Synthesis and In Vitro Evaluation of Novel Acyclic and Cyclic Nucleoside Analogs with a Thiadiazole Ring

The synthesis of six thiadiazole nucleoside analogs is reported: 5-diacetylamino-1,2,4-thiadiazol-3-one (1), 5-amino-2- (tetrahydrofuran-2-yl)-1,2,4-thiadiazol-3-one (2), 5-amino-3-[(2′-hydroxyethoxy)methyl]-1,3,4-thiadiazol-2-one (3), 5-amino-3-(4′-hydroxy-2′-hydroxymethyl-butyl)-1,3,4-thiadiazole-2-thione (4), (R)-5-amino-3-(2′,3′-dihydroxypropyl)-1,3,4-thiadiazole-2-thione (5), and (S)-5-amino-3-(2′,3′-dihydroxypropyl)-1,3,4-thiadiazole-2-thione (6). The synthesis, characterization, and properties of these new synthesized thiadiazole derivatives are discussed. A dimerization of 5-amino-3H-1,3,4-thiadiazole-2-thione (14) by sodium nitrite resulting in di-(5-amino-1,3,4-thiadiazol-2-yl) disulfide (19) is also reported. The preliminary in vitro evaluation of these newly synthesized compounds is discussed.

For this reason, 5-amino-2H-1,2,4-thiadiazol-3-one and 5amino-3H-1,3,4-thiadiazol-2-one can be considered as the analogs of cytosine.Based on this analogy, within the framework of our systematic studies, we have synthesized some novel acyclic or cyclic nucleoside analogs with a thiadiazole ring instead of a pyrimidine ring.

Preparation of 5-Amino
The literature shows two other possible synthetic approaches.Rüfenacht [30] used potassium methyl xanthate and hydrazine hydrate to form O-methylester. Phosgene was introduced to O-methyl ester to produce 5-amino-2-methoxy-1,3,4-thiadiazole by a cyclization reaction.Subsequently, the hydrolysis of 5-amino-2-methoxy-1,3,4thiadiazole yielded 13 as the product.Clarkson and Landquist [31] reported a similar approach, but they used cyanogen chloride for the cyclization reaction.Considering that both phosgene and cyanogen chloride are war gases and not easy to handle, solid cyanogen bromide was used in our synthesis to perform this cyclization reaction.Following Clarkson and Landquist's procedure, 2-amino-5-ethoxy-1,3,4-thiadiazole (12) was successfully synthesized in an 80% yield.The hydrolysis of 12 with a trace of hydrochloric acid gave 13 as white crystals in 53% yield.The structures of compounds 11-13 were identified by their analytical and spectral data.These data were also compared with literature values for identification.

Preparation of Bis-(5-amino-1,3,4-thiadiazol-2-yl) Disulfide (19) by Dimerization.
In our attempts to carry out a diazotization reaction, we discovered an interesting dimerization reaction.The reaction was designed to use sodium nitrite and an acid to prepare nitrous acid in situ, which could react with 14 to form a diazonium salt.Then, this diazonium group could be replaced by a functional group such as OH.Various experimental conditions and acids have been used to study this reaction.After treating 14 with sodium nitrite and acetic acid in an ice bath, a yellow solid was produced in 73% yield.Surprisingly, the NMR and mass spectrometry have shown that this yellow product was not the compound we proposed from a diazotization reaction, but a disulfide, bis-(5-amino-1,3,4-thiadiazol-2-yl) disulfide (19) (Scheme 5).The primary amino group on the aromatic thiadiazole ring was not affected by the diazotization reaction at all.Besides acetic acid, another reagent-stannic chloride (SnCl 4 ⋅5H 2 O) was also used with sodium nitrite to yield 19 successfully in 68% yield.
It is of interest to report that sodium nitrite with an acid or stannic chloride can oxidize 14 to produce disulfide 19.The preparation of 19 was reported by Mahieu et al. in 1986 with sulfonyl chloride as the oxidant [39].Another oxidizing agent reported for this reaction was sodium chlorite, which was described by Ramadas and Srinivasan [40] and Ramadas et al. [41].Our new reaction conditions can represent a good addition to the synthesis of disulfide 19.The structure of 19 was determined by IR, 1 H NMR, 13 C NMR, and the melting point.These data were in agreement with not only the proposed disulfide structure but also the literature values [39,40] for identification and confirmation.

