In Vivo Nitric Oxide Synthase Inhibitors Can Be Deprived of This Activity: Unexpected Influence of the Tetrachloroplatinate(II) Counteranion. Crystal Structures of Bis(S-Methyl-Isothiouronium)-N,N′-Bis(3-Guanidinopropyl)Piperazinium and Hexamidinium Tetrachloroplatinates(II) Salts

The synthesis and crystal structures of bis(S-methylisothiouronium) (MSTUH)+, N,N′-bis((3- guanidinopropyl)piperazinium (PipeC3GuaH4)4+ and hexamidinium (HexaH2)2+ tetrachloro platinate(ll) salts ( called hereafter PtMSTU, PtPipeC3Gua and PtHexa respectively ) were investigated. These compounds contain the “amidine” function ( - C(=NH)NH2 ) in which the H atoms supplied by the acid have become attached to the imino group of each terminal amidino function. Moreover, in PtPipeC3Gua, the nitrogen atoms of the chair-piperazine moiety are also protonated. The influence of tetrachloroplatinate(ll) counteranion ( versus sulfate, nitrate and diisethionate ) in the in vivo nitrite inhibition by the (MSTUH)+, (PipeC3GuaH4)4+ and (HexaH2)2+ cations was investigated. The three tetrachloroplatinate(ll) salts, unexpectedly, do not inhibit significantly the in vivo nitrite production in comparison with the other salts (sulfate, nitrate and diisethionate and their corresponding previous countercations) which exhibit NO synthase inhibition, especially bis(S-methylisothiouronium) sulfate, a selective and potent inducible NO synthase (iNOS) inhibitor commonly used as standard.


Introduction
The free radical and paramagnetic molecule nitdc oxide (NO) is produced enzymatically in physiological and pathological conditions by a family of NO synthases NOS which catalyze the oxidation of L-arginine from the guanidino group to L-citrulline. These enzymes include constitutive cNOS and inducible iNOS forms. NO from endothelial constitutive isoform (ecNOS) is a potent vasodilator and was identified with the endothelium dependent relaxing factor EDRF which mediates vascular relaxation in response to acetylcholine and substance P ]. NO is also implicated in the regulation of blood pressure and inhibits platelet aggregation and leukocyte adhesion 2 ]. Endothelial overproduction of NO is implicated in atherosclerosis, diabetes and hypertension 3 ]. The continuous release of NO from the ecNOS keeps the vasculature in a continuous state of active vasodilatation.
The neuronal enzyme is found in brain and in peripheral nervous cells. In addition to its role in blood vessels, NO is a neurotransmitter found in high density in emotion-regulating brain regions [4 ]. NO from the constitutive neuronal isoform is implicated in neurodegeneration and neuroinjury. The inducible form iNOS is expressed in response to immunological stimuli in macrophages and other cells smooth muscle cells, hepatocytes and produces large amounts of NO which can act as cytotoxic molecule 5 ]. The cytotoxicity of NO from activated macrophages In Vivo Nitric Oxide Synthase Inhibitors Can Be Deprived of this Activity: Unexpected Influence of the Tetrachloroplatinate(II) Conteranion. plays a role in their antimicrobial activity. NO synthesized by lymphocytes is greatly implicated in apoptosis phenomena. Recent studies showed that treatment with exogenous NO produces DNA strand breaks 6 ]. The generation of endogenous NO in large amount is implicated in circulatory shock secondary to the release of the pr0iflammatory cytokines tumor necrosis factor TNFx and interleukine ILl 7 ]. Antiinflammatory cytokines including IL4, ILl0 and IL13 inhibit the induction of iNOS. Overproduction of NO by iNOS has been implicated in the chronic inflammation, vascular dysfunction in diabetes and transplant rejection. Novel therapeutic approaches could be designed to modulate the NO biosynthesis drugs with high selectivity for iNOS inhibition may be useful for treatment of neurodegenerative disorders, chronic inflammatory diseases and septic shock whereas drugs inhibiting brain NO synthase bNOS may be protective against neuroinjury.
Many types of NOS inhibitors are known. The generation of NO can be inhibited by analogues of the substrate L-arginine No-propyI-L-arginine was recently described as a potent and neuronal nitric oxide synthase inhibitor 8 ]. Other classes of compounds that are not amino acids but containing the amidine function C = (NH) NH2 ), such as amidines 9 ], Salkylisothioureas 10 and substituted aminoguanidines and aminoisothioureas 11 ], are reported to be inhibitors of NOS. Bis(S-methylisothiouronium) sulfate is known to be a potent and preferential inhibitor of human and rat iNOS ;it exerts beneficial effects and improves survival in a rodent model of septic shock [ 12 ]. More recently, 1H-pyraz01e-l-carboxamidine hydrochloride (or PCA and its substituted derivatives have been shown to inhibit NOS with an increased selectivity toward iNOS when the pyrazole ring is substituted, as in . In the course of our investigations, we have previously examined the ability of some N,N'-(alkane-l,0-diyl)bis(guanidinium) and N,N'-bis (3-guanidinopropylpiperazine) dinitrates to inhibit nitrite production in rat pleural cavity 14 ]. Moreover, in a previous paper, we have demonstrated that N,N'-(octane-l,8-diyl)bis(guanidinium) tetrachloroplatinate(ll) exhibits cytostatic effects against trophozoites of an Acanthamoeba strain, in contrast to the tetrachloropalladate(ll) salt which lacks this effect 15 ]. This prompted us to study the possible modifications of pharmacological properties of NOS inhibitors when pharmacologically inert anions sulfate, nitrate, chloride were replaced by other counteranions such as tetrachloroplatinate(ll) which might reverse or enhance the expected properties.
Hexamidine diisethionate is a drug well-known for its antimicrobial effects which result from cationic surface active properties as a consequence of the bipolar structure of its cation. To our knowledge, its NOS inhibitor properties have not been described.
The aim of this work was to compare the anti-NO synthase properties of bis(Smethylisothiouronium) sulfate, a compound chosen as a reference NO inhibiting drug in our previous experiments ), N,N'-bis(3-guanidinopropylpiperazine) dinitrate and hexamidine diisethionate versus the corresponding tetrachloroplatinate(ll) salts, called hereafter PtMSTU, PtPipeC3Gua and PtHexa respectively. All these compounds contain the amidine function.

