Development of Pyrazolopyrimidine Anti-Wolbachia Agents for the Treatment of Filariasis

Anti-Wolbachia therapy has been identified as a viable treatment for combating filarial diseases. Phenotypic screening revealed a series of pyrazolopyrimidine hits with potent anti-Wolbachia activity. This paper focuses on the exploration of the SAR for this chemotype, with improvement of metabolic stability and solubility profiles using medicinal chemistry approaches. Organic synthesis has enabled functionalization of the pyrazolopyrimidine core at multiple positions, generating a library of compounds of which many analogues possess nanomolar activity against Wolbachia in vitro with improved DMPK parameters. A lead compound, 15f, was selected for in vivo pharmacokinetics (PK) profiling in mice. The combination of potent anti-Wolbachia activity in two in vitro assessments plus the exceptional oral PK profiles in mice puts this lead compound in a strong position for in vivo proof-of-concept pharmacodynamics studies and demonstrates the strong potential for further optimization and development of this series for treatment of filariasis in the future.

Elemental analyses (%C, %H, %N) were determined by the University of Liverpool Microanalysis Laboratory, reported percentages are within error limits of ±0.5 %. LCMS analysis was carried out using SHIMADZU LCMS-2020 with a Chromolith@Flash RP-18e (25 x 2.0 mm) column at 25 o C and a mixture of eluent A) 0.0375% TFA in water (v/v) and eluent B) 0.01875% TFA in Acetonitrile (v/v) as mobile phase, with run time: 1.5 or 2 min. *Compounds 2, 5d, 6a-b, 6d-e and 9a-b are commercially available.

Synthesis of ethyl 2-(methylsulfonyl)-3-oxobutanoate (6c)
To a mixture of ethyl 3-oxobutanoate (2.0 g, 15.4 mmol) in THF (20 mL) was added NaH (922.08 mg, 23.1 mmol, 1.5 eq) in portions at 0°C under N 2 protection. The mixture was allowed to stir at 0°C for 30 min. Then methanesulfonyl chloride (3.89 g, 30.7 mmol, 2 eq) was added and the mixture was warmed to 25°C and stirred for 2 h (followed by TLC). The mixture was diluted with ethyl acetate (60 mL) and washed with saturated brine (40 mL x 2), dried with Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by column chromatography eluting with 5-15% ethyl acetate in petroleum ether to afford 6c as a yellow liquid (1.00 g, crude), which was used directly in the next step without further purification.

Synthesis of ethyl 2-methyl-3-oxopropanoate (6f)
A solution of ethyl propanoate (25.0 g, 244.8 mmol) and ethyl formate (36.27 g, 489.6 mmol, 2 eq) in DCM (200 mL) at 0°C was added TiCl 4 (92.86 g, 489.4 mmol, 2 eq) followed by triethylamine (59.45 g, 587.5 mmol, 2.4 eq) dropwise under N 2 protection. The reaction mixture was allowed to stir at 0°C for 1 h and at 10-15 °C for another 1 h (followed by TLC). The reaction mixture was poured into ice water and extracted with DCM (100 mL x 3). The organic layer was washed with brine (100 mL x 3), dried over Na 2 SO 4 , filtered and concentrated to give 6f as a brown oil (50.0 g, crude), which was used directly for next step without further purification.
General Procedure 1 -Synthesis of pyrazolo[1,5-a]pyrimidin-7-ol (1c, 7a-f) (A) To the corresponding 3-amino-1,2-pyrazole (5 mmol) in AcOH (8 ml) was added the appropriate β-keto ester (20 mmol, 4 eq) and the reaction was allowed to stir at 110 o C for 3-12 h (followed by TLC). The reaction was cooled to room temperature and diluted with Et 2 O causing precipitation of the desired product which was filtered and washed with further Et 2 O. The product was dried under high vacuum and was used without further purification.
(B) To the corresponding 3-amino-1,2-pyrazole (5 mmol) in toluene (10 ml) was added the appropriate β-keto ester (5 mmol, 1 eq) and TsOH (0.1 eq). The reaction was allowed to stir at 110 o C for 2-12 h (followed by TLC). The reaction was cooled to room temperature and filtered and washed with petroleum ether. The filtrate collected was concentrated in vacuo to give the desired product. The product was dried under high vacuum and was used without further purification.
To a mixture of ethyl 3-((1H-pyrazol-3-yl)imino)-2-methylpropanoate (52.0 g, 266.4 mmol, 1 eq) in THF (300 mL) obtained from last step at 0°C was added t BuOK (29.9 g, 266.4 mmol, 1 eq) in portions and the mixture was allowed to stir at 10-15 °C for 30 min. Solids were precipitated (followed by TLC). The mixture was cooled to 0°C and acified with HCl/EtOAc (4 M) to pH7. The mixture was concentrated to give 7f as a light yellow solid (60.0 g, crude) contained KCl. And it was used directly for next step without further purification.

