Short and Efficient Synthesis of the Antituberculosis Agent Pretomanid from (R)-Glycidol

An efficient gram-scale synthesis of the antituberculosis agent pretomanid using straightforward chemistry, mild reaction conditions, and readily available starting materials is reported. Four different protecting groups on the glycidol moiety were investigated for their technical feasibility and ability to suppress side reactions. Starting from readily available protected (R)-glycidols and 2-bromo-4-nitro-1H-imidazole, pretomanid could be prepared in a linear three-step synthesis in up to 40% isolated yield. In contrast to most syntheses reported so far, deprotection and cyclization were performed in a one-pot fashion without any hazardous steps or starting materials.


INTRODUCTION
With an enormous infection prevalence of around one-quarter of the entire global human population, tuberculosis (TB), commonly caused by Mycobacterium tuberculosis, is one of the most significant health threats of our age.Although most of the infections remain latent, TB is still one of the leading causes of death through infection worldwide. 1Compared to other infectious diseases, TB currently has a high morbidity and mortality (in 2020, 10 million people contracted active TB and 1.8 million people died from the disease). 2,3Particularly in combination with the AIDS pandemic, it is still one of the major causes of death in Africa. 4,5retomanid (1) belongs to the class of nitroimidazoles which dates back to the 1960s and enjoys a revived attention in recent years.The antibiotic and antiprotozoal drug metronidazole (2), one of the earliest representatives of this drug class, is still in use as an important medication and is on the WHO's "List of Essential Medicines". 6It can be used as an antibiotic and as an antiprotozoal medication.As diseases caused by protozoans remain a considerable problem in low-income countries, nitroimidazoles like metronidazole (2) and megazol (3) are highly important medicines.Besides pretomanid (1), the related compound delamanid (4) belongs to antituberculosis agents that are highly effective against multiresistant strains (Figure 1). 7retomanid (1), first described in 1997 by Barry and Baker (Pathogenesis Corp.), 8 belongs to a novel class of antituberculosis agents. 9−24 The first route was reported by Barry and Baker (Pathogenesis Corp.) in 1997. 8Starting from the explosive 2,4-dinitroimidazole, pretomanid (1) was prepared in five steps and 17% overall yield.The synthesis also includes other potentially hazardous or undesirable conditions like the use of NaH/DMF, which can be problematic for scale-up in a technical process due to the associated explosion risk. 25In 2013, Read and Fairlamb 19 reported a modified version of the initial route.Their synthesis starts with the safe and readily available 2-bromo-4-nitroimidazole (7), which undergoes a nucleophilic substitution with a TBS-protected glycidol.This is followed by installation of the aryl moiety, cleavage of the protecting group, and final cyclization to 1.Over four steps, pretomanid (1) was obtained in 10% yield, and the sequence contained multiple chromatographic purifications.

Organic Process Research & Development
preemptive to reduce the potential migration of the protecting group, which results in the undesired regioisomer in the Obenzylation.Another key aspect of this work was to establish an easily applicable, technically feasible, and scalable synthesis without the need for chromatographic steps.

Preparation of 2-Bromo-4-nitro-1H-imidazole (7).
The published process for preparing the starting material 2bromo-4-nitroimidazoline (7) 26 revealed optimization potential with respect to process safety and isolated yield.The use of molecular bromine, difficult on a larger scale, could be replaced with the in situ formation of bromine from hydrobromic acid and hydrogen peroxide.
The intermediate 2,5-dibromo-4-nitroimidazole does not require isolation but could be debrominated in a single pot to yield 7 in moderate yields (Scheme 3A).However, on scaling this one-pot procedure, a decrease in product purity was observed due to intractable salt contamination, so the final procedure run on kilogram scale involved isolation of 7b and debromination with NaI and TFA to yield 2-bromo-4nitroimidazoline (7) in good yields and very high purity (Scheme 3B).Although the conditions described herein have been run on a 1−2 kg scale, the authors wish to point out that further safety studies will be required to scale this reaction further. 27.2.Preparation of Protected (R)-Glycidols.Preparation of the protected (R)-glycidol derivatives 16−20 was performed according to literature procedures (Table 1).Glycidols 16, 17, and 19 were purified by distillation, whereas compounds 18 and 20 were used for the next step without prior purification.

