Crystal structure of nafamostat dimesylate

The crystal structure of nafamostat mesylate is reported, which is a serine protease inhibitor, and has been applied clinically as an anticoagulant and anti-inflammatory agent.


Chemical context
Nafamostat mesylate (I) is the bismethanesulfonic salt of 6amidino-2-naphthyl-4-guanidinobenzoate. It shows broadspectrum serine protease inhibition effect, and is also a reversible competitive inhibitor as camostat mesylate (II) (Tamura et al., 1977;Fujii & Hitomi, 1981;Matsumoto et al., 1989). Although nafamostat mesylate has been applied clinically with success as an effective anticoagulant and antiinflammatory agent during hemodialysis and for treatment of severe acute pancreatitis (Takeda et al., 1989), the crystal structure has not previously been reported.
In addition, nafamostat has attracted attention as an inhibitor for the activity of transmembrane protease serine 2 (TMPRSS2), a host cell serine protease that mediates viral cell incursion for influenza virus and coronavirus, thereby inhibiting viral infection and replication (Yamamoto et al., 2016(Yamamoto et al., , 2020Hoffmann et al., 2020). Since nafamostat contains flex- ISSN 2056-9890 ible moieties, it is necessary to determine the conformation to understand the structure-activity relationships. The crystal structure of nafamostat mesylate (I) is reported herein. From the crystallographic study, the phenylguanidine groups in nafamostat and camostat are essentially similar except for the direction of residual groups.

Structural commentary
The nafamostat moiety in the title compound ( Fig. 1) shows a divalent cation with a screw-like motif, which consists of four planar parts: the amidino group, the naphthyl group (rings A and B), phenyl ring C and the guanidinium group (shown in Fig.1). The dihedral angles between the amidino and naphthyl groups, the naphthyl group and ring C, and ring C and guanidinium group are 11.35 (13), 44.66 (10) and 51.11 (15) , respectively. The guanidinium group is approximately perpendicular to the naphthyl group, subtending a dihedral angle of 84.30 (14) .
The overlay of nafamostat (green) and camostat (red) is presented in Fig. 2, in which the r.m.s. deviation is 0.027 Å for phenylguanidinium groups. The partial structures are essentially similar, except for the direction of residual groups. Very recently, the crystal structure of human TMPRSS2 in a covalent complex with nafamostat has been solved (Fraser et al., 2021). The nafamostat in the complex is hydrolysed, and results in phenylguanidino acylation of Ser441 (yellow) in the active site. It was considered that the nafamostat moiety may be easily nucleophilic-attacked, approaching from the O13 atom side without steric hindrance.

Supramolecular features
In the crystal, the naphthyl groups of nafamostat form hydrophobic columnar structures, shown in Fig. 3

Figure 2
Overlay of the crystal structures of nafamostat moiety (green), camostat moiety (red) and covalently binding partial structure (yellow) of mature nafamostat with Ser441 in the active site from pdb7MEQ (Fraser et al., 2021), using Mercury (Macrae et al., 2020).

Figure 1
The title compound nafamostat mesylate (I) showing the atom and ring labelling. Displacement ellipsoids are drawn at the 50% probability level.

Figure 3
Part of the crystal structure of nafamostat mesylate (I). The naphthyl groups related by the inversion center (yellow) and equivalent (grey) are stacking along the b-axis direction, forming a columnar structure. The methanesulfonate groups containing the S27 and S32 atoms are represented in blue and red, respectively. H atoms have been omitted for clarity. thyl groups correlated with the inversion center (yellow) are stacking along the b-axis direction, in which the perpendicular distances of the centroid of the naphthyl ring system and those at (Àx, 1 À y, 1 À z) and (Àx, 2 À y, 1 À z) are 3.4208 (8) and 3.5134 (8) Å , respectively. The columnar structures are surrounded by a hydrophilic region consisting of the methanesulfonate ions and the guanidinium, imidamidium and ester groups. The two independent methanesulfonate ions play different roles. The columnar structure intercalates the methanesulfonate group (blue) containing the S27 atom, and is linked to three neighbouring guanidinium groups and one diamine group. Hydrogen bonds [N25-H25AÁ Á ÁO28 = 2.827 (3) and N26-H26BÁ Á ÁO29 vii = 2.925 (3) Å ; Table 2] link the molecules, forming an infinite C 2 2 (8) chain, with other hydrogen bonds [N25-H25BÁ Á ÁO29 vi = 2.931 (3) and N26-H26AÁ Á ÁO31 vi = 2.916 (3) Å ] forming an R 2 2 (8) ring. The columnar structures are also consolidated by the other methanesulfonate ion (red) containing the S32 atom, which is linked by two opposing amidino groups [N15-H15AÁ Á ÁO33 iii =2.854 (3) and N15-H15BÁ Á ÁO34 iv = 2.830 (3) Å ], related by the inversion center, into an R 4 4 (12) ring. A weak C-HÁ Á Á interaction is also observed ( Table 2).

Synthesis and crystallization
Nafamostat mesylate (CAS No. 82956-11-4) was purchased from Tokyo Chemical Industry Co. Ltd (TCI). A small portion (ca 10 mg) was dissolved in a small volume of hot water (ca 100 mL), and acetone (ca 900 mL) was added slowly until it became cloudy white. On slow cooling to ambient temperature, colourless octahedral crystals suitable for X-ray measurements were obtained.

Refinement
Crystal data, data collection and structure refinement details at a low temperature (95 K) are summarized in Table 3. All the H atoms were located in difference-Fourier maps. In the NH or NH 2 groups, H atoms were freely refined. The C-bound H atoms were included in calculated positions and treated as riding atoms: C-H = 0.95-0.98 Å with U iso (H) = 1.2-1.5U eq (C).