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. Author manuscript; available in PMC: 2021 May 5.
Published in final edited form as: Methods Enzymol. 2020 May 5;638:69–87. doi: 10.1016/bs.mie.2020.04.051

Gram-scale preparation of the antibiotic lead compound salicyl-AMS, a potent inhibitor of bacterial salicylate adenylation enzymes

Nihar Kinarivala a, Lisa C Standke b, Tezcan Guney a, Cheng Ji a, Naoyoshi Noguchi c,d, Yasutomi Asano c,e, Derek S Tan a,b,f,*
PMCID: PMC7367610  NIHMSID: NIHMS1594779  PMID: 32416922

Abstract

Salicyl-AMS (1) is a potent inhibitor of salicylate adenylation enzymes used in bacterial siderophore biosynthesis and a promising lead compound for the treatment of tuberculosis. An optimized, multigram synthesis is presented, which provides salicyl-AMS as its sodium salt (1·Na) in three synthetic steps followed by a two-step salt formation process. The synthesis proceeds in 11.6% overall yield from commercially available adenosine 2´,3´-acetonide and provides highly purified material.

Keywords: antibiotic, tuberculosis, drug development

1. Introduction

Tuberculosis remains a global public health crisis, with an estimated 10 million new cases and 1.5 million deaths in 2018 (2019; Furin et al., 2019). Current treatments are effective against drug-sensitive infections, but multi-drug resistant and extensively-drug resistant tuberculosis present major threats, and an estimated 484,000 new infections resistant to the first-line drug rifampicin were reported in 2018. Thus, there is a great need for new antibiotics to treat drug-resistant tuberculosis infections. Mycobacterium tuberculosis, the bacteria that causes tuberculosis, acquires the essential nutrient Fe3+ using iron-chelating molecules called siderophores (Quadri & Ratledge, 2005; Ratledge, 2004; De Voss et al., 1999; De Voss et al., 2000). Siderophores bind iron with very high affinity and are used to transport iron across the cell envelope. Accordingly, inhibition of siderophore biosynthesis to limit bacterial iron uptake has been advanced as promising new antibacterial strategy (Lamb, 2015).

In collaboration with the laboratory of Luis Quadri, we have previously reported the development of the first designed siderophore biosynthesis inhibitor, salicyl-AMS (5‛-O-[N-salicylsulfamoyl]adenosine) (1) (Figure 1a) (Ferreras et al., 2005; Bythrow et al., 2019). Salicyl-AMS is a potent inhibitor of MbtA, a salicylate adenylation enzyme that catalyzes the first step in the synthesis of the M. tuberculosis mycobactin siderophores 2 (Quadri et al., 1998; McMahon et al., 2012). Salicyl-AMS mimics a tightly-bound reaction intermediate, salicyl-AMP (4), and potently inhibits MbtA biochemical activity as well as M. tuberculosis siderophore production and bacterial growth in cell culture. In collaboration with the laboratory of William Bishai, we demonstrated promising in vivo efficacy of salicyl-AMS in a mouse model of tuberculosis (Lun et al., 2013). Aldrich and coworkers have also reported extensive structure–activity relationship studies of salicyl-AMS analogues (Duckworth et al., 2012; Somu et al., 2006a; Vannada et al., 2006; Somu et al., 2006b; Qiao et al., 2007a; Qiao et al., 2007b; Neres et al., 2008; Gupte et al., 2008; Engelhart & Aldrich, 2013; Dawadi et al., 2015; Nelson et al., 2015; Dawadi et al., 2016; Krajczyk et al., 2016; Dawadi et al., 2018).

Fig. 1.