Alkylation of 2-Bromo-4-nitro-1H-imidazole (7).
The alkylation of imidazole 7 using the p-anisoyl-protected PMBz-(R)-glycidol 16 was carried out in toluene at slightly elevated temperatures using DIPEA as a base (Table 2, entry 1).The desired product 21 precipitates during the reaction, causing a beige suspension after 48 h.Filtration and washing with toluene were insufficient as a significant amount of product was lost in the mother liquor.Therefore, toluene was removed prior to redissolving the residue in ethyl acetate to obtain a homogeneous solution.HPLC data from this solution revealed 99% conversion of imidazole 7 to produce 79% of the desired isomer 21 (HPLC, 315 nm).Observed side products were assumed to be the N′-and O-regioisomers as well as cyclized compounds on the basis of HPLC-MS data (see the Supporting Information for more details).After washing the ethyl acetate phase with aqueous HCl and NaHCO 3 solution, crude 21 was obtained as a yellowish-orange solid showing a purity of 83% (HPLC, 315 nm).Purification attempts by precipitation from ethyl acetate/n-heptane only led to a minor increase in purity (86%).Due to a loss of product, and in order to achieve a high overall yield, crude 21 was used for further transformations.
When using Bz-glycidol 17 (Table 2, entry 2) for the alkylation, similar results in terms of conversion were observed.

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In contrast to the crystalline PMBz-derivative 21, crude imidazole 22 was however isolated as a viscous oil (HPLC purity 81%, 315 nm).Adding MTBE (methyl tert-butyl ether) to the solution of crude 22 in ethyl acetate led to precipitation of contaminants which increased the purity of imidazole 22 remaining in the organic layer to 87% (HPLC, 315 nm).
Trityl-glycidol 18 (Table 2, entry 3) was found to be less reactive in the alkylation of imidazole 7. Thus, the temperature had to be increased to 70 °C, which gave a 98% consumption of 18 (81% conversion to 23, HPLC, 315 nm) after 46 h.Crude imidazole 23 was obtained as a brownish solid and was used in the next step without further purification.
Crude TBS-protected nitroimidazole 8 (Table 2, entry 4) was obtained as an orange viscous oil under similar conditions as the Bz-derivative.In contrast to the other protecting groups tested here, most of the impurities could be separated from the desired product 8 by recrystallization.However, the use of crude 8 for the following steps proved superior in terms of the final yield.Alkylation with the TMS-protected glycidol 20 did not give the desired product and only side product formation was observed instead.
Further investigations revealed that the alkylation of 7 could also be performed in water using K 2 CO 3 as a base.In the case of PMBz-glycidol 21, similar conversions as with toluene/ DIPEA were observed (Table 3, entry 1).Crude alcohol 21 also precipitated out during the reaction.As only traces of 21 could be detected in the liquid fraction of the reaction mixture, the product was isolated by filtration.Washing the solid with water furnished crude 21 of improved HPLC purity (87%), which could be further increased to 98% by precipitation from ethyl acetate/n-heptane.
Trityl-glycidol 18 as well as TBS-glycidol 19 were not suitable for the alkylation reaction in water (Table 3, entries 3 and 4) due to their poor solubility.For trityl-glycidol 18, a high temperature (70 °C) had to be applied to observe conversion.Mainly side product formation was detected under these conditions, presumably due to undesired hydrolysis of the epoxide ring.Similar observations were made with TBSglycidol 19.