Bioactivity Results and Discussion
All new synthesized thiadiazole acyclic and cyclic nucleoside analogs (1-7) and disulfide 19 were tested for cytotoxicity against P388 murine leukemia cells, PANC-1 human pancreatic cancer cells, A549 nonsmall cell lung adenocarcinoma cells, DLD-1 human colon cancer cells, and NCI/ADR drugresistant human breast cancer cells.These structures of thiadiazole nucleoside analogs did not prove to be significantly cytotoxic to the tumor tissue cultures at concentrations of 5 g/mL.This result did not surprise us because most of our target compounds are acyclic nucleosides, which are mostly antiviral agents and not anticancer agents.
Compounds 1-7 and 19 were also tested for their antimicrobial activity against Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA).Better results were obtained in screening for antimicrobial activity.Two compounds, 3 and 19, were active against Staphylococcus aureus and MRSA at 50 g per disc.The minimum inhibitory concentration (MIC) for compound 3 toward S. aureus was >50 g/mL, and toward MRSA was >50 g/mL as well.MIC of disulfide 19 toward S. aureus was 50 g/mL and toward MRSA was 25 g/mL.
In summary, the thiadiazole acyclic and cyclic nucleoside analogs 1-7 and disulfide 19 are not active against five available kinds of cancer cells.Although they are not cytotoxic, it was noted that 3 and 19 possess considerable antibacterial activity against S. aureus and MRSA.

Experimental
The melting points were determined on a Fisher-Johns melting point apparatus (W.H. Curtin & Co.) or Mel-Temp (Electrothermal). 1 H and 13 C NMR spectra were recorded with a Varian 400-MHz spectrometer.Infrared spectra were measured on a 4020 GALAXY series FT-IR spectrometer (Mattson Instruments) (potassium bromide disk), or on an Avatar 320 FT-IR spectrometer (Nicolet Instruments).UV spectra were measured on a Cary 3 UV-visible spectrophotometer.Thin layer chromatography (TLC) used silica gel 60 F-254 precoated plates, and the spots were located in the UV light or by iodine vapor.Low resolution MS spectra were recorded on an M-8000 Hitachi mass spectrometer with an L-7100 pump and ion trap mass analyzer.All low resolution mass spectra were obtained in ESI positive mode.High resolution mass spectra were determined by Mass Spectrometry Services at University of Florida, Gainesville, FL, USA.Elemental analyses were performed by Desert Analytics, Tucson, AZ, USA.Specific rotation measurements were conducted at Perkin-Elmer model 141 polarimeter by Dr. David A. Lightner at the University of Nevada, Reno, NV, USA.All solvents used were reagent grade, except for dimethyl sulfoxide, chloroform, acetone, and methanol used in NMR spectroscopic measurements.(8) [41].Potassium thiocyanate was predried with anhydrous tetrahydrofuran (THF) by stirring overnight.Then, the white powder was filtered off and dried under vacuum on the rotary evaporator to remove THF.A solution was prepared by the addition of 48 g (0.49 mol) potassium thiocyanate in 600 mL toluene.To this solution, 60 mL (0.50 mol) benzoyl chloride was added dropwise with stirring.The solution became milky white after the addition of benzoyl chloride.The mixture was refluxed for 4 hours under argon.The color changed from white to orange.Then, the solution was cooled to room temperature, the white precipitate was filtered off, and the amber filtrate was refluxed with 24.0 g urea (0.40 mol) for 5 hours.Then the reaction mixture was cooled to room temperature and placed in an ice bath for 2 hours to form the crystals.The solution was stirred periodically, and the walls of the flask were scratched when the solution was in the ice bath.After crystallization, bright yellow crystals (33.39 g) were filtered off from the cold solution and dried.This was the crude product, mp 168-171 ∘ C. Recrystallization from acetonitrile yielded 32.06 g of bright yellow solid, yield 36%, mp 174-175 ∘ C (lit.mp: 175 ∘ C [38]).Thiobiuret (9).N-ureidocarbothioyl benzamide (11 g, 49 mmol) was added to a solution of 220 mL methanol with 0.5 mL of concentrated HCl.The mixture was refluxed for 55 hours.The reaction mixture was cooled to room temperature, and then all the solvent was evaporated.The yellow residue was washed with 5 mL of hexane twice to remove the methyl benzoate and then dried under reduced pressure.Recrystallization from water yielded 4.34 g of light yellow powder, yield 75%, mp 176-177 ∘ C. The second crystallization from acetonitrile gave a white powder, mp: 186-188 ∘ C (lit.mp: 189-193 ∘ C [38]).3251 (m, N-H), 1717 (s, C=O), 1580 (s, NH 2 bending), 1490 (s, N-H bending) cm −1 .