Fig. 1

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(a) Salicyl-AMS (1) is a potent inhibitor of the salicylate adenylation enzyme MbtA, and mimics a tightly-bound salicyl-AMP intermediate (4), blocking production of the mycobactin siderophores (2). R represents various fatty acyl chains (mycobactin variants) or chains terminating in a carboxylate or methyl ester (carboxymycobactin variants). (b) Structure of highly toxic fragment 5´-O-sulfamoyladenosine (AMS, 6). (c) Potential decomposition of salicyl-AMS (1) leading to N3–5´-cycloadenosine (7) and N-salicylsulfamate (8).

To enable further preclinical evaluation of salicyl-AMS, we report herein an optimized, scalable protocol for synthesis, purification, and analysis of salicyl-AMS. Importantly, this protocol provides salicyl-AMS that is free of the des-salicyl fragment 5´-O-sulfamoyladenosine (AMS, 6) (Figure 1b) (<0.0006 mol%, below the lower limit of detection), which is known to be highly cytotoxic (NCI-60 mean GI50 = 1.8 nM; NSC133114) (Jaffe et al., 1970; Bloch & Coutsogeorgopoulos, 1971; Rengaraju et al., 1986), as well as an N3–5´-cycloadenosine byproduct (7), which forms under basic conditions via a precedented pathway (Figure 1c) (Shuman et al., 1969; Duckworth et al., 2011; Shi et al., 2013). Moreover, salicyl-AMS is highly acidic at the acylsulfamate moiety (pKa ≈ 1.5) (Cisar & Tan, unpublished results; Somu et al., 2006a), and Aldrich has reported previously that conversion to the triethylammonium salt (1·Et3N) provides increased storage stability (Somu et al., 2006a). Our protocol affords salicyl-AMS as the corresponding sodium salt (1·Na), which is more commonly used in pharmaceutical formulations (Paulekuhn et al., 2007), to enable its use in preclinical and potentially clinical studies.

2. Synthesis of salicyl-AMS, sodium salt

Salicyl-AMS (1) was synthesized in three steps from commercially available adenosine 2´,3´-acetonide (13), in up to 10-g scale and an overall yield of 14.4% (Figure 2). The route incorporates numerous improvements to our original route (Ferreras et al., 2005), based on subsequent reports from our group and Aldrich and coworkers. Notably, only a single protecting group manipulation is required. Sulfamoyl chloride (10) was prepared by treatment of commercially available chlorosulfonylisocyanate (9) with formic acid (Figure 2a) (Brodsky & Du Bois, 2005; Heacock et al., 1996; Appel & Berger, 1958). Salicylic acid, N-hydroxysuccinimide ester (12) was prepared by activation of commercially available salicylic acid (11) with dicyclohexylcarbodiimide and coupling with N-hydroxysuccinimide (Van Brussel & Van Sumere, 1978) (Figure 2b).

Fig. 2.

Fig. 2

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(a) Preparation of sulfamoyl chloride (10). (b) Preparation of salicylic acid, N-hydroxysuccinimde ester (12). (c) Synthesis of salicyl-AMS (1) and conversion to the corresponding sodium salt (1·Na). CSA = camphorsulfonic acid; DCC = N,Ń-dicyclohexylcarbodiimide; DMA = N,N-dimethylacetamide; DMF = N,N-dimethylformamide; NHS = N-hydroxysuccinimde.

Adenosine acetonide 13 reacted with sulfamoyl chloride (10) selectively at the 5´-hydroxyl in N,N-dimethylacetamide (Okada et al., 2000; Cisar et al., 2007; Ferreras et al., 2008) to form 5´-O-sulfamoyladenosine 2´,3´-acetonide (14) in 62.5% yield (Figure 2c). Acylation with salicylic acid, N-hydroxysuccinimide ester (12) using cesium carbonate base in N,N-dimethylformamide (Somu et al., 2006a) provided salicyl-AMS 2´,3´-acetonide (15) in 84.5% yield. Finally, deprotection was achieved most efficiently using camphorsulfonic acid in methanol to afford salicyl-AMS (1) in 27.3% yield (14.4% overall yield over three steps). This deprotection method provided higher yields compared to a variety of other conditions evaluated (5:2 trifluoroacetic acid [TFA]/H2O; 3 equiv TFA in 1:1 MeOH/H2O; 3 equiv toluenesulfonic acid in MeOH; 1 equiv BF3·Et2O in CH2Cl2; 2 equiv FeCl3 in MeOH).