Alkylation of the Secondary Alcohol Using 4-(Trifluoromethoxy)benzyl Bromide.
For the O-alkylation step, the purity of crude alkylated imidazoles 8, 21−23 was sufficient, and it was possible to use them without cumbersome purification steps which also prevented a loss of overall yield (Table 4).A solution of alkylated imidazole 8, 21−23 in NMP was added slowly over 2 h to a suspension of 4-(trifluoromethoxy)benzyl bromide and NaH in toluene/ NMP.Careful addition and temperature control were crucial to suppress undesired cyclized side products.In general, the reaction proceeded with high overall conversion (96−99%, HPLC, 315 nm) of alkylated imidazoles 8, 21−23, followed by quenching the reaction mixture with acetic acid/water and extraction with toluene to furnish the benzylated products (75−90%, HPLC, 315 nm).
2.5.Deprotection and Cyclization to Pretomanid.A one-pot deprotection/cyclization reaction to pretomanid (1) was achieved using an excess of K 2 CO 3 in MeOH in the case of PMBz-and Bz-protected starting materials 24−25.It was important to perform the reaction at low temperatures and follow the conversion carefully by HPLC because 10 was observed to react with in situ generated methoxide to form side product 27 (Scheme 4, see the SI for more details).Moreover, pretomanid (1) was prone to ring-opening by methoxide at longer reaction times.
A methanolic solution of crude 24 or 25 was cooled to −10 °C and an excess of K 2 CO 3 was added at once.After 2 h, HPLC data showed complete saponification to 10. Keeping this temperature overnight led to the predominant formation of side product 27.Warming to room temperature led to the same result.When the reaction mixture was warmed to 0 °C and kept overnight (14 h), only 10−12% of side product 27 was formed along with 60−70% of pretomanid 1.For workup, the reaction was quenched by the addition of water.While stirring overnight at ambient temperature, crude pretomanid (1) precipitated.After filtration and drying, the solid was suspended in hot MTBE to remove impurities.Pretomanid (1)

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was obtained as a colorless solid (HPLC purity: ≥99%, 315 nm) in 30−40% overall isolated yield.One of the major intentions of investigating the trityl and TBS routes was to replace methanol as required for the ester cleavage due to the risk of nucleophilic substitution of bromine in 7. Crude imidazole 26 (80 area %, HPLC, 315 nm) was readily deprotected in the presence of HCl yielding crude alcohol 10 (79 area %, 315 nm).
Cyclization attempts using NaH in THF were unsuccessful.Only 21% consumption (12% conversion to 1) could be observed when 3.0 equiv of NaH were added.Further addition of NaH led to a complex product mixture.
To use the conditions applied for the Bz and PMBz route (K 2 CO 3 in MeOH), precursor 26 first had to be deprotected using methanolic HCl followed by K 2 CO 3 addition at −10 °C.
For the one-pot deprotection and cyclization of TBSprotected alcohol 9 to pretomanid (1), various conditions were investigated (Table 5).In order to replace MeOH, various solvents as well as different conditions for the deprotection of intermediate 10 and subsequent cyclization to 1 were tested.
As in the trityl route, HCl in 1,4-dioxane was used to remove the TBS group.For the cyclization, potassium carbonate was used first, but side product formation was observed.By switching to sodium hydride, only the displacement of the primary hydroxy group by chloride was observed.
When using potassium fluoride to deprotect and cyclize in a one-pot fashion, DMF and THF were tested as solvents, but the conversion to pretomanid (1) was slow.By addition of TBACl, complete deprotection could be observed after 12 h at 40 °C.Heating the reaction mixture for cyclization led to slow product formation but side products formed as well due to the long reaction times required.Using a TBAF solution (1 M in THF) at −5 °C, complete deprotection could be observed after 2 h.Direct cyclization after deprotection could be achieved by heating the reaction mixture to reflux for 3−5 days (Table 5 entry 7).In the case of DMF (Table 5 entry 8), side products were formed.When THF was used as a solvent, side product formation was only observed in traces.After workup, the desired product still contained tetrabutylammonium salts and t-butyldimethylsilanol as impurities.After the recrystallization from 2-propanol/heptane and washing with water, tetrabutylammonium salts were only present in traces but unfortunately, t-butyldimethylsilanol could not be removed without column chromatography.Pretomanid (1) was obtained in 31% overall yield.

CONCLUSIONS
In variation of Fairlamb's approach, the synthesis of pretomanid (1) from (R)-glycidol and 2-bromo-4-nitro-1Himidazole (7) using different O-protecting groups was investigated with the aim of avoiding purification of intermediates and reducing product loss.Despite the carryover of impurities through each step, pretomanid (1) could be isolated in 30−40% yield over three steps in a purity over 99% (Scheme 6).
In this direct comparison, the PMBz route proved to be the most practical route so far with an overall yield of 40% and a purity of 99.7% (HPLC, 315 nm).
Most of the overall yield is lost in the alkylation and the onepot deprotection/cyclization step.Improvement of these transformations is challenging due to the readily formed Nand O-regioisomers and the entropically favored competing cyclization involving the secondary hydroxy group to form the undesired five-membered ring.