General Procedure 2 -Synthesis of 7-chloropyrazolo[1,5-a]pyrimidines (1d, 8a-f)
To a solution of pyrazolo[1,5-a]pyrimidin-7-ol (5 mmol) in acetonitrile (10 mL) was added POCl 3 (4 eq). The reaction mixture was allowed to stir at reflux temperature for 1-16 h (followed by TLC). The mixture was cooled to room temperature and poured into ice-water slowly. The mixture was basified with sat. NaHCO 3 (aq) to pH 7, The mixture was extracted with ethyl acetate (x 3), the combined organic layer was washed with water, followed by brine, dried over MgSO 4 , filtered and concentrated in vacuo. The crude was then purified by column chromatography using 10-50% ethyl acetate in hexane to give the desired product.

Synthesis of 7-chloro-6-methylpyrazolo[1,5-a]pyrimidine (8f)
7f was treated according to General Procedure 2 to give 8f as a brown solid (62%), which was used directly in the next step without any purification.

Synthesis of 7-chloro-3-iodo-5,6-dimethylpyrazolo[1,5-a]pyrimidine (11a)
8d (463 mg, 2.6 mmol) and NIS (573 mg, 2.6 mmol) in anhydrous MeCN (25 mL) were allowed to stir at room temperature for 1 h (followed by TLC). Volatiles were removed in vacuo and the residue was diluted with DCM (20 mL). The solution was washed with water (10 mL) and brine (10 mL). The organic layer was dried over MgSO 4 , filtered and evaporated to dryness. The crude was purified by column chromatography using dichloromethane to isolate 11a as a white solid (

General Procedure 3 -the substitution of 7-chloro-pyrazolo[1,5-a]pyrimidines (1, 3, 10ad, 12a, 12i, 12k)
(A) To a solution of 7-chloropyrazolo [1,5-a]pyrimidine (1 mmol) in anhydrous DMF (5 mL) was added 2-picolylamine (1.2 eq) and triethylamine (2 eq). The reaction mixture was allowed to stir at 60-110 o C for 1-4 h (followed by TLC). Water (5 mL) was slowly added to the mixture. Precipitates formed were collected by filtration. If no precipitate was formed, the mixture was extracted with ethyl acetate, the organic layer was washed with brine, dried over Na 2 SO 4 , filtered and concentrated. The crude solid/residue was then purified by column chromatography or prep-HPLC to give the desired product.
(B) To a solution of 7-chloropyrazolo [1,5-a]pyrimidine (1 mmol) in anhydrous DMF (5 mL) was added 2-picolylamine (1 eq) and K 2 CO 3 (2 eq). The reaction mixture was allowed to stir at 60 o C/reflux for 2 -24 h (followed by TLC). The solvent was removed in vacuo and the residue was then extracted with ethyl acetate, washed with water, followed by brine, dried over Na 2 SO 4 , filtered and concentrated. The crude was then purified by column chromatography or prep-HPLC to give the desired product.
A mixture of 14j_int (100 mg, 0.23 mmol) and 10% Pd/C (10 mg) in MeOH (3 mL) was allowed to stir at room temperature for 1 h under H 2 (15 psi) (followed by TLC). The mixture was filtered through celite and the filtrate was concentrated in vacuo to give a residue. The residue was purified by prep-TLC using 50% ethyl acetate in petroleum ether to give 14j as a colourless oil (100 mg, crude), which was used directly in the next step without further purification.

