LABORATORY EXPERIMENT pubs.acs.org/jchemeduc
A Safer and Convenient Synthesis of Sulfathiazole for Undergraduate Organic and Medicinal Chemistry Classes Jeff Boyle, Sandra Otty, and Vijayalekshmi Sarojini* School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
bS Supporting Information ABSTRACT: A safer method for the synthesis of the sulfonamide drug sulfathiazole, for undergraduate classes, is described. This method improves upon procedures currently followed in several undergraduate teaching laboratories for the synthesis of sulfathiazole. Key features of this procedure include the total exclusion of pyridine, which has potential health hazards, from the first step of the synthesis and a simplified procedure for converting the intermediate p-acetamidobenzenesulfonamide to the final product by acid hydrolysis. Characterization of the synthetic sulfathiazole obtained by the modified route was achieved using MS, NMR, and antibacterial activity testing of the synthetic product against Escherichia coli DH5α. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Organic Chemistry, Safety/Hazards, Hands-On Learning/ Manipulatives, Bioorganic Chemistry, Drugs/Pharmaceuticals, Medicinal Chemistry, NMR Spectroscopy
T
he discovery of Prontosil by Gerhard Domagk in 1932 marked the beginning of the modern chemotherapeutic era.1 3 Since then, over 5000 sulfonamides have been prepared and tested for the prevention and cure of bacterial infections in humans.4 Synthesis of the sulphonamide drug sulfathiazole has been part of the medicinal chemistry course since 2002. This experiment is executed in two laboratory sessions (6 h in total). Students complete the synthetic part of sulfathiazole in the first session. In the second session, students purify the crude product by recrystallization, record IR spectra, and carry out antibacterial testing of the purified product against Escherichia coli. This laboratory is also appropriate for an organic or biochemistry laboratory.
or ingestion causing nausea, insomnia, headache, and abdominal pain.6 Pyridine and some of its derivatives are also known to induce infertility in male rats and is considered as a potential carcinogen.7 11 The quantity of pyridine used in this experiment has been minimal such that the health and safety regulation limits are complied with. However, given the potential health concerns stated above, an attractive alternative for large classes would be to exclude pyridine from this experiment. Recovery of the product sulfathiazole 4 from the reaction mixture after alkaline hydrolysis of intermediate 3 posed another practical difficulty for students. In the original procedure, shown in Scheme 1, sulfathiazole 4 was precipitated out of the alkaline reaction mixture by optimizing the pH to near neutral using a combination of concentrated and dilute (2 N) hydrochloric acids. Optimal pH of 7 8 is necessary for obtaining good quality product in reasonable yield. At the same time it is important to keep the volume as minimum as possible so that the product precipitates out of solution when the pH is between 7 and 8. This fine balance of optimal pH and volume is especially important for a small-scale synthesis for obtaining satisfactory results. In a typical procedure, students add 1 mL of conc HCl dropwise to an ice-cold solution of the reaction mixture with constant swirling and monitoring of the pH. After adding 1 mL of concd HCl, they switch to 2 N HCl and continue the neutralization process until a pH of 7 8 is reached. Addition of one extra drop of HCl is usually enough to protonate the free amino group
’ OLD SYNTHETIC SCHEME The synthetic procedure based on available literature is shown in Scheme 1.5 In this procedure, 4-acetamidobenzenesulfonyl chloride 1 is slowly added with stirring to a solution of 2-aminothiazole 2 dissolved in dry pyridine. The reaction mixture, after refluxing for 25 min, is poured into ice and the intermediate p-acetamidobenzenesulfonamide 3, which precipitates out, is collected by vacuum filtration. Intermediate 3 is then subjected to hydrolysis using 2 M NaOH for 50 min. The reaction mixture is neutralized carefully with hydrochloric acid to precipitate the product sulfathiazole 4, is collected by vacuum filtration, and is recrystallized from hot ethanol. One major drawback of this procedure is the use of pyridine during the first step of the synthesis. Pyridine is a toxic compound with an unpleasant odor and can enter the body by inhalation Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.