Salicyl-AMS was then converted to the corresponding sodium salt via a two-step procedure. Aldrich and coworkers have reported formation of the triethylammonium salt of salicyl-AMS directly from silica flash chromatography in the presence of 1% triethylamine (3:1 ethyl acetate/methanol) (Somu et al., 2006a). However, in our hands this led to incomplete conversion to the triethylammonium salt. Thus, we first attempted direct conversion of the free acid of salicyl-AMS (1) to the sodium salt 1·Na using 1 equivalent of aqueous sodium hydroxide or 1 equivalent of aqueous sodium bicarbonate at various temperatures (25, 0, –20 °C). However, all of these reactions resulted in formation of varying amounts of an N3–5´-cycloadenosine byproduct (7), which has been reported previously for related sulfamoyladenosines (Figure 1c) (Shuman et al., 1969; Duckworth et al., 2011; Shi et al., 2013).

Accordingly, we turned to a two-step salt formation process involving initial conversion of salicyl-AMS to the stable, isolable triethylammonium salt, followed by salt exchange to the desired sodium salt. Thus, the free acid of salicyl-AMS (1) was treated with excess, freshly distilled triethylamine at –20 °C to form the triethylammonium salt 1·Et3N. Purification by silica flash chromatography (4:1 ethyl acetate/methanol with 1% Et3N) removed the cycloadenosine byproduct 7 and afforded the triethylammonium salt of salicyl-AMS in 85.8% yield. Notably, increasing the reaction temperature from –20 °C to 0 °C or 25 °C resulted in increased formation of the cycloadenosine byproduct 7.

Next, conversion of the triethylammonium salt 1·Et3N to the sodium salt 1·Na was achieved using cation exchange on a Dowex 50WX8 column, which was washed rigorously with water, methanol, and acetonitrile, then pretreated with aqueous sodium hydroxide to generate the sodium form. A solution of salicyl-AMS, triethylammonium salt (1·Et3N, 0.1 M) was passed through the column and the collected solution was lyophilized to provide the salicyl-AMS, sodium salt (1·Na) in 94.4% yield (81.0% overall yield from free acid; 11.6% overall yield from adenosine acetonide 13). This salt formation protocol has been applied successfully to both small-scale (100 mg) and large-scale syntheses (2.5 g). Elemental analysis indicated a sulfur/sodium ratio of 1.43:1, comparable to the theoretical value of 1.39:1.

LC-MS/MS analysis of the salicyl-AMS free acid (1) indicated 0.31 mol% AMS as an impurity. In contrast, analysis of the final salicyl-AMS, sodium salt (1·Na) indicated <0.0006 mol% AMS, below the lower limit of detection. With appropriate minor modifications, this protocol has been applied to sodium salt formation with other salicyl-AMS analogues (Bythrow et al., 2019), and may also be useful for the preparation of acyl-AMS inhibitors of other targets in the adenylate-forming enzyme superfamily (Lux et al., 2019).

2.1. Equipment

  • Round bottom flask

  • Stir bar

  • Magnetic hot plate

  • Septum

  • Vacuum pump

  • Vacuum/argon manifold

  • Thermometer

  • Sonicator (water bath)

  • Chromatography columns (glass, various sizes)

  • Distillation apparatus

  • Rotary evaporator (Buchi)

  • Lyophilizer

  • Automated chromatography instrument (CombiFlash; Teledyn Isco)

  • NMR spectrometer (400 or 500 MHz; Bruker)

  • High-performance liquid chromatograph–mass spectometer (LC-MS; Waters)

  • High-performance liquid chromatograph–triple quadrupole mass spectrometer (LC-MS/MS; Agilent)

2.2. Chemicals

Reagents were obtained from Aldrich Chemical (www.sigma-aldrich.com), Acros Organics (www.fishersci.com), Alfa Aesar (www.alfa.com), or TCI America (www.tcichemicals.com) and used without further purification unless otherwise indicated. Optima or HPLC grade solvents were obtained from Fisher Scientific (www.fishersci.com), degassed with Ar, and purified on a solvent drying system unless otherwise indicated.