2.
Results and discussion.

2.1.
Descriptions of the structures.
In the three tetrachloroplatinate(ll) salts, the amidine function C(=NH)NH2 was protonated with the H atoms supplied by the acid at the imino group of each terminal amidino function. (Tables 1 and 2 2.1.1. Description of the PtMSTU structure. Figure la The asymmetric unit consists of half of a square planar [PtCI] anion and one [MSTUH] cation. The S(1) C(2) bond length of 1.782 (7) A is in good agreement with the length of a pure $ C single bond 1.82 A while the S(1) C(1) bond length (1.730 (6) A is significantly shorter and indicates arl approximately 50% double-bond character. The atoms $(1), C(1), N(1) and N (2) of the isothiouronium cation were coplanar and the methyl carbon. C(2) is 0.17 A out of this plane, to which the S(1) C (2) bond is inclined at an angle of 5.6". Between the two N C $ bond angles, there is a difference of 7.6". The larger angle is the one to which the remote substituent 128 on the sulfur atom stands in a syn-relationship as shown by the torsion angles of 6.02 for the N(2) C(1) S(1) C(2) and of-174.48 for the N(1)-C(1) S(1) C(2). Similar observations have been made on the crystal structures of bis(S-methylisothiouronium) sulfate 16 and Smethylthiouronium p-chlorobenzoate 17 ].
All the amidinium H atoms are involved in hydrogen bonding to C1(1), C1(1), C1(2) and C1(2"). The most noticeable feature is that there is one bifurcated hydrogen bond involving H(102) attached to N(1) with C1(1 ) and C1(2) symmetry code i'-x, y, z ii" x, y, z iii :x, l+y,z). The asymmetric unit consists of two half crystallographically independent and square planar [PtCI4] 2 anions and one half [PipeC3GuaH4] 4* cation where PipeC3GuaH4 is the tetraprotonated form of the organic ligand N,N'-bis (3-guanidinopropyl)piperazine. The crystal structure shows that i) the organic cation presents a center of symmetry, ii) the four H atoms supplied by the acid have become attached to the two nitrogen atoms of the piperazine moiety and, to the imino group of each terminal amidino function. The three equivalent guanidinium C N bonds vary from 1.31(1) to 1.34 (2) ,& which are in agreement with those found in N,N'-(octane-l,8-diyl)bis(guanidinium) tetrachloroplatinate(ll), the so-called [OBG]2*[PtCI4]2 from 1.295(9) to 1.314 (8) ,& 15 ]. The atoms C (11), N (12), N (13) and N(14) are coplanar and the propyl carbon C(10)is 0.14 ,& out of this plane, to which the N(14) C(10) is inclined at an angle of 5.40. The diprotonated piperazinium moiety exhibits a chair conformation, with the N(1) and its centrosymmetrically related atom being 0.70 A out of the C(2) C(3) C(2=) C(3=) equatorial plane symmetry code :i 2 x, y, z ). The torsion angles in the N(14) C (10) 5, No. 3, 1998 In Vivo Nitric Oxide Synthase Inhibitors Can Be Deprived of this Activity: Unexpected Influence of the Tetrachloroplatinate(II) Conteranion.