Published: November 04, 2011 141
dx.doi.org/10.1021/ed101120b | J. Chem. Educ. 2012, 89, 141–143
Journal of Chemical Education
LABORATORY EXPERIMENT
Scheme 1. Synthesis of Sulfathiazole (Old Method)
Scheme 2. Synthesis of Sulfathiazole (New Method)
Table 1. Results from the Hydrolysis of p-Acetamidobenzenesulfonamide to Sulfathiazole
of sulfathiazole resulting in very low yield because the protonated form of sulfathiazole is soluble in water. Many students found this neutralization step quite tricky to handle, especially near the end point and, often, after acidification led to a dramatic decrease in the yield and quality of the product sulfathiazole 4.
’ NEW SYNTHETIC SCHEME Replacement of Pyridine
To avoid using the toxic chemical pyridine in the large undergraduate teaching laboratory, alternative routes were explored for the synthesis of sulfathiazole that delivered the same learning objectives. Different organic as well as inorganic bases such as triethylamine (TEA), N-methyl morpholine (NMM), and anhydrous potassium carbonate were explored as alternatives for pyridine for the synthesis of sulfathiazole. Neither TEA nor NMM generated p-acetamidobenzenesulfonamide 3 as a solid product from the condensation of sulfonyl chloride 1 with amino thiazole 2 in dry acetonitrile. Instead, in both cases, a black tar resulted that was not worthy of any further analysis. On the other hand, condensation of sulfonyl chloride 1 with amino thiazole 2 to form the intermediate p-acetamidobenzenesulfonamide 3 in dry acetonitrile using anhydrous K2CO3 resulted in a light brown solid, which was confirmed to be the desired intermediate 3 based on NMR and MS data. The use of TEA or NMM for this reaction was not explored any further. The reader is encouraged to refer to other literature methods for the synthesis of sulfonamides under different conditions involving the use of various solvents and bases, as well as microwave assisted syntheses.12 14 The hydrolysis of the p-acetamido group of intermediate 3 by aqueous acid followed by neutralization with solid sodium bicarbonate would simplify the neutralization step and serve as a better option for undergraduate students. Use of solid sodium bicarbonate for the neutralization step eliminates the complications of over acidification as well as over dilution leading to poor yield and lower quality product. This modified route for the synthesis of sulfathiazole is shown in Scheme 2. The only concern was if the heterocyclic ring would be stable to acid hydrolysis conditions. To test this, the laboratory instructors carried out the synthesis of sulfathiazole 4 using the modified procedure shown in Scheme 2. The quality of the sulfathiazole synthesized following the modified protocol was confirmed by 1H and 13C NMR and MS analysis. The NMR data of the synthetic sulfathiazole obtained by this modified route was found to match perfectly with that of the commercial sample. The synthetic sulfathiazole
a
Reflux
p-Acetamidobenz-
Sulfathiazole,
Time/min
enesulfonamide, 3/g
4/g
Rfa
M + H+
20
0.502
0.174
0.57
256.0195
30
0.506
0.185
0.57
256.0193
40 50
0.502 0.503
0.178 0.206
0.57 0.57
256.0194 256.0191
TLC solvent system was DCM-MeOH (9:1).
obtained by the modified route was also tested for antibacterial activity against E. coli in comparison to the commercial sample. NMR and MS spectra and results of the antibacterial activity tests are provided as part of the Supporting Information. Shortening the Hydrolysis Time
To determine if shortening the hydrolysis time would have detrimental effects on product yield or quality, the progress of the acid-catalyzed hydrolysis of the intermediate, p-acetamidobenzesulfonamide 3, to sulfathiazole 4 was monitored as a function of time every 10 min starting from 20 to 50 min. Four parallel reactions were set up using the intermediate 3 synthesized from the same batch. The product isolated at 20, 30, 40, and 50 min, respectively (described in the Supporting Information), recrystallized from hot ethanol, and were characterized by TLC and ESIMS. There was no detrimental effect on the quality or yield of the final product between the four reactions. Product yields, Rf values, and ESIMS results obtained from these four reactions are summarized in Table 1. The 30 min hydrolysis time was chosen, this reaction was repeated several times, and reproducible results were obtained. Thus, a reflux time of 30 min is recommended for the acid-catalyzed hydrolysis of the intermediate p-acetamidobenzenesulfonamide 3 to sulfathiazole 4 for undergraduate classes.