  • Chlorosulfonylisocyanate

  • Formic acid

  • Petroleum ether

  • Salicylic acid

  • N-Hydroxysuccinimide

  • 1,4-Dioxane

  • N,N´-Dicyclohexylcarbodiimide

  • N,N-Dimethylacetamide

  • Sodium bicarbonate

  • Sodium sulfate

  • N,N-Dimethylformamide

  • Cesium carbonate

  • (1S)-(+)-10-Camphorsulfonic acid

  • Triethylamine

  • Dowex 50WX8 (hydrogen form)

  • 1 N Sodium hydroxide solution

  • Guanosine

  • Methanol

  • Ethyl acetate

  • Water

  • Acetonitrile

  • Silica gel 60 thin-layer chromatography (TLC) plates with fluorescent indicator (F254)

  • Silica gel 60 (230–400 mesh)

  • Celite

  • Phosphate buffered saline

  • Dimethylsulfoxide

3. Protocol

All reactions were performed in flame-dried glassware under positive Ar pressure with magnetic stirring unless otherwise noted. Liquid reagents and solutions were transferred thru rubber septa via syringes flushed with Ar prior to use. Cold baths were generated as follows: 0 °C, wet ice/water; –25 °C, dry ice/acetone.

TLC was performed on 0.25 mm E. Merck silica gel 60 F254 plates and visualized under UV light (254 nm) or by staining with potassium permanganate (KMnO4), or cerium ammonium molybdenate (CAM). Silica flash chromatography was performed manually on E. Merck 230–400 mesh silica gel 60 or on an ISCO CombiFlash Rf+ instrument with RediSep silica gel normal phase columns or RediSep Gold silica gel normal phase columns with UV detection at 254 nm.

NMR spectra were recorded on a Bruker Ultrashield Plus 400 MHz Avance III NMR or UltraShield Plus 600 MHz Avance III NMR with DCH CryoProbe at 24 °C in methanol-d4 or DMSO-d6.

3.1. Preparation of sulfamoyl chloride (10)

3.

  1. In a 100-mL round bottom flask, chlorosulfonylisocyanate (9) (20 g, 141.31 mmol, 12.27 mL, 1.0 equiv) was cooled to 0 °C.

  2. Formic acid (6.5 g, 141.31 mmol, 5.33 mL, 1.0 equiv) was added dropwise to the reaction mixture at 0 °C.

  3. The reaction mixture was allowed to warm to 25 °C and stirred for 1 hour.

  4. The reaction mixture was diluted with petroleum ether (50 mL), then filtered.

  5. The filter cake was collected and dried under vacuum to give sulfamoyl chloride (10) (15 g) as a white solid, which was used without further purification.

  6. Note: Chlorosulfonylisocyanate and sulfamoyl chloride are highly sensitive to moisture and react violently with water.

3.2. Preparation of salicylic acid, N-hydroxysuccinimide ester (12)

3.

  1. In a 2-L round bottom flask, salicylic acid (11) (42 g, 304.08 mmol, 1.0 equiv) and N-hydroxysuccinimide (35 g, 304.08 mmol, 1.0 equiv) were dissolved in 1,4-dioxane (700 mL) and cooled to 0 °C under nitrogen.

  2. N,N´-Dicyclohexylcarbodiimide (62.74 g, 304.08 mmol, 61.51 mL, 1.0 equiv) was dissolved in 1,4-dioxane (300 mL) and the solution was added to the reaction mixture.

