Organic Process Research & Development 2008, 12, 183–191
Practical Synthesis of a Peptide Deformylase (PDF) Inhibitor Yugang Liu,* Mahavir Prashad, Lech Ciszewski, Kevin Vargas, Oljan Repicˇ, and Thomas J. Blacklock Process Research & DeVelopment, NoVartis Pharmaceuticals Corporation, One Health Plaza, East HanoVer, New Jersey 07936, U.S.A.
Abstract: A practical chromatography-free synthesis of an N-formylated hydroxylamine peptide deformylase inhibitor LCD320 is described. A diastereoselective Michael reaction of (4S)-3-[2-(cyclobutylmethyl)-1-oxo-2-propenyl]-4-(phenylmethyl)-2-oxazolidinone with O-benzyl hydroxylamine was used to establish the key stereogenic center. We found that traces of residual Li+ from a previous step had a great impact on the diastereoselectivity of this reaction. A very efficient amidation coupling reaction of proline derivative (2S,4R)-4-fluoro-1,2-pyrrolidinedicarboxylic acid 1,1-dimethylethyl ester with weakly nucleophilic 3-pyridazinamine using methanesulfonyl chloride in the presence of 1-methylimidazole in DMF was also developed that proceeded without racemization. Introduction LCD320 (1) belongs to a novel class of antimicrobial agents known as peptide deformylase (PDF) inhibitors.1 It is a followup compound to LBM415.2,3 Peptide deformylase is a highly conserved bacterial enzyme that plays a key physiological role in bacterial cell growth and sustained viability. PDF modulates a step in prokaryotic protein synthesis not generally found in the cytosolic protein synthesis of eukaryotes of mammalians, thus providing a new target for the development of a novel class of antibacterial agents with an entirely new mechanism of action. Respiratory tract infections (RTIs) comprise the single largest market for all antibacterials (>$15 billion annually). The major pathogen that causes RTIs, Streptococcus pneumoniae, is now becoming resistant to many existing therapies on the market. The need for a new class of antibacterials that are active against resistant strains and have no cross-resistance with currently used agents is the number one priority in antibacterial research. Large quantities of LCD320 (1) were needed for toxicology and clinical studies, which required a practical synthesis that was suitable for scale-up. Results and Discussion On the basis of our experiences with the β-lactam route2 to LBM415, we decided to use the same strategy for LCD320. * To whom correspondence should be addressed. Tel: 862-778-3470. Fax: 973-781-4384. E-mail:
[email protected].
(1) (a) For a discussion of PDF inhibitors, see: Huntington, K. M.; Yi, T.; Wei, Y.; Pei, D. Biochemistry 2000, 39, 4543 and references therein. (b) Pratt, L. M.; Beckett, R. P.; Davies, S. J.; Launchbury, S. B.; Miller, A.; Spavold, Z. M.; Todd, R. S.; Whittaker, M. Bioorg. Med. Chem. Lett. 2001, 11, 2585. (2) Jiang, X.; Prasad, K.; Prashad, M.; Slade, J; Repicˇ, O.; Blacklock, T. J. Synlett 2006, 18, 3179. (3) Slade, J.; Parker, D. J.; Girgis, M.; Mueller, M.; Vivelo, J.; Liu, H.; Bajwa, J. S.; Chen, G. P.; Carosi, J.; Lee, P.; Chaudhary, A.; Wambser, D.; Prasad, K.; Bracken, K.; Dean, K.; Boehnke, H.; Repicˇ, O.; Blacklock, T. J. Org. Process Res. DeV. 2006, 10, 78. 10.1021/op700265n CCC: $40.75 2008 American Chemical Society Published on Web 01/23/2008