’ HAZARDS 4-Acetamidobenzenesulfonyl chloride is corrosive and is harmful by inhalation, in contact with skin, and if swallowed. Acetonitrile is a highly flammable liquid and may be harmful by inhalation, ingestion, or skin absorption. 2-Aminothiazole may cause skin and eye irritation and may be harmful if swallowed. Hydrochloric acid is corrosive and causes burns to all body tissue. Potassium carbonate and sodium bicarbonate can cause eye 142
dx.doi.org/10.1021/ed101120b |J. Chem. Educ. 2012, 89, 141–143
Journal of Chemical Education
LABORATORY EXPERIMENT
Table 2. Student Yields of Sulfathiazole Using the New Synthetic Route Year
(6) International Chemical Safety Cards, ICSC: 0323, CAS 110-86-1. (7) Gross, G. A.; Turesky, R. J.; Laurent, B. F.; Stillwell, W. G.; Skipper, P. L.; Tannenbaun, S. R. Carcinogenesis 1993, 14, 2313–2318. (8) Elosua, C.; Bariain, C.; Matias, I. R.; Rodriguez, A.; Colacio, E.; Castillo, A. S.; Antonio, S. C.; Gutierrez, A. F. Sensors 2008, 8, 847–859. (9) Pyridine safety data. http://www.atsdr.cdc.gov/toxfaqs/tfacts52.pdf (accessed Oct 2011). (10) Pyridine safety data. http://cartwright.chem.ox.ac.uk/hsci/ chemicals/pyridine.html (accessed Oct 2011). (11) O’Morain, C.; Smethurst, P.; Dore, C. J.; Levi, A. J. Gut 1984, 25, 1078–1084. (12) Caddick, S.; Wilden, J. D.; Judd, D. B. J. Am. Chem. Soc. 2004, 126, 1024–1025. (13) Sharma, A. K.; Das, S. K. Synth. Commun. 2004, 34, 3807–3819. (14) Rad, M. N. S.; Khalafi-Nezhad, A.; Asrai, Z.; Behrouz, S.; Amini, Z.; Behrouz, M. Synthesis 2009, 3983–3988.
Student Yield (%)
2009
41
35
39
48
37
47
42
48
32
28
34
2010
46
50
42
41
45
48
47
44
45
32
44
irritation and ingestion of large amounts may cause diarrhea, nausea, vomiting, and respiratory irritation.
’ STUDENT RESULTS This modified procedure for the synthesis of sulfathiazole was executed in our medicinal chemistry undergraduate laboratory in 2009 and 2010. A range of percentage yields of sulfathiazole reported by students from the academic years 2009 and 2010 are summarized in Table 2. ’ CONCLUSION The modified synthetic procedure described here has the advantages of excluding the toxic chemical pyridine from undergraduate laboratories as well as the use of a simplified procedure for the hydrolysis of the p-acetamido group of intermediate 3, reaction work up, and recovery of the final product. Reaction time for the hydrolysis step has also been shortened without any detrimental effect on overall yield or quality of the product. To the best of our knowledge, the syntheses of sulfathiazole and its derivatives reported in the literature as well as followed in most undergraduate teaching laboratories use pyridine as a solvent and base for the condensation of sulfonylchloride with amino thiazole.4 This is the first report of the synthesis of sulfathiazole that excludes pyridine from the procedure and standardized in a way for convenient execution in large undergraduate teaching laboratories. ’ ASSOCIATED CONTENT
bS
Supporting Information Laboratory notes for the students; additional notes for the instructors and bioassay results; NMR and MS spectra. Answers to the postlab questions are available from the author. This material is available via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT Financial support from The University of Auckland in the form of a Teaching Improvement Grant is gratefully acknowledged. ’ REFERENCES (1) Sulek, K. Wiad. Lek. 1968, 21, 1089. (2) Domagk, G. Wien. Med. Wochenschr. 1954, 104, 817. (3) Domagk, G. Ann. N.Y. Acad. Sci. 1957, 69, 380. (4) Northey, E. H. The sulfonamides and allied compounds, American Chemical Society Monograph Series; Van Nostrand Reinhold: New York, 1948. (5) Roberts, R. M., Gilbert, J. C., Rodewald, L. B., Wingrove, A. S. Modern Experimental Organic Chemistry, 3rd ed.; Holt, Reinhart and Winston, Inc.: New York, 1979; p 361 377. 143
dx.doi.org/10.1021/ed101120b |J. Chem. Educ. 2012, 89, 141–143