  3. The reaction mixture was allowed to warm to 25 °C and stirred for 12 hours.

  4. The reaction mixture was filtered to provide salicylic acid, N-hydroxysuccinimide ester (12) (76 g, crude) as a white solid, which was used without further purification.

  5. 1H-NMR (DMSO-d6, 400 MHz): δ 10.63 (br.s, 1H), 7.85 (dd, J1 = 1.6 Hz, J2 = 6.4 Hz, 1H), 7.56 (td, J1 = 1.6 Hz, J2 = 5.6 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.98–6.96 (m, 1H), 2.87 (s, 4H).

3.3. Synthesis of 5´-O-sulfamoyladenosine 2´,3´-acetonide (14)

3.

  1. In a 1-L round bottom flask, adenosine 2´,3´-acetonide (13) (46 g, 149.69 mmol, 1.0 equiv) was dissolved in N,N-dimethylacetamide (500 mL) and cooled to 0 °C under nitrogen.

  2. Sulfamoyl chloride (10) (34.59 g, 299.38 mmol, 2.0 equiv, from section 3.1) was added to the reaction mixture at 0 °C.

  3. The reaction mixture was allowed to warm to 25 °C and stirred for 12 hours with monitoring by LC-MS analysis of aliquots.

  4. Upon completion, the reaction was quenched by addition of saturated aqueous sodium bicarbonate until the pH reached 7–8.

  5. The mixture was extracted with ethyl acetate (5 × 500 mL), then the organic layers were combined, dried over sodium sulfate, filtered, and concentrated by rotary evaporation to provide a yellow residue.

  6. The residue was purified by silica flash chromatography (ethyl acetate → 20:1 ethyl acetate/methanol) to afford 5´-O-sulfamoyladenosine 2´,3´-acetonide (14) (43 g, 84% purity: 93.48 mmol, 62.5% yield [adjusted for purity]) as a yellow oil.

  7. LC-MS: tR = 1.092 min, purity: 84%, m/z 387.0 [M+H]+

  8. 1H-NMR (CD3OD, 400 MHz): δ 8.27 (s, 1H), 8.22 (s, 1H), 6.25 (d, J = 2.4 Hz, 1H), 5.43 (dd, J1 = 2.0 Hz, J2 = 6.0 Hz, 1H), 5.14 (dd, J1 = 2.8 Hz, J2 = 6.0 Hz, 1H), 4.52–4.50 (m, 1H), 4.32–4.27 (m, 2H), 1.61 (s, 3H), 1.39 (s, 3H).

3.4. Synthesis of 5´-O-(N-salicylsulfamoyl)adenosine 2´,3´-acetonide (15)

3.

  1. In a 1-L round bottom flask, 5´-O-sulfamoyladenosine 2´,3´-acetonide (14) (43 g, 84% purity: 93.48 mmol, 1.0 equiv [adjusted for purity], from section 3.3) was dissolved in N,N-dimethylformamide (450 mL) and cooled to 0 °C under nitrogen.

  2. Salicylic acid, N-hydroxysuccinimide ester (12) (52.35 g, 222.58 mmol, 2.38 equiv, from section 3.2) and cesium carbonate (108.78 g, 333.87 mmol, 3.57 equiv) were added to the reaction mixture at 0 °C.

  3. The reaction mixture was allowed to warm to 25 °C and stirred for 12 hours with monitoring by LC-MS analysis of aliquots.

  4. Upon completion, the reaction mixture was filtered and concentrated by rotary evaporation to provide a white residue.

  5. The residue was purified by silica flash chromatography (1:1 petroleum ether/ethyl acetate → 20:1 ethyl acetate/methanol) to afford 5´-O-(N-salicylsulfamoyl)adenosine 2´,3´-acetonide (15) (40.2 g, 99.5% purity: 78.97 mmol, 84.5% yield [adjusted for purity]) as an off-white solid.