Description of the PtHexa structure. Figure lc
The two phenyl dngs of the hexamidinium are twisted by 14.4" with respect to each other, compared to 35* in the pentamidine complex formed with the dodecanucleotide d( CGCGAATTCGCG )2 18 ]. The torsion angles show that the central -(CH2)6-linker chain exhibits an all trans configuration, as in free pentamidine 19 ], in contrast with the pentamidinedodecanucleotide complex in which the deviations from 180" pure staggered values are responsible for the shortening of the chain length to 9.13 . compared to 9.75 A value found in the crystal structure of free pentamidine 19] ). The amidinium groups are twisted out of the planes of the phenyl dngs by 25.0 and 155.0" compared to 27* in free pentamidine and to 3 and -6* in the pentamidine complex. Moreover, in PtHexa, the dihedral angle between the two amidinium groups is 132.5".
The crystal packing is characterized by intermolecular bgnds invol.v.ing C1 (2)  NO generation was assessed 0.5 and 7 hours after the induction of the inflammatory reaction. According to previous studies 20 this production correlated to the activation of constitutive and inducible NO synthases respectively.
The three non-platinum salts tested demonstrated a significant inhibition of NO generation during an experimental inflammatory reaction. This inhibition was observed on constitutive NOS (in the early time of inflammation and on inducible NOS in the later time and may be beneficial in some pathological situations[ 21 ]. Moreover, the NO synthase antiinhibitor properties of these new tetrachloroplatinate(ll) salts is described here for the first time, to our knowledge. These tetrachloroplatinate(ll) salts did not, however, enhance nitrite production as shown in Figure 2 A and Figure 2 B. As NO exerts an ambivalent role on many physiological functions, control of its over-production may be of therapeutic value 22 ]. This property, demonstrated for an antiseptic compound such as hexamidine, may add some cytotoxic activity to its other properties. On the contrary, if this anti-NO property is unsuitable, formation of tetrachloroplatinate(ll) salts deprived of action on NOS may be appropriate.

Experimental
Synthesis of the tetrachloroplatinate(ll) salts.

3.2.
Structure determination. The refined cell constants and other relevant crystal data are presented in Table 1, together with details of the intensity measurements. The crystals were mounted, using glass fibers, on an ENRAF-NONIUS CAD4 diffractometer equipped with a graphite monochromator. The lattice parameters were refined using 25 reflections. The data were collected using the 0-2e scan technique and with Mo Ko radiation % = 0.71073 ,& ).
During the data collection, three intensity control reflections were monitored every two hours, showing no loss of intensity. The data were corrected for Lorentz and polarisation effects. The structure was solved by a combination of direct methods using SIR procedure 23 and heavy-atom techniques and refined by full-matrix least-squares method based on F, using CRYSTALS 24 ]. An empirical absorption correction with the program DIFABS 25 was used. Anisotropic displacement parameters were assigned to all non-H atoms. The hydrogen atoms were introduced in calculated idealized positions (d(C-H) 0.98 ,& and their atomic coordinates were recalculated after each cycle. They were given isotropic thermal parameters 20% higher than those of the carbon to which they are attached. Least-squares refinements were performed by minimizing the function .w(IFol-lFol) =, where Fo and Fc are the observed and calculated structure factors. The weighting scheme used in the last refinement cycles was w Carrageenan X was obtained from Pierrefitte Auby Neuilly/Seine, France ). Hanks' solution without phenol red ), N-(1-naphthyl)ethylenediamine, and sulfanilamide were purchased from Sigma Chemical Co. St. Louis, Mo, USA ). Phosphoric acid was purchased from Merck (Darmstadt, Germany and bis(S-methylisothiouronium)sulfate was purchased from Aldrich Steinheim, Germany ).

Animals and treatments
Male Sprague-Dawley rats weighing 180-200 g Depre, Saint-Doulchard, France were used for all experiments 29 ]. Protocols were submitted to and approved by the local ethics committee.
Pleurisy was induced by intrapleural injection of 0.1 mL of 10 g/L carrageenan X suspension in saline 30 ].
Bis(S-methylisothiouronium) sulfate, PipeC3Gua dinitrate and hexamidinium diisethionate on the one hand and the corresponding tetrachloroplatinate(ll) salts on the other hand were administered 0.1 mL with the inflammatory 14 ]. Animals were euthanized with ether, and the right pleural cavity was opened for pleural exudate collection after 30 minutes or seven hours. Samples were centrifuged at 500 g for 5 min, and the supernatants were then centrifuged at 1000 g for 20 min in order to remove exudate fibrin. Samples were then adjusted to a final volume of ml with Hank's solution and stored at 80C until nitrite determination was carded out. 3.3.3. Nitrite determination in pleural exudate. Nitrite was measured in pleural exudate as a parameter of NO formation 31 ]. Aliquots of 0.1 mL were incubated in individual wells of a 96-well plate, with 0.1 mL of Griess reagent 0.5 g/L N-(1-naphthyl)ethylenediamine and 5 g/L sulfanilamide in phosphoric acid 50 g/L )), at room temperature for 10 min. The absorbance was measured at 550 nm using a microplate reader (Dynatech, MRX ), in comparison with the incubating medium Hank's solution without cells.
Sodium nitrite was used to establish a nitrite standard curve.