Synthesis and Crystal Structure Studies of Mitomycin C Dihydrate

The absolute configuration of mitomycin C was determined by X-ray single crystal diffraction (CuKα), and a new crystalline dihydrate of mitomycin C had been prepared. The experiment result provides a definitive answer to the real absolute configuration of mitomycin C and may put an end to the dispute that baffles researchers for decades. At the same time, some contentious structures about the mitomycin C in American Pharmacopoeia USP36-NF31, Chinese pharmacopoeia 2015 edtion and numbers of literatures are marked. The absolute configuration is also verified by 1D (H and C) and 2D (HSQC, HMBC, H-H COSY and NOESY) NMR studies indirectly. Powder X-ray diffraction (PXRD) pattern of the mitomycin C dihydrate is similar to that calculated for it, which suggests that the purity of sample is excellent.

American Pharmacopoeia USP36-NF31 and Chinese pharmacopoeia 2015 edtion define configuration B as the standard structure of mitomycin C; instead, European Pharmacopoeia EP7.0, British Pharmacopeia BP2017 and Japanese Pharmacopeia JP17 define configuration A. Then, we probed further and found Crystal Structure Theory and Applications out that two drugs Mitosol and Mitozytrex, approved by FDA, both record the structure as configuration B (Table 1). In Cambridge Structural Database System, S.K. Arora [10] reported different kinds of single crystal of mitomycin C, but they failed to reach a mutual agreement. The reason contributed to this question may be due to limitation of early crystallography techniques, for example, they were inclined to use molybdenum target radiography, by which radiography is hardly to obtain believable absolute configuration of compound, rather than copper target.
In this paper, we determined the absolute configuration of mitomycin C by X-ray single crystal diffraction (CuKα), analyzed its structure by 1D and 2D NMR spectra, and prepared a new crystalline. Most importantly, the result of our experiment can provide a definitive answer and put an end to the dispute about the real absolute configuration of mitomycin C.

Reagents and Instruments
All reagents were obtained from commercial sources and used without further purification. 1 H NMR and 13 C NMR spectra were recorded on a Varian Unity 400 MHz spectrometer. 2D NMR were recorded by a Varian Unity 100 MHz NMR spectrometer and used to assist in structure elucidation. DMSO-d 6 was used as the solvent in both of 1D and 2D NMR spectra (DMSO-d 6 : 2.49 ppm for 1 H and 39.9 ppm for 13 C). The powder X-ray diffraction (PXRD) pattern was recorded on crushed single crystals in the 2θ range of 6˚ -55˚ with a CuKα radiation.

Preparation of Single Crystal
Dissolve mitomycin C (200 mg) in water (40 ml), then the solution was stirred in room temperature for 20 minutes [12]. After the violet insoluble substance was filtered, chloroform (10 ml) was added into the violet aqueous solution slowly.
Keep the mixture in a sealed container stored in a dark place at room temperature and many purple-dark prismatic crystals appeared on the container wall after 3 days.

NMR Spectra
The 1 H, 13 C, HSQC, HMBC, 1 H-1 H COSY NMR spectra allowed the assignments of all the protons to their bonding carbons (Table 2), which confirmed the planar structure of the molecule correct. There was a striking contrast between the signals of two protons linking to C 3 (C 13 ) in 1 H NMR spectrum (δH 3 : 4.00, 3.35; δH 13 : 4.53, 4.10) and this phenomenon could be ascribed to the chiral structure of C 2 and C 10 .
The relative configuration of compound was assigned by NOESY spectra, in particular, by comparing the 1 H-1 H COSY ( Figure 2) and NOESY (Figure 3) spectra, we observed that H 15 had no 1   different orientation with C 10 -C 13 and C 1 -N 1 .

Y. Q. Feng et al.
Unfortunately, the 1D and 2D NMR spectra did not provide enough information to ascertain the absolute configuration of mitomycin C [13] [14]. Thus, a single crystal of mitomycin C was prepared.

Description of the Single Crystal Structure
The absolute configuration of the title compound is shown in Figure 4. These configurations at C 10 , C 11 , C 1 and C 2 are, respectively, (S), (R), (S) and (S) in our crystal, which structure is in accordance with configurations A, but is exactly opposite to configurations B ( Figure 5).  is 56.36 (0.27)˚. This structure is demarcated by the benzoquinone cycle, carbamoyloxymethyl side chain on one side, and two water molecules on other side ( Figure 3). The selected bond lengths and bond angles are given in Figure 6.
We can observe an interesting phenomenon from Figure 7 that water molecules become the center of this unit cell, which are surrounded by the order array of mitomycin C. The interacting with each other by hydrogen bonds (Table   3) of these mitomycin C forming a construction of "well", at the same time these water molecules are inlaid in the "well" steady because of the hydrogen bonds between water and mitomycin C.

Powder X-Ray Diffraction (PXRD) Analysis
The mitomycin C dihydrate is also characterized by Powder X-ray diffraction (PXRD). As shown in Figure 8, the PXRD pattern is almost consistent with its simulated spectra, indicating the sample has good purity.

Conclusion
In this research, it has been found that the absolute configuration of mitomycin C at C 10 , C 11 , C 1 and C 2 is, respectively, (S), (R), (S) and (S). This conclusion is confirmed by X-ray single crystal diffraction with Cu radiation, which could give Crystal Structure Theory and Applications In addition, the structure of mitomycin C also is confirmed and described in detail by 1D and 2D NMR spectra. What's more, a novel crystal morphology called mitomycin C dihydrate is obtained, which has the advantages of simple preparation and stable quality, and shows a promise to be a new drug crystal morphology.