Carmofur was obtained from Shanghai Oriental Pharmaceutical Science and Technology Co. Ltd. Acetonitrile (CH₃CN), Methanol (CH₃OH), Ethanol (CH₃CH₂OH) and Acetic acid (CH₃COOH) were purchased from Shanghai crystal pure biological technology co., Ltd. Trifluoroacetic-acid (TFA), Formic acid (HCOOH), Triethylamine (Et₃N) and ammonium solution (NH₃, 25%) were purchased from Shanghai Ling Feng Chemical Reagent Co., Ltd. Sodium hydroxide (NaOH) is purchased from Sinopharm Chemical Reagent Co., Ltd.

In our study, some instruments were used as follows DMX 500 Nuclear Magnetic Resonance Spectrometer (Brucker Germany), Shimadzu UV-1601PC instrument (Japan Shimadzu), AB135-s electronic balance (Switzerland METTLER TOLEDO), Color plate (Yixing crystal optical instrument co., LTD.) and pipetting gun (Shanghai Hanlin Experimental Instrument Co., Ltd.).

Scanning wavelength: 200 nm - 400 nm; Absorbance range: 0.0 - 2.0.

All the Experimental data were recorded at 298 K.

Solvent: Acetonitrile (CH₃CN), Methanol (CH₃OH), Ethanol (CH₃CH₂OH).

Acid: Trifluoroacetic-acid (TFA), Acetic acid (CH₃COOH), Formic acid (HCOOH), Base: Triethylamine (Et₃N), Sodium hydroxid solution (0.2 g/ml), Ammonium solution (NH₃, 25%).

UV measurements were made on a spectrometer. Firstly, we measured precisely1ml carmofur solution (1 mg carmofur dissolved in 10 ml acetonitrile) by the pipetting gun, addition to 2 ml solvent. Then, a certain amount of acid was added to the above acetonitrile solution of carmofur respectively. The base was added to carmofur dissolved in acetonitrile containing a certain equivalent of acid.

The variable?temperature ¹H and ¹³C NMR of carmofur were recorded on the same equipment in Acetonitrile: Trifluoroacetic-acid (9:1) at −10˚C, 10˚C, 20˚C, 40˚C, 60˚C, 70˚C.

In the process of UV absorption spectra test, we found that UV spectroscopy of carmofur could change clearly in CH₃CN. Upon titration of the carmofur solution with TFA, the absorption band centered at λ_max = 213 nm gradually disappears (Figure 2). Because of the addition of trifluoroacetic acid, the solvent polarity and dipole increased, resulting in the polarity of the solute increased. It also caused the maximum wavelength red shift and decreased the absorbance. The result showed that π bonding orbital of carmofur structure jumped into anti- bonding orbital. When Et₃N was added to the protonated solution, the original spectrum centered at λ_max = 213 nm is steadily regained. This is because anti-bonding orbital of carmofur structure jumped back into π bonding orbital. From this change phenomenon, it is apparent to show that the acid/base switching process in CH₃CN is fully reversible.

The result indicated the addition of TFA in CH₃CN could induce conformational alteration of carmofur and Et₃N could make the altered conformation of carmofur back to the original conformation. The acid/base induced amide conformational alteration process was reversible. We found that NaOH (0.2 mg/cm³) and NH₃ also could make the altered conformation of carmofur back to the original conformation. (Figure 2)

In Figure 3, the UV spectra indicated the addition of CH₃COOH in CH₃CN could induce conformational alteration of carmofur. In the protonated solution by CH₃COOH, Et₃N/NaOH (0.2 mg/cm³)/NH₃ could make the altered conformation of carmofur back to the original conformation.

In Figure 4, the UV spectra indicated the addition of HCOOH in CH₃CN could induce amide conformational alteration of carmofur. In the protonated solution by HCOOH, Et₃N/NaOH (0.2 mg/cm³)/NH₃ could make the altered conformation of carmofur back to the original conformation.

There are the same phenomenon of carmofur in solvents such as CH₃CH₂OH and CH₃OH. The addition of HCOOH/TFA/CH₃COOHin CH₃CH₂OH/CH₃OH could induce conformational alteration of carmofur. In the

protonated solution, Et₃N/NaOH/NH₃could make the altered conformation of carmofur back to the original conformation.

In addition, we also did likewise experiment with inorganic acid, such as sulfuric acid, hydrochloric acid and phosphoric acid. When sulfuric acid, hydrochloric acidor phosphoric acid was added to the carmofur solution respectively, UV spectroscopy of carmofur had no obvious change. Inorganic acid (sulfuric acid, hydrochloric acid and phosphoric acid) could not induce conformational alteration of carmofur.

In the variable temperature ¹³C-NMR spectrum of carmofur in Acetonitrile: Trifluoroacetic-acid (9:1), we found that ¹³C-NMR spectrum of carmofur contains two kinds of data at room temperature (Figure 5). The result showed that there were two conformers of carmofur in Acetonitrile: Trifluoroacetic-acid (9:1) at room temperature, which is due to the partially double-bond character of carmofur’s amide bond at 20˚C. Meawhile, in the variable temperature ¹H-NMR spectrum of carmofur in Acetonitrile: Trifluoroacetic-acid (9:1), ¹H-NMR spectrum of carmofur appeared only a set of data at room temperature (Figure 6). The phenomena showed ¹H-NMR spectrum of two conformers of carmofur were the same.

Along with the gradual increase in temperature to 70˚C, ¹³C-NMR spectrum of carmofur revealed that these two kinds of data remain the same proportion and have no any other changes (Figure 7). When discreasing temperature to −10˚C, these two kinds of data still had no any changes (Figure 7). As we can see from Figure 6, ¹H-NMR spectrum of carmofur appeared no any changes at the different temperature. The above experimental

phenomena testified that temperature did not have any effect on these two conformers of carmofur in Acetonitrile: Trifluoroacetic-acid (9:1). These two conformers of carmofur were structurally stable in Acetonitrile: Trifluoroacetic-acid (9:1).