthalimide, followed by removal of the phthalimide protecting group with N-methyl hydrazine, Scheme 2.  zole first and then with tert-butyl hydroperoxide to oxidize the P III to P V . The chlorophosphates 5 a-c reacted with N-methyl imidazole or triethylamine or DIPEA; following the procedures suggested in the literature.
As a final step, the cleavage of the dimethoxybenzyl group was achieved with 1-5 % TFA in DCM (Scheme 6). Using these very mild conditions, it was possible to obtain the target prodrugs 3 a-f with moderate to good yields. This marks the development of chemistry to cleave a protecting group subsequent to the masked phosphate (prodrug moiety) being introduced into the molecule.
Attempts at cleavage of the monomethoxy benzyl group were not successful, as the higher stability of this required more acidic conditions and longer reaction time (5 % TFA in DCM); under these conditions, decomposition of the starting prodrug was observed. An alternate method for removing a 4-methoxybenzyl group is with oxidative conditions (for example, DDQ). However, in our case, DDQ was not able to remove the 4-methoxybenzyl group and, consequently this protecting group was not further investigated.

Stability studies
The stability of the six prodrugs synthesised was evaluated in phosphate buffer saline at 37 8C by LC-MS and 31 P NMR spectroscopy. The prodrugs were dissolved in buffer, (DMSO was also added in case of poor aqueous solubility), incubated, and analysed hourly until total decomposition was observed.
In the LC-MS experiment the disappearance of the molecular ion for the starting prodrug was observed and the declining intensity of the ion current peak was plotted against time, producing the decomposition curve. The half-life of the prodrugs was determined as the time when the intensity of the starting peak was fallen to half of the starting value, Figure 2 and Table 1.
In the case of the p-nitro phosphoramidate 3 c it was also possible to detect one of the by-products produced by the decomposition of the prodrug in phosphate buffer. This was the corresponding phosphoramidate with the loss of the p-nitro phenyl group. Such results could be explained by the effect of the nitro group, which stabilizes the negative charge in the phenolate anion, thus making the hydrolysis of the group easier and therefore this phosphoramidate prodrug has a shorter half-life compared to the corresponding phosphoramidate with the simple phenyl ester 3 b.
Unfortunately in the other cases, although some new peaks, with lower molecular ions compared to the parent prodrugs were identified, a conclusive structure could not be attributed to them and further analysis is undergoing. 31 P NMR spectroscopy also showed the decomposition of the prodrug by the disappearance of the signal for the phosphate prodrugs with time courses comparable with those found by LC-MS. Figure 3 shows the 31 P NMR spectra for the case of the phosphoramidate 3 b. The two peaks at d = 3.3 and 3.1 ppm for the chiral phosphorous of the starting phosphoramidate 3 b are still present after 24 h incubation at 37 8C (bottom spectrum) and only one new little peak at d = 1.2 ppm is detected probably due to the 4-phospho-d-erythronohydroxamic acid C [7] (the main peak at 0.5 ppm is due to the phosphate buffer).

Biological evualuation
The prodrugs 3 a-f and some of the protected hydroxamic intermediates were assayed for in vitro activity against T. b. brucei (Bs427) and in a counter screen for cytotoxicity against a mammalian cell line (HEK 293T). The IC 50 values are presented Table 2. The compounds showed activity against the parasite. Whilst further work is required to prove that the killing is by inhibition of 6-PGDH, this result could indicate that the compounds are now able to permeate the cell-membrane, be converted from the prodrug to the active hydroxamate, and then kill the parasite by inhibiting 6-PGDH. Compounds 3 d, 3 a, and 3 b showed the highest activity against T. b. brucei in decreasing order of activity. Compounds 3 e and 3 c had moderate activities whereas 3 f showed no trypanocidal activity even at 100 mm. There seemed to be a correlation between stability of the compounds in aqueous buffer and in vitro activity.
Interestingly some of the masked hydroxamate analogues (24 and 27) also showed improved activity on T. brucei strains, which could indicate cleavage of the dimethoxybenzyl moiety under cellular conditions.
Finally none of the compounds tested showed appreciable cytotoxicity against the mammalian cell line HEK293T; indicat-ing good selectivity against trypanosomes. This would be predicted by the selectivities observed for compounds A-C, which were very selective for the parasite enzyme over the corresponding mammalian one.