  6. LC-MS: tR = 1.173 min, purity: 99.5%, m/z 507.1 [M+H]+

  7. 1H-NMR (CD3OD, 400 MHz): δ 8.46 (s, 1H), 8.15 (s, 1H), 7.91 (dd, J1 = 1.6 Hz, J2 = 7.6 Hz, 1H), 7.33–7.29 (m, 1H), 6.82–6.79 (m, 2H), 6.25 (d, J = 2.8 Hz, 1H), 5.39 (dd, J1 = 2.8 Hz, J2 = 6.0 Hz, 1H), 5.16 (dd, J1 = 2.0 Hz, J2 = 6.0 Hz, 1H), 4.59–4.58 (m, 1H), 4.34 (dd, J1 = 2.8 Hz, J2 = 4.0 Hz, 2H), 1.60 (s, 3H), 1.27 (s, 3H).

3.5. Synthesis of salicyl-AMS (free acid) (1)

3.

  1. In a 1-L round bottom flask, 5´-O-(N-salicylsulfamoyl)adenosine 2´,3´-acetonide (15) (40.2 g, 99.5% purity: 78.97 mmol, 1.0 equiv [adjusted for purity], from section 3.4) was dissolved in methanol (500 mL) and cooled to 0 °C under nitrogen.

  2. Camphorsulfonic acid (55.04 g, 236.93 mmol, 3.0 equiv) was added at 0 °C in one portion.

  3. The reaction mixture was allowed to warm to 25 °C and stirred for 12 hours with monitoring by LC-MS analysis of aliquots.

  4. Upon completion, the reaction mixture was quenched by addition of solid sodium bicarbonate until the pH reached 7–8, then concentrated by rotary evaporation.

  5. The residue was purified by silica gel chromatography (ethyl acetate → 5:1 ethyl acetate/methanol) to afford 25 g of partially purified product.

  6. The partially purified product was further purified by preparative HPLC (column: Phenomenex Gemini C18 250 × 50 mm, 10 μm; mobile phase: water (0.05% v/v ammonia hydroxide) with 1–18% acetonitrile, 28 min). Lyophilization afforded salicyl-AMS (free acid) (1) (10.05 g, 99.9% purity: 21.53 mmol, 27.3% yield [adjusted for purity]) as a white solid.

  7. 1H-NMR (CD3OD, 400 MHz): δ 8.57 (s, 1H), 8.17 (s, 1H), 7.93 (dd, J1 = 1.6 Hz, J2 = 8.0 Hz, 1H), 7.29 (td, J1 = 1.6 Hz, J2 = 7.2 Hz, 1H), 6.80–6.75 (m, 2H), 6.09 (d, J = 6.4 Hz, 1H), 4.72 (t, J = 5.6 Hz, 1H), 4.42–4.40 (m, 2H), 4.37 (dd, J1 = 3.2 Hz, J2 = 11.2 Hz, 1H), 4.33–4.31 (m, 1H).

3.6. Conversion to triethylammonium salt (1·Et3N)

3.

  1. In a 250-mL round bottom flask, salicyl-AMS (free acid) (1) (2.5 g, 5.36 mmol, from section 3.5) was dissolved in 200 mL methanol (27 mM).

  2. The mixture was sonicated in a water bath for 5–10 minutes to increase solubilization.

  3. The mixture was filtered using a 0.2 μM syringe filter.

  4. The filtered solution was transferred to a 500-mL round bottom flask and cooled to –20 °C with stirring for 10 minutes.

  5. Note: Salicyl-AMS may precipitate at temperatures below –25 °C.

  6. Triethylamine (11.2 mL, 80.4 mmol, 15 equiv) was added dropwise at –20 °C and the solution was stirred for 30 minutes at –20 °C.

  7. Note: Freshly distilled triethylamine was used.

  8. The reaction mixture was allowed to warm to room temperature and concentrated by rotary evaporation.

  9. The residue was purified by silica flash chromatography (4:1 ethyl acetate/methanol + 1% triethylamine) to afford salicyl-AMS, triethylammonium salt (1·Et3N) (2.61 g, 4.60 mmol, 85.8% yield) as a white solid.

  10. Note: The crude product was dry-loaded onto the silica gel column by dissolving in 200 mL methanol, addition of 10 g Celite, rotary evaporation, and transfer of the Celite onto the column.

  11. 1H-NMR (CD3OD, 600 MHz): δ 8.41 (s, 1H), 8.13 (s, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 6.72–6.87 (m, 2H), 6.08 (d, J = 6.4 Hz, 1H), 4.69 (t, J = 5.6 Hz, 1H), 4.24–4.34 (m, 4H), 3.17 (q, J = 7.2Hz, 6H), 1.33 (t, J = 7.2 Hz, 9H) (Somu et al., 2006b).

3.7. Conversion to sodium salt (1·Na)

3.

  1. Dowex 50WX8 (hydrogen form) (1,000 g) was placed in a glass column (3 in inside diameter x 12 in length, with 2,000 mL reservoir).

  2. The resin on the column was sequentially washed with water (5 × 1,000 mL), methanol (5 × 1,000 mL), water (5 × 1,000 mL), acetonitrile (5 × 1,000 mL), water (5 × 1,000 mL) and 1 N aqueous sodium hydroxide (5 × 1,000 mL).

  3. The resin on the column was washed repeatedly with water until the eluent reached pH 7.

  4. Salicyl-AMS, triethylammonium salt (1·Et3N) (2.61 g, 4.60 mmol, from section 3.6) was dissolved in minimal amount of 1:1 water/acetonitrile (≈50 mL).

  5. The solution was loaded onto the Dowex column and incubated with the resin for 10 min.

  6. The column was eluted with water (1,000 mL) and 25-mL fractions were collected and analyzed by spotting on a TLC plate and visualizing under UV light (254 nm).

  7. Fractions that were UV active were combined and lyophilized to afford salicyl-AMS, sodium salt (1·Na) (2.12 g, 4.34 mmol, 94.4% yield, 99.9% purity) as a fluffy white solid.

  8. 1H-NMR (CD3OD, 600 MHz): δ 8.55 (s, 1H), 8.19 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.3 Hz, 1H), 8.70–6.84 (m, 2H), 6.12 (d, J = 6.4 Hz, 1H), 4.73 (t, J = 5.5 Hz, 1H), 4.34–4.44 (m, 4H).

  9. Elemental analysis was performed to determine the percentage of carbon, hydrogen, nitrogen, sulfur, and sodium (Robertson Microlit). The sulfur:sodium ratio was calculated at 1.43:1, comparable to the theoretical ratio of 1.39:1 (1:1 molar ratio).

3.8. LC-MS/MS analysis of salicyl-AMS, sodium salt (1·Na) to quantify AMS impurity

3.8.1. Preparation of samples:

  1. Preparation of salicyl-AMS test sample solution: salicyl-AMS, sodium salt (1·Na) (2 mg) was dissolved in 0.4 mL of 9:1 phosphate buffered saline (PBS)/dimethylsulfoxide (DMSO) to generate a 5 mg/mL stock solution.

  2. Note: The salicyl-AMS, sodium salt (1·Na) solution should be freshly prepared before use.

  3. Preparation of guanosine internal standard solution: guanosine (2.5 mg) was dissolved in 0.5 mL DMSO to generate a 5 mg/mL stock solution. The stock solution was diluted 1,000-fold with PBS to generate a 5 μg/mL working solution.

  4. A calibration curve of AMS was prepared as follows:

    1. 5´-O-Sulfamoyladenosine (AMS, 6) (Ferreras et al., 2005) (2 mg) was dissolved in 0.4 mL DMSO to generate a 5 mg/mL stock solution.

    2. The AMS stock solution was diluted 100-fold with PBS to generate a 50 μg/mL working solution.

    3. The 50 μg/mL AMS working solution was serially diluted two-fold with PBS 12 times to generate calibration curve solutions at 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195, 0.0977, 0.0488, 0.0244, 0.0122 μg/mL. A 0 μg/mL blank was also generated with PBS, for a total of 14 samples.

3.8.2. Preparation of 96-well plate

  1. 100 μL of each salicyl-AMS calibration curve solution (from section 3.8.1, step 4) was added to each well of 96-well plate, along with 100 μL of the internal standard working solution (from section 3.8.1, step 3). The experiment was conducted in triplicates.

  2. Final concentrations:

    1. Guanosine (internal standard): 2.5 μg/mL in all wells

    2. AMS calibration curve: 0.061–25 μg/mL

  3. Note: When calculating the concentrations of the internal standard and of AMS in the samples, a dilution factor of 2 needs to be applied compared to the working solutions.

3.8.3. Method for LC-MS/MS analysis

LC-MS/MS analysis was carried out on an Agilent Technologies 6410 triple quadrupole LC-MS/MS system with autosampler in electrospray ionization (ESI) mode, with an Agilent Zorbax Eclipse XBD-C18 reverse phase column (50 × 4.6 mm, 5 μm) using a flow rate of 0.5 mL/min and an isocratic mobile phase of 10% acetonitrile in 0.1% aqueous formic acid over 10 min. Positive electrospray in multiple reaction monitoring (MRM) mode was employed to quantify AMS, using the ion transitions indicated below.

Compound Precursor Ion Product Ion Fragmentor Collision Energy
AMS (6) 347.1 136.1 128 20
Guanosine 284.1 152.1 71 8
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4. Concluding remarks

We have developed an efficient protocol for the multigram-scale preparation of the anti-tuberculosis lead compound salicyl-AMS as its sodium salt (1·Na). The process proceeds in three synthetic steps, followed by a two-step salt formation protocol, to afford the target compound in 11.6% overall yield from commercially available adenosine 2´,3´-acetonide (13). The two-step salt formation process includes an additional chromatographic purification and removes both a cycloadenosine byproduct (7) and a toxic trace impurity AMS (6), providing highly purified material suitable for preclinical evaluation. This protocol has been applied to milligram to multigram-scale syntheses, and with minor modifications to other salicyl-AMS analogues (Bythrow et al., 2019). We envision that this protocol may also be useful in the preparation of other acyl-AMS inhibitors of the adenylate forming enzyme superfamily (Lux et al., 2019).

Table 1.

Elemental analysis of salicyl AMS, sodium salt (1·Na).

Sample %C %H %N %S %Na
Experimental 38.65 3.89 15.54 6.39 4.47
Calculated 41.81 3.51 17.21 6.56 4.71
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Acknowledgments

We thank Kazuyoshi Aso (Tri-Institutional Therapeutics Discovery Institute and Axcelead Drug Discovery Partners) and Makoto Fushimi (Tri-Institutional Therapeutics Discovery Institute and Takeda Pharmaceuticals) for helpful discussions and for coordinating large-scale synthesis of salicyl-AMS (free acid) at WuXi AppTec. Generous financial support was provided by the National Institutes of Health (R01 AI118224 to D.S.T.; F31 AI129244 and T32 GM073546-Gross to L.C.S.; and CCSG P30 CA008748 to C. B. Thompson) and the Tri-Institutional Therapeutics Discovery Institute.

Footnotes

Conflicts of Interests

D.S.T. is an inventor on U.S. Patents 8,461,128 and 8,946,188, and D.S.T. and L.C.S. are inventors on U.S. Patent Application PCT/US2019/068107 based on this work.

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