Thionation of bicyclic β-lactam compounds by Lawesson ’ s reagent

We have developed a study on the thionation of cephalosporins and penicillins using Lawesson’s reagent. Best results were obtained in the preparation of 7α-methoxy-8-thioxocephalosporins (70-98% isolated yield), and 6α-methoxy-7-thioxopenicillins (14% isolated yield).


Introduction
It has been more than seven decades since Fleming observed the antibacterial action of penicillin 1 and about sixty years since penicillin was introduced into clinical practice.The subsequent development of other classes of β-lactam antibiotics (cephalosporins, cephamycins, carbapenems, monobactams) has made these compounds one of the most successful therapeutic agents to date and the most commonly prescribed antimicrobials.The remarkable biological effect of β-lactam antibiotics results from their capacity to disrupt the biosynthesis of the bacterial cell wall.Through the years, the effectiveness of β-lactam antibiotics has decreased due to the rapid evolution of bacterial resistance not only to single but also to multiple antibiotics, thanks to mutation and gene exchange. 2Bacterial resistance to antibiotic drugs is also aggravated by the overuse of antibiotics in humans and animals and the noncompliance to the course of treatment by patients.The most common form of bacterial resistance to the β-lactam antibiotics is the production of β-lactamase enzymes in some bacteria. 3These enzymes efficiently hydrolyze the amide bond of the β-lactam ring to give products that are devoid of antibacterial activity.Therefore, a current challenge in medicinal chemistry is the generation of new structures which will overcome the defense mechanisms of the bacteria.
To this end, β-thiolactams surge as an interesting class of non-β-lactamic analogues of penicillins and cephalosporins.Thionation, the conversion of a carbonyl group into a thiocarbonyl group, can be performed by different methodologies.Phosphorus pentasulfide and boron sulfide were the most common thionating reagents until the development of 2,4-bis(4methoxy-phenyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide, known as Lawesson's reagent (LR) (1) (Figure 1), an excellent reagent for converting carbonyl into thiocarbonyl compounds. 4hionation of monocyclic β-lactams (monobactams) has been studied by different groups. 5owever, synthesis of bicyclic β-lactams such as penicillins and cephalosporin, has been scarcely reported.In 1975, Wojtkowski et al. 6 reported the thionation of some cephalosporins to form the corresponding 8-thioxocephalosporin using boron sulfide.The highest yield obtained was 20%.Similarly, thionation of penicillin derivatives was preformed with only 1% yield.Recently, the synthesis of the 8-thioxocephalosporin (2) was reported in good yield using Lawesson's reagent (Figure 1).In order to obtain a general access to a variety of 8-thioxocephalosporins and 7thioxopenicillins for biological screening and mechanistic studies, we became interested in the development of a reliable synthesis of these compounds.In this paper, we present a study about the scope and limitations of thionation of cephalosporins and penicillins using Lawesson's reagent.

Results and Discussion
First, we selected cephalosporin 3 as the substrate for thionation studies (Scheme 1).Compound 3 was synthesized according to Chauvette et al. 8 Different conditions for thionation were attempted, starting material was recovered unless high temperature (100°C), two equivalents of Lawesson's reagent, and two hours of reaction were applied.Under these conditions, a compound was isolated in excellent yield (89%) after column chromatography.The isolated material was characterized as the thioamide 4 by comparison with the 1 H NMR data reported by Wojtkowski. 6Due to their strained β-lactam ring, penicillins and cephalosporins have the normal amide resonance restricted.This causes an increase in the double bond character between the carbon and oxygen atoms, making their chemical behavior closer to that of a ketone group.Then, preparation of 4 was in agreement with Nishio et al., 9 that established that the amide group tends to be more reactive to LR than ketones.The corresponding β-thiolactams 5 and 6 could not be obtained under any of the conditions employed.
Esters 9-11a were subjected to thionation with LR under different conditions.Best results were obtained when 9-11a were treated with 1.8 equivalents of LR for 3 h at 90°C, hence, the corresponding 7α-methoxy-8-thioxocephalosporins (12-14a) were obtained in isolated yields ranging from 70 to 98%.

Scheme 3
Together with cephalosporins, penicillins are the most successful class of β-lactam antibiotics.Considering that the best yield reported for the synthesis of a 7-thioxopenicillin was 1%, 6 we decided to test the reaction of a series of penicillins with LR (Figure 2).Generally speaking, penicillins demonstrated to be less reactive than cephalosporins to LR. Benzyl 6,6dihydropenicillanate 1,1-dioxide (15) and methyl 6,6-dibromopenicillanate (16), remained unreactive under conditions optimized for cephalosporins (toluene, 1.8 equiv. of LR, 90°C, 3 h) and, when conditions were more severe, an extended decomposition was observed.Furthermore, under the optimized conditions, thionation of benzyl 6α-chloropenicillanate (17) led to decomposition products.