Conclusions
We have developed a new procedure for the synthesis of several classes of phosphate prodrugs in the presence of other potentially interfering groups (that is, hydroxamic acid in our case). The use of the 2,4-dimethoxybenzyl protecting group allowed the introduction of the five masked phosphate groups at the penultimate step of the overall synthesis. The cleavage of the hydroxamate protecting group using very mild conditions (1-2 % TFA in DCM in 15 min) was compatible with all the masking groups allowing us to achieve chemoselectivity between the alcohol function and the hydroxamic moiety in the total synthesis.
The stability studies showed that some of the prodrugs have relatively short half-lives in aqueous phosphate buffer at 37 8C. Comparison of the measured half-lives with those reported by AzØma et al [21] for a series of enzyme-labile aldolase inhibitors containing masked phosphates and other data reported for both SATE [18,22] and phosphoramidate [23] nucleosides indicates that the prodrugs reported herein have shorter half-lives than reported for other compounds where these phosphate masking groups are used. This is presumably due to particular features of the structures of the compounds reported herein. It is possible that the hydroxamic acid or one of the other hydroxyl groups promotes hydrolysis.
Although the mechanism of action has yet to be proven, the activity against the parasites correlates with the stability studies, showing that the compounds with the longest half-lives (the phosphoramidate 3 b, the mixed, and the bis-SATE 3 a and 3 d) are the most active in vitro, whereas the least stable are the least active, indicating that their trypanotoxicity is limited by the fact that they could decompose prior to entry into the parasite cells. Future work to address issues of stability will be required before these compounds might be considered for antimicrobial use in vivo.
Biological evaluation shows that the prodrug approach has drastically improved the in vitro activity of the parent compound B. [7] This data also provides further (chemical) validation of 6-PGDH as a drug target, although additional work is required to establish that the cellular mode of action of these compounds is indeed specific inhibition of the enzyme.
In conclusion the chemical synthesis of five different classes of phosphate prodrugs has been achieved. All but one of these compounds show trypanotoxicity Experimental Section 1 H NMR, 13 C NMR, 31 P NMR, and 2D-NMR spectra were recorded either on a Bruker Avance DPX 300 spectrometer or on a Bruker Avance DPX 500 spectrometer. Chemical shifts (d) are expressed in ppm. Signal splitting patters are described as singlet (s), broad sin-     13 (13). Pivaloyl chloride (3.2 mL, 26 mmol) was added to a stirred solution of 2-mercaptoethanol (1.8 mL, 26 mmol) and triethylamine (3.6 mL, 26 mmol) in CH 2 Cl 2 , cooled at À78 8C. The mixture was stirred at À78 8C for 1 h, then allowed to warm to room temperature and stirred further for 1 h. Water (30 mL) was added, the organic layer was separated, and the aqueous phase extracted with CH 2 Cl 2 (3 20 mL). The combined organic extracts were dried over Na 2 SO 4 and concentrated in vacuo. The oily residue was purified by flash column chromatography (SiO 2 , hexane/EtOAc 90 %!75 %) to afford the title compound as colourless oil, 4.02 g, 95 %. R f 0.44 in (70 % EtOAc/hexane). 1    Diisopropylamino dichloro phosphine (22). A solution of diisopropylamine (10.5 mL, 75 mmol) in THF (30 mL) was added dropwise into a vigorously stirred solution of PCl 3 (3.25 mL, 32.5 mmol) in THF (30 mL) at À78 8C, under atmosphere of Argon. The white suspension was stirred at room temperature for 2 h. The hydrochloride salt was filtered off and washed with THF (15 mL). The filtrate was concentrated to a colourless oil, with the rotary evaporator, and was then purified by distillation under vacuum (76-78 8C, 5 mbar ca) avoiding any contact with air. The title compound was obtained as colourless liquid (3.46 g, 53 %) which solidifies at 4 8C.   (25 a). To a cooled (À78 8C) solution of 4 a (31 mg, 0.1 mmol) and 5 c (111 mg, 0.4 mmol) in dry THF (2 mL), NMI (0.048 mL, 0.6 mmol) was added dropwise with a syringe over 1 min. The reaction was stirred for 5 min at À78 8C, then 6 h at room temperature and it was kept for the weekend in the freezer. TLC still revealed presence of the starting materials and the mixture was stirred for further 8 h at room temperature. The solvent was removed under reduced pressure. The crude yellowish residue was taken in DCM (10 mL) and washed with HCl 0.1 m (2 10 mL). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The resulting colourless oil was purified by chromatography (SiO 2 , MeOH 0 %!2 % in DCM). Compound 25 a was obtained as a yellow oil, 44 mg, 79 %. 1  The reaction was at stirred room temperature for 48 h. The solution was washed with HCl 0.1 n (3 10 mL). The organic phase was dried over Na 2 SO 4 , concentrated under reduced pressure, and purified by chromatography. SiO 2 eluted with MeOH 0 %!0.1 % in Chloroform. The title compound was obtained as yellowish oil, slightly contaminated of starting alcohol 4 b, 60 mg (29 %) and was used for the next step without further purification. 1