EXPERIMENTAL SECTION
Chemicals were obtained from commercial suppliers and were used without further purification.Deuterated solvents were purchased from Deutero GmbH (Kastellaun, Germany).Dry solvents were purchased from Acros Organics.Column chromatography was performed using cyclohexane and ethyl acetate which were purchased in technical grade and distilled prior to use.All air-or moisture-sensitive reactions were performed under an inert atmosphere (nitrogen or argon) in oven-dried glassware using Schlenk techniques.Reaction temperatures refer to the temperature of the particular cooling or heating bath.Thin-layer chromatography (TLC) was performed on silica plates (TLC Silica 60 F 254 , Merck).UV active compounds were visualized using UV light (λ = 254 nm and λ = 365 nm).All NMR spectra were recorded on the following spectrometers: Bruker Avance-III HD ( 1 H-NMR: 300 MHz, 13 C-NMR: 75.5 MHz, 19 F-NMR: 282 MHz), Bruker Avance-II ( 1 H-NMR: 400 MHz, 13 C-NMR: 100.6 MHz, 19 F-NMR: 377 MHz).Chemical shifts are referenced to residual solvent signals ([D]chloroform: 7.26 and 77.16 ppm, for 1 H-NMR and 13 C-NMR, respectively) and reported in parts per million (ppm) relative to tetramethylsilane ( 1 H, 13 C) and trichlorofluoromethane ( 19 F).Infrared spectra were recorded on an FTIR spectrometer (Bruker Tensor 27) equipped with a diamond ATR unit.Electrospray ionization (ESI) mass spectra were recorded on a 1200-series HPLC system or a 1260-series Infinity II HPLC system (Agilent-Technologies) with binary pump and integrated diode array detector coupled to an LC/ MSD-Trap-XTC ion trap mass spectrometer (Agilent-Technologies) or an LC/MSD Infinitylab LC/MSD (G6125B LC/ MSD) quadrupole mass spectrometer.High-resolution mass spectra were recorded on a 6545 Q-ToF-mass spectrometer.For analytical HPLC, an Agilent 1260 Infinity system equipped with a binary pump, a diode array detector, and an LC/MSD InfinityLab LC/MSD (G6125B LC/MSD) mass spectrometer was used.An Ascentis Express C18 column (2.7 μm, 2.1 mm × 30 mm, 40 °C) or an ACE C18 PFP column (3 μm, 4.6 mm × 150 mm, 40 °C) with gradient elution using acetonitrile/water (+0.1% formic acid) and a flow rate of 1.0 mL/min was used.Melting points were measured at a Kruss-Optronic KSP 1 N digital melting point meter.

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was quenched by adding water (50 mL) while cooling and transferred into a separating funnel.The organic phase was washed with saturated NaHCO 3 solution (50 mL), dried over Na 2 SO 4 , and filtered.All volatiles were removed in vacuo at 40 °C, and the residue was distilled under high vacuum.The desired product (16) was collected as a colorless liquid (11.(21).A round-bottom flask was charged with imidazole (7) (95%, 1.00 g, 4.95 mmol, 1.00 eq.), K 2 CO 3 (0.068 g, 0.495 mmol, 0.10 equiv), and water (20 mL).The suspension was heated to 55 °C and stirred for 5 min before (S)-oxirane-2-yl-methyl 4-methoxybenzoate (16) (1.44 g, 6.92 mmol, 1.40 equiv) was added in one portion.The slight greenish suspension was stirred for 44 h at 55 °C.After cooling to room temperature, the mixture was vacuum-filtered and carefully washed with water (4 × 4 mL).The slight yellowish solid was dried in the air overnight and then in a desiccator for 2 days.The crude product 21 (1.99 g, HPLC purity: 87%, 315 nm) was used for the next step without any further purification.Characteristic NMR signals: