Synthesis of typical sulfonamide antibiotics with [ 14 C]- and [ 13 C]-labelling on phenyl ring for environmental studies

As a kind of used antibiotics, sulfonamide (SAs) has become ubiquitous environmental contaminants that caused The of in complex need to be cost SAs. Using commercially available uniformly [ l4 C]- and [ l3 C]-labelled aniline as starting material, we synthesized [phenyl-ring- 14 C]- and [phenyl-ring- l3 C]-labelled sulfamethoxazole (SMX), sulfamonomethoxine (SMM), and sulfadiazine (SDZ) using four-step (via condensation of labelled N-acetylsulfanilyl chloride and aminoheterocycles) or ve-step (via condensation of labelled N-acetylsulfonamide and chloroheterocycles) reactions in good yields (5.0−22.5% and 28.1−54.1% for [ 14 C]-and [ 13 C]-labelled SAs, respectively) and high purities (> 98.0%). of low nucleophilic activity. This study provided synthetic methods for effective laboratory preparation of commercially unavailable labelled SAs, which benet to studies on fate and behavior of SAs in both natural and engineered environments and biological systems.


Background
Sulfonamide antibiotics (SAs) are widely used in the treatment of human disease and in modern livestock. Due to their low biodegradation and insu cient removal in waste water treatment plant [1,2], high concentrations of sulfonamides, such as sulfadiazine (SDZ), sulfamethoxazole (SMX), and sulfamonomethoxine (SMM), are widely detected in environmental media such as agroecosystem, sediments, and rivers [3][4][5]. After entering the environment, SAs exert adverse effects on organisms [6][7][8][9], and the environment hazards and risk of SAs have caused a widely concern. A comprehensive understanding of SAs in environment, including its adsorption, biodegradation, transformation, formation of non-extractable residues (NERs), and transport, helps assess their environment risks.
Techniques using [ 14 C]-radioactive and [ 13 C]-stable isotopes are often used to study the environmental behavior of pollutants. For example, [ 14 C]-tracer with low detection limit and convenient handling with complex environmental samples is used to investigate the environmental fate especially the mineralization and NERs of organic pollutants such as pesticides, brominated ame retardants, alkylphenols, and polycyclic aromatic hydrocarbons [10][11][12][13][14]. Using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy as analytical tools, stable isotopes (e.g., 13 C, 15 N) helps quantify and identify metabolites of the pollutants in complex matrices [15][16][17][18][19][20]. Phospholipid fatty acid analysis and DNA stable-isotope probing using [ 13 C]-tracers are powerful tools for analysis of microbial biomass and community composition [21,22]. Therefore, [ 14 C]-and [ 13 C]-labelled SAs are increasing in need, however are either commercially unavailable or commercially too expensive. E cient and easyoperating ''in house'' syntheses of [ 14 C]-and [ 13 C]-SAs, especially on micro-scales with good yields, are highly desired.
Compared with conventional synthetic method, a successful synthesis of [ 14 C]-SAs on a micro-scale requires stable solvents, suitable reaction conditions, and simple puri cation methods of each product [23]. The conventional synthetic method of unlabelled SAs was a four-step route, including acetylation of aniline using acetic anhydride, chlorosulfonation of N-acetylaniline with ClSO 3 H, condensation of sulfonyl chlorides with nucleophiles such as amines, and alkaline hydrolysis of the acetyl-protecting group [24][25][26][27]. It is however notable that the synthetical conditions in the route described above was suitable for synthesis of SAs at gram-level and cannot be applied to synthesis of SAs on a micro-scale (milligram-level) due to the di culty of mixing in solvent-free condition and crystalizing of products. In a previous study, [ 14
The extract was evaporated and the product was puri ed by ash chromatography with an elution gradient (Table S1), resulting in 8a (1.81 × 10 8 Bq) in 98.3% yield with a purity of 99.0% as analyzed by TLC using petroleum ether: ethyl acetate (1:4 / v: v), containing 0.2% CH 3 COOH as eluent (R f value of 8a = 0.26) coupled to autoradiography.
Uniformly [phenyl-ring-l4 C]-labelled SDZ ( 10a ) The crude 9a (9.25 × 10 7 Bq, 6.29 × 10 8 Bq/mmol, 57.0% radiochemical purity) was reacted with NaOH solution (10%, 5 mL) for 3 h at 100°C and neutralized with 6 M HCl to pH 6. The products were extracted with ethyl acetate (15 mL each) eight times. The extracts were dried with anhydrous Na 2 SO 4 , evaporated to around 0.5 mL. The crude product was then recrystallized from boiling methanol. The precipitates were centrifuged and washed three times with methanol, resulting in 10a (3.11 × 10 7 Bq, 6.29 × 10 8 Bq/mmol). The purity of 10a was 98.3% as determined by HPLC (t R = 5.73 min. For details, see SI). The supernatant was further extracted ve times with ethyl acetate (15 mL each), which was dried with anhydrous Na 2 SO 4 and evaporated to dryness, giving solids containing 10a. The solid product was mixed with unlabelled SDZ (54 mg) and then recrystallized from boiling methanol. The precipitate was washed three times with methanol, resulting in another portion of 10a with a low speci c activity (1.10 × 10 7 Bq, 7.40 × 10 7 Bq/mmol) with a radiochemical purity of 98.3%. The total amount of 10a was 4.21 × 10 7 Bq with a total yield of 79.9%. The chemical structure of 10a were characterized by 1 H-NMR, 13  Uniformly [phenyl-ring-l3 C]-labelled N-acetylaniline ( 2b ) To 13 C-labelled aniline hydrochloride (1b, 3.00 g, 99% of 13 C atom) in a 200-mL ask were K 2 CO 3 solution (0.32 g/mL, 30 mL) and acetic anhydride (4.70 g) added sequentially with stirring at 25°C. The mixture was further stirred at 25°C for 1 h and then extracted ve times with ethyl acetate (15 mL each). The extract was washed with 20 mL of H 2 O and then evaporated, resulting in 2b (3.01 g, 99% of 13 C atom, 99.0% purity (For detail, see SI.4) in 95.7% yield.
The mixture was heated at 58°C for another 2 h and cooled down to room temperature. White crystals were formed after dropwise addition of ice-cold water (10 mL) to the mixture and were washed twice with ice-cold water (each 10 mL) by ltration, resulting in 3b (4.29 g, 99% of 13 C atom, 96.0% purity (SI.4)) in 82.8% yield.
Then acetone was removed by evaporation. After addition of ice-cold water and adjusting with 6 M HCl to pH about 6, 8b (672 mg, 99% of 13 C atom, 98.0% purity (SI.4)) was obtained in 73.0% by ltration and washing with ice-cold water.

Results And Discussion
Labelled SMX, SMM, and SDZ with uniformly labelling of l3 C and l4 C on the phenyl ring were prepared from commercially available labelled aniline, via four-step or ve step syntheses (Fig. 1). The yields and radiochemical or chemical purities of the products are summarized in Table 1. Three unlabelled SAs and intermediates were synthesized in the same way and characterized by HPLC-Q-TOF-MS/MS and NMR, shown in SI, Table S2. Chlorosulfonation of aniline on the para-position of the amino group is the key step for the synthesis of SAs. Prior to the chlorosulfonation with ClSO 3 H, acetylation of aniline is needed to prevent possible oxidation of the amino group and bis-sulfonation on the ring during chlorosulfonation. We performed the acetylation in aqueous solution with addition of K 2 CO 3 to improve nucleophilic activity of aniline (1a), resulting in acetylaniline (2a) in a good yield of 90.0%. The method with less procedures was more convenient than the previous report [29].  (Table 1). Our method not only completely converted 2a, but also reduced decomposition of 3a by hot H 2 SO 4 , which was generated by hydrolysis of excess ClSO 3 H.
Water inhibits the condensation of 3a with amino heterocyclic compounds (e.g., 11 and 12). To avoid the water interference, molecular sieves were applied to adsorb the water during the condensation. With this method, we obtained 4a and 6a in good yield of 51.0% and 15.6%, respectively ( Table 1).
The condensation of 3 with amino heterocycles was a nucleophilic substitution. Compound 11 had a higher nucleophilic activity than compound 12, according to their electron cloud density, which was in agreement with the higher yield of 4a than 6a (51.0% vs. 15.6%, respectively) and 4b than 6b (73.8% vs. 42.3%, respectively) ( Table 1). Condensation of 3 with other heterocyclic compounds could be used to prepare other [ 14 C]-or [ 13 C]-labelled sulfonamides, such as with 2-aminopyrimidine for SDZ [28]. However, owing to the low nucleophilic activity of 2-aminopyrimidine, the yield of 10a at micro-scale was very low (7.4%, and overall yield of from 1a to 10a was 2.4%) and the yield of 9b was also lower than 4b and 6b (21.0% vs. 73.8% and 42.3%, respectively) ( Table 1). Therefore, for the preparation of 10a and 10b, we used a ve-step synthetic pathway (Fig. 1). We used two steps to synthesize 9 instead of one step. We rstly synthesized 8 by condensation of 3 with ammonium hydroxide, which has a high nucleophilic activity and is a base capable of neutralizing the by-product H 2 SO 4 , in good yield of 98.3% for 8a and 73.0% yield for 8b. Coupling of 8 to 13 gave both 9a and 9b in good yield (36.0% and 74.8%, respectively). The synthesis of 9 from 3 via this two-step pathway not only completely converted 3 to 8 with a higher stability, to avoid the decomposition of 3, but also gave a much higher overall yield than the one-step reaction (35.4% vs. 7.4% for 9a, 54.6% vs. 21.0% for 9b).  [30]. The peaks of C-atoms in 13 C-labelled compounds are split into triplets due to 13 C− 13 C coupling and have much higher intensity than those in the non-labelled compound with natural 13 C-atom abundance (1.1%), thus the triplet signals can be used to identify chemical nature of labelled carbon atoms, e.g., the residues of pesticides (e.g., cyprodinil), humus monomers (e.g., catechol), and emerging pollutants (e.g., tetrabromobisphenol A) bound to soil humic substances [31][32][33], which provide more clear information about incorporation into humic substances of pollutants with labeling on single or double carbon atoms (e.g., SDZ, nonylphenol, chlorophenol) [34][35][36].

Characteristics of the synthetic methods
The main advantages of our synthetic methods over those previously reported are the success to Puri cation of products is important for product quality. We also provide feasible methods for puri cation of small amount of [ 14 C]-products and obtained [ 14 C]-compounds with high purity. Crystallization in water as puri cation procedure or direct use of reaction mixture of previous synthetic step without further puri cation could be appliable to synthesis of unlabelled SAs at gram-scale [26,27], which are however not appliable to the synthesis of [ 14 C]-labelled SAs at milligram-scale, because recrystallization may recover much less products. In this study, we used classic chromatographic separation method, such as ash column chromatography and preparative TLC, to purify small amount of [ 14 C]-products.

Conclusions
This study describes optimized methods for synthesis of SAs labelled with 14 13 C, relatively to aniline). The methods consist of four-step (via condensation of 3 and aminoheterocycles) or ve-step (via condensation of 8 and chloroheterocycles) reactions. The four-step pathway is suitable for synthesis of large amount of SAs (e.g., gram level) or SAs containing aminoheterocyles of high nucleophilic activity, while the ve-step pathway is especially appliable to synthesis of SAs (e.g., SDZ) at milligram scale containing an aminoheterocycle of low nucleophilic activity. This study provided synthetic methods for effective laboratory preparation of commercially unavailable labelled SAs, which bene t to studies on fate and behavior of SAs in both natural and engineered environments and biological systems.