General Procedure 5 -Boc deprotection (15a-k)
(A) To the Boc-protected amine (1 mmol) in dichloromethane (8 mL) was added trifluoroacetic acid (2 mL). The reaction mixture was allowed to stir at room temperature overnight (followed by TLC). The solvent was removed in vacuo, the residue was diluted with ethyl acetate, washed with sat. NaHCO 3 (aq) (x 2), followed by brine, then dried over MgSO 4 , filtered and concentrated. The crude was purified by column chromatography to give the desired product. (B) To the Boc-protected amine (0.2 mmol) in ethyl acetate (2 mL) was added 4M HCl in ethyl acetate (4 mL), the resulting solution was allowed to stir at room temperature for 1 -12 h (followed by TLC). The mixture was filtered and the filter cake was further washed with ethyl acetate (2 mL). The solid collected was dissolved in methanol (5 mL), and the solution was basified with strong base anion exchange resin and concentrated in vacuo.
The residue was then purified by prep-HPLC or prep-TLC to give the desired product.

Synthesis of 5,6-dimethyl-N-(pyridin-2-ylmethyl)-3-(tetrahydro-2H-pyran-4-yl)pyrazolo [1,5-a]pyrimidin-7-amine (15j)
have a complex cytoplasm texture (high SER texture score), whereas Wolbachia-uninfected cells (doxycycline-treated positive control) have a uniform cytoplasm texture (low SER texture score). From this analysis, the following readouts were calculated per well: cell number, SER texture score, and percentage of Wolbachia-infected cells. Z' factor (Z') validation of each plate was calculated using the percentage of Wolbachia-infected cells value from the vehicle and positive controls. 14 Vehicle controls have a high Wolbachia load and therefore a high percentage of cells classed as infected with Wolbachia. Positive control (doxycycline-treated) cells have a low Wolbachia load and therefore a low percentage of Wolbachia-infected cells. All compound sample wells were then analysed and normalized (along with the positive controls) against the vehicle (untreated) control to give a percentage reduction of Wolbachia-infected cells. In addition, using the cell number analysis, any compounds with a host cell number amounting to less than 50% of the vehicle control were classed as toxic and retested at a reduced compound concentration. All compounds that were >90% of the positive control's percentage reduction of Wolbachia-infected cells were classed as strong hits (because they were similar to or greater than the 5 µM doxycycline positive control). Compounds that yield infection rates between 50% and 90% of the positive control were classed as moderate hits [because they were similar to the suboptimal (50 nM) doxycycline control]. All hit compounds were then reconfirmed in a full dose response to define their potency.

In vitro microfilariae (mf) B. malayi assays 2
Within the in vitro mf assay; compounds are incubated with 8000 mf B. malayi per well (five wells per compound) for 6 days, before DNA is extracted and qPCR performed to compare wsp:gst ratio of drug treated vs control wells. Details for the sources of mf B. malayi are described below.

Animals
Male BALB/c SCID were purchased from Harlan Laboratories, UK, while male CB.17 SCID mice and BALB/c WT mice were purchased from Charles River, UK. Male Meriones unguiculatus (Mongolian gerbils; jirds) were purchased from either Charles River, UK or Janvier Laboratories, France. Rodents shipped to REFOTDE, Buea, Cameroon, were maintained in conventional housing with (Halliday et al. Parasites & Vectors 2014, 7:472 Page 2 of 14 http://www.parasitesandvectors.com/content/7/1/472) daily cage cleaning and changing of food. Food, water and bedding were sterilised by autoclaving. For B. malayi experiments, animals were kept at the Biomedical Services Unit (BSU), University of S30 Liverpool, UK in specific pathogen-free (SPF) conditions. All experiments carried out in Cameroon were approved by the Animal Care Committee, REFOTDE. All experiments on animals in the UK were approved by the ethical committees of the University of Liverpool and LSTM, and were conducted according to Home Office (UK) requirements. The life cycle of B. malayi (Bm) was maintained in mosquitoes and susceptible Meriones gerbils at LSTM.
To generate infective Bm larvae (BmL3) female adult Aedes aegypti mosquitoes were fed with Bm microfilariae (mf) collected from infected gerbils by catheterisation, as previously described, 3 followed by mixing with human blood and feeding through an artificial membrane feeder (Hemotek®). Blood-fed mosquitoes were reared for 14 days to allow for development to L3. The L3 were collected from infected mosquitoes by crushing and concentration using a Baermann's apparatus and RPMI medium.

In vitro drug metabolism/pharmacokinetic assays
The DMPK data described in the manuscript were measured once through a high-throughput platform provided by AstraZeneca UK. The methods of the five assays, including LogD7.4, aqueous solubility, plasma protein binding, human microsome and rat hepatocyte clearance measurements, have been reported in detail previously. 4