We first synthesized β-lactam intermediate 12 and investigated the ring-opening reaction with proline derivative 13 (vide infra). Compound 4, which was made by the alkylation of diethyl malonate with bromomethyl cyclobutane (vide infra), was decarboxylated in a minimal amount of 1-methyl-2-pyrrolidinone (NMP) or DMF at 110 °C for 2 h to afford compound 5 in quantitative yield (Scheme 1). Other solvents such as toluene and ethanol did not give any decarboxylated product 5. Acid 5 was treated with trimethylacetyl chloride and triethylamine to form the corresponding mixed anhydride, which reacted with (S)-(-)-4-benzyl-2-oxazolidinone in the presence of LiCl4 to afford the desired product 6 in 90% yield. Asymmetric hydroxymethylation of 6 was accomplished using s-trioxane and TiCl4 in the presence of N,N-diisopropylethylamine in dichloromethane to afford 7 in 85% yield as a single diastereomer. Quite interestingly, racemization of the newly generated stereogenic center occurred if the reaction mixture was not quenched promptly. To obtain high diastereoselectivity, the reaction had to be quenched at ∼95% conversion. Hydrolysis of 7 using LiOH/H2O2 afforded chiral acid 8 in 94% yield. Acid 8 was coupled with O-benzylhydroxylamine hydrochloride (9) in aqueous sodium hydroxide solution in the presence of 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) to give amide 10 in 77% yield, which upon treatment with methanesulfonyl chloride in the presence of pyridine afforded mesylate 11 in 79% yield. β-Lactam 12 was produced in 90% yield by treatment of 11 with aqueous potassium carbonate in isopropyl acetate in the presence of 10 mol % of tetrabutylammonium bromide. With the desired β-lactam 12 in hand, we investigated the ring-opening reaction with 13. Unfortunately, a mixture of the desired product 14 and bicyclic product 15 were formed in ∼1:9 ratio under a variety of conditions when the conversion of 12 was >95%. We also identified that byproduct 15 was produced via the intermediacy of desired compound 14, since the ratio increased with reaction time. Although not anticipated when we first selected this approach, now it became obvious that the pyridazin-3-ylamino group was such a good leaving group that cyclization of 14 to produce 15 (4) Ho, G.-J.; Mathre, D. J. J. Org. Chem. 1995, 60, 2271. Vol. 12, No. 2, 2008 / Organic Process Research & Development
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Scheme 1 a
a Reagents and conditions: (a) NaOMe, NaOH; (b) HCl, 69%; (c) NMP, 110 °C, quantitative; (d) pivalyl chloride, LiCl, (S)-(-)-4-benzyl-2-oxazolidinone, THF, -15 °C, 90%; (e) s-trioxane, TiCl4, N,N-diisopropylethylamine, CH2Cl2, 0 °C, 85%; (f) LiOH monohydrate, H2O2, THF, 0 °C, 94%; (g) 9, NaOH, EDCI, H2O, 77%; (h) CH3SO2Cl, DMF, pyridine, 79%; (i) iPrOAc, K2CO3, H2O, Bu4NBr, 90%; (j) 13, NaOH, rt; (k) 2-ethylhexanoic acid, THF.
seemed to be inevitable. Therefore, another approach had to be developed. We decided to use the Michael addition route3 that was originally used for LBM415. This alternative synthesis started with a simple alkylation reaction of diethyl malonate with bromomethyl cyclobutane (2; Scheme 2). For molecules that lack chromophores, it is generally challenging to develop a simple and reliable HPLC method for in-process controls. By monitoring the reaction at 220 nm using HPLC, we were able to determine the end-point of this reaction reliably. The product 4 was isolated as a solution in isopropyl acetate and was carried directly to the next step. This avoided purification by silica gel chromatography. The yield of 4 was 69% as determined by concentrating a known amount of the solution to dryness and subsequent 1H NMR analysis of the residue. Since conversion of 4 to 16 had to be run in ethanol, a solvent exchange from isopropyl acetate to ethanol was performed prior to the reaction. Then the ethanol solution of 4 was added to a mixture of piperidine (1.1 equiv) and aqueous formaldehyde solution (4.5 equiv), and the mixture was heated at reflux until the reaction was complete (∼2.5 h). Ethanol was removed, and the product 16 was extracted into isopropyl acetate. Acid 16 was not isolated, and the isopropyl acetate solution was used directly in the next step. Thus, purification of 16 by silica gel 184
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chromatography was not necessary. The yield of 16 was 78% as determined by concentrating a known amount of the solution to dryness. The HPLC purity of 16 was 95.6%. The coupling of 16 with (S)-(-)-4-benzyl-2-oxazolidinone was achieved using an improved literature procedure.4 Thus, 16 (1 equiv) was mixed with trimethylacetyl chloride (0.95 equiv) in the presence of triethylamine (2.5 equiv) in tetrahydrofuran at -15 °C to form the corresponding mixed anhydride, and then (S)-(-)-4-benzyl-2-oxazolidinone (0.9 equiv) and anhydrous lithium chloride (1 equiv) were added sequentially. The reason for using 99% purity. The key stereogenic center was created by a diastereoselective Michael reaction of O-benzyl-hydroxylamine with 17.3 The reaction was rather slow in THF at 45 °C, requiring 67 h to complete. Higher reaction temperature resulted in decreased diastereoselectivity. To reduce the reaction time, we executed the reaction under fairly concentrated conditions by adding the fluffy solid 17 to a solution of 2 equiv of O-benzyl hydroxylamine in a minimal amount of toluene, heating the slurry to
Scheme 2 a
a Reagents and conditions: (a) diethyl malonate (3), NaOMe, NaOH; (b) HCl, 69%; (c) piperidine, HCHO, 78%; (d) pivalyl chloride, LiCl, (S)-(-)-4-benzyl-2oxazolidinone, THF, -15 °C, 82.2%; (e) BnONH2 hydrochloride (9), toluene, NaHCO3, rt (f) TsOH monohydrate, EtOAc, rt, 60%; (g) NaOH, EtOAc, rt; (h) LiOH monohydrate, H2O2, THF, 0 °C, 74%; (i) Ac2O, HCO2H, 0 °C; (j) EtOAc, -10 °C (k) toluene/heptanes, dicyclohexylamine, rt, 79%; (l) 22, 1-methylimidazole, CH3SO2Cl, DMF, -10 °C, 77.4%; (m) conc. HCl, CH3CN, rt, 96.5%; (n) toluene, citric acid, rt; (o) 1-methylimidazole, CH3SO2Cl, DMF, -12 °C; (p) 10% Pd/C, HCO2NH4, EtOH, 60 °C, 60.1%.
48 °C for 3 h to obtain a clear solution and then distilling part of the toluene to further accelerate the Michael addition reaction. The reaction was usually complete in 40 h. The reaction mixture was then diluted with ethyl acetate and reacted with ptoluenesulfonic acid monohydrate to form the salts. The O-benzyl-hydroxylamine tosylate salt was insoluble in ethyl acetate and was filtered off. The mother liquor that contained 18 was concentrated, and tert-butyl methyl ether was added as an antisolvent to precipitate the desired salt 18. Although the diastereoselectivity in the reaction mixture was ∼3–4:1, the undesired diastereomer remained in the mother liquor, allowing the isolation of the desired product 18 as a solid in 60–64% yield containing only 3.0 kg of LCD320 (1). Conclusions A practical chromatography-free synthesis of the N-formylated hydroxylamine peptide deformylase inhibitor 1 was achieved by using a diastereoselective Michael reaction of 17 with O-benzylhydroxylamine to establish the key stereogenic center. We found that traces of Li+ had great impact on the diastereoselectivity of this reaction. The very expensive and not readily available Ghosez reagent used in the coupling reaction of 21 with 22 was replaced by a cheap and readily available reagent methanesulfonyl chloride in the presence of 1-methylimidazole. Dichloromethane was replaced with DMF, and lowering the reaction temperature eliminated epimerization of C-2 in 22. The coupling reaction using these new conditions was much cleaner, and intermediate 23 was isolated as a solid. In the coupling reaction 20 + 13 f 24, HATU was replaced with methanesulfonyl chloride as well, and the crude 24 could be carried to the next step without purification. Intermediate 24 was hydrogenated with 10% Pd/C and ammonium formate to afford 1, which was crystallized from isopropyl acetate/ ethanol, thus avoiding purification by silica gel chromatography. Experimental Section Materials were obtained from commercial suppliers and were used without purification. Melting points were obtained on Thomas-Hoover capillary melting-point apparatus. NMR data were obtained on a Bruker instrument operating at 500 MHz for 1H and 126 MHz for 13C. High resolution mass spectra were recorded on a Micromass LCT mass spectrometer with an Agilent 1100 LC system, or with a Micromass GCT mass spectrometer with field ionization direct probe. All reaction temperatures given refer to internal temperatures. (4S)-3-[2-(Cyclobutylmethyl)-1-oxo-2-propenyl]-4-(phenylmethyl)-2-oxazolidinone (17). An inerted 800-L reactor was charged with diethyl malonate (3, 30.4 kg, 189.77 mol) and ethanol (111 kg). Then 30% w/w sodium methoxide in methanol (34.2 kg, 189.77 mol) was added (exothermic) via an addition funnel over 10 min while maintaining the internal temperature at 20–30 °C. The reaction mixture was heated to 70 ( 3 °C over a period of 40 min and was stirred at this temperature for an additional 1 h. The reaction mixture was cooled to 65 ( 3 °C, and bromomethyl cyclobutane (2, 23.6 kg, 157.95 mol) was added over 20 min while maintaining the internal temperature at 62–73 °C. The yellow solution was heated to 70 ( 3 °C (reflux) and stirred at this temperature for 6 h. A solution of sodium hydroxide (19.0 kg, 475.0 mol) in deionized water (318 kg) was added over a period of 30 min while maintaining the internal temperature at 65–75 °C. The reaction mixture was heated to an internal temperature of 80 ( 3 °C over 10 min and stirred for an additional 3 h. It was Vol. 12, No. 2, 2008 / Organic Process Research & Development
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cooled to 25–30 °C over a period of 30 min and concentrated under reduced pressure (130–200 mbar) to ∼350 L batch volume. Isopropyl acetate (103 kg) was added. The mixture was stirred for 5 min, and the bottom aqueous layer was separated. The aqueous layer was cooled to 0 ( 3 °C over 20 min, and concentrated hydrochloric acid (50.4 kg) was added over a period of 20 min to adjust the pH to 2 ( 0.5 while maintaining the internal temperature at 0–20 °C. Isopropyl acetate (61 kg) was added to the batch, and the mixture was stirred for 10 min while warming to 20 ( 5 °C. The upper organic layer was separated and washed with 10% (w/v) aqueous sodium chloride solution (15 kg) to afford ∼90 kg of compound 4 solution in isopropyl acetate (containing 18.8 kg of 4). The solvent was removed under reduced pressure (130–200 mbar) to a batch volume of approximately 27 L, and ethanol (200 proof, 55 kg) was added. The solution was distilled under reduced pressure (110–150 mbar) to a batch volume of approximately 32 L, and the residue was cooled to 25 ( 5 °C over 20 min. Ethanol (200 proof, 89 kg) was added followed by 37% aqueous formaldehyde solution (44.2 kg, 546.92 mol). The mixture was stirred at 20 ( 5 °C, and piperidine (11.2 kg, 130.84 mol) was added over 30 min while maintaining the internal temperature at 20–35 °C to obtain a slurry. The slurry was heated to 80 ( 3 °C (reflux, CO2 evolution) over 50 min and stirred at this temperature for an additional 2.5 h. The reaction mixture was a solution at this point. The reaction mixture was cooled to 20 ( 3 °C over 40 min. The solvent was distilled under reduced pressure (90–120 mbar) to a batch volume of ∼34 L. Isopropyl acetate (74 kg) was added, and the batch was distilled under reduced pressure (130–200 mbar) to ∼34 L in volume. The distillation residue was diluted with isopropyl acetate (48 kg) and cooled to 20 ( 3 °C over 15 min. Then 2 N HCl (56 kg) was added over a period of 30 min while maintaining the temperature at 20–35 °C to adjust the pH to