Figure 2
Only in the case of using benzyl 6α-methoxypenicillanate (18) as starting material, the expected β-thiolactam was isolated.Different conditions were attempted, mostly leading to decomposition; however, when compound 18 reacted with two equivalents of LR in toluene at Experimental Section General Procedures.Chemical reagents were purchased from commercial sources and were used without further purification unless noted otherwise.Solvents were analytical grade or were purified by standard procedures prior to use.Infrared spectra (IR) were recorded on a Shimadzu Prestige 21 spectrophotometer and only partial spectral data are listed. 1H NMR spectra were recorded on a Bruker AC200 at 200 MHz in CDCl 3 unless otherwise stated, in the presence of TMS (0.00 ppm) as the internal standard.Conventional and gel-phase 13 C NMR spectra were recorded on the same apparatus at 50 MHz with CDCl 3 as solvent and reference (76.9 ppm), unless otherwise stated. 13C NMR assignments were made on the basis of chemical shifts and proton multiplicities (from DEPT spectra).Analytical thin-layer chromatography (TLC) was carried out with silica gel 60 F 254 pre-coated aluminum sheets (Merck).Flash column chromatography was performed using Merck silica gel 60 (230-400 mesh).
[α] D 25 = +57.9(c 1.25, CHCl 3 ); IR (film) 1769 cm -1 (C=O, ester), 1714 cm -1 (C=O, lactam) , 1223 cm -1 (ester II band); To a solution of the crude 7-α-methoxy-3'-deacetoxycephalosporanic acid (663.3mg) in anhydrous DMF (5.0 mL), triethylamine (870 µl, 631 mg, 6,2 mmol, 2,1 equiv.)and p-methoxybenzyl chloride (700 µl, 770 mg, 4,9 mmol, 1,7 equiv.)were successively added dropwise at 0°C.The temperature was increased to 45ºC, and the reaction mixture was stirred for 4.5 h.The reaction was treated with water and extracted with AcOEt (5 x 25 mL).The combined organic layers were washed with 1N HCl (2 x 10 mL), 0.5N NaOH (2 x 10 mL), brine and dried over Na 2 SO 4 .The solvent was removed under reduced pressure to give a brown oil (1.12 g) as crude reaction product.The residue was purified by flash column chromatography eluting with hexane and AcOEt.Two fractions were obtained: the 7-βepimerized product 11b (75.1 mg, 5% from 7-ADCA) and the expected product 11a (283.9 mg, 30% from 7-ADCA  .6β-aminopenicillanic acid (6-APA) (1.0 g, 4.6 mmol) was suspended in anhydrous DMF (8.0 mL) at room temperature and triethylamine (1.3 mL) was added dropwise.The reaction mixture was heated at 45ºC and once the suspension becomes a solution (~20 min), benzyl chloride was added dropwise.The mixture was stirred at the same temperature for 4.5 h and then allowed to reach room temperature.Ethyl acetate (10 mL) was added and the triethylammonium chloride was filtered.The organic layer was washed with water (3 x10 mL), dried over Na 2 SO 4 and concentrated to a half of its original volume.After that, a solution of pTsOH (880 mg) in AcOEt (15 mL) was added at 0ºC to give a yellow solid which was filtered and washed successively with AcOEt and ethyl ether to afford, after under reduced pressure, 1.17g (51%) of the ammonium salt of the benzylated 6-APA as a white solid.A portion of this white solid (337.9 mg, 0.71 mmol) was placed in a round bottom flask and water (20 mL) and CH 2 Cl 2 (20 mL) were added with vigorous stirring.Then, NaNO 2 (800 mg, 11.6 mmol) and pTsOH (160 mg , 0.93 mmol) were added dropwise at 0ºC.After 10 min at 0ºC, the layers were separated in a separatory funnel.The organic layer was washed with brine and dried over Na 2 SO 4 to give 338.9 mg of the benzyl 6-diazopenicillanate as a deep yellow oil.This oil was dissolved in methanol and stirred overnight at room temperature.Solvent was evaporated under reduced pressure and the residue was purificated by chromatography column to give 74.0 mg (33% from the ammonium salt) of pure product 18.[ .penicillin 18 (90.8mg, 0.28 mmol) was placed in a round bottom flask, the system was purged with anhydrous nitrogen and the compound dissolved in anhydrous toluene (4.5 mL).Lawesson's reagent (1) (113 mg, 0.28 mmol, 2.0 equiv.) was previously dried in a vacuum desiccator over phosphorus pentoxide for 1 h, and then added to the solution of 18.Once the reagent was added, the reaction mixture was stirred at room temperature for 15 min and then for 1 h at 85ºC.Solvent was removed under reduced pressure.The crude product was filtered over silica gel using CH 2 Cl 2 as eluting solvent.The solvent was evaporated and then the residue was purified by chromatography column (hexane-AcOEt) to provide 12.2 mg (14%) of pure product 19.