Synthesis, Characterization and Antiviral Properties of Pd(II) Complexes With Penciclovir

With the aim to improve and extend the antiviral activity of the antiherpic drug penciclovir, to a wider spectrum of viruses, we have synthesized and characterized new binary and ternary complexes of Pd(II) of formulae cis-(pen)2PdCl2 and cis,[(nucl)2Pd(pen)2]Cl2, where nucl = guanosine, inosine, cytidine or penciclovir. The characterization was mainly based on IR and 1H NMR spectroscopy, and the results showed that in all prepared complexes, penciclovir coordinates to the metal through N7. The far-i.r. spectrum of the complex cis-(pen)2PdCl2 confirmed the cis- geometry around Pd(II). All the prepared complexes were markedly active against HSV-1 and HSV-2 strains, but not against thymidine kinase-deficient HSV-1 strains.

Acyclic nucleoside analogues are well known for their antiviral activity[1 ]. The antiherpic drug, acyclovir (ACV), was the first acyclic nucleoside analogue shown to be antivirally effective [2]. Various other guanosine analogues have been synthesized, among which penciclovir or 9(4-hydroxy-3-(hydroxymethyl)but-l-yl)guanine (Fig. 1). Like acyclovir, penciclovir acts through a selective inhibition of viral DNA synthesis and replication [3]. For the acyclic nucleoside analogues to be antivirally active they must be enzymatically metabolized within the herpes virus-infected cells [4].
Thus the interactions of metal ions with acyclic nucleosides and their derivatives present a great interest, because the majority of enzymes, in virus-infected and uninfected cells, require metal ions for their activity [5]. Although several metal complexes of acyclovir have been synthesized, characterized and tested against a variety of viruses [6][7][8][9][10], to our knowledge, until today, there are not reports on the interaction ofpenciclovir with metal ions.
Herein we report on the synthesis, characterization and antiviral properties of some Pd(II) complexes with penciclovir. The nucleosides guanosine, inosine, cytidine were purchased from Sigma and used without further purification. Palladium(II) chloride was obtained from Fluka A.G. Penciclovir [11], and the complexes (guo)2PdCl2, (ino)2PdCl2 and (cyd)2PdCl2 were prepared according to the literature [12]. The reaction of H2PdCI4 with penciclovir at molar ratio 1:2, in strong acidic solutions (0.SN HCI), produces the c_is-(pen)2PdCl2, because the trans-influence of pen is comparable to that of pyridine [13] (eq 1). The ci___ configuration around the metal was confirmed by a Kurnakofftest [14]. A mixture of the above complex and excess of thiourea, were mixed in the solid state and dissolved in D20. The H NMR spectrum of the mixture showed the presence of free penciclovir.
The i.r. spectra of the complexes showed a strong band invariably at 1697 to 1726 cm q assigned to free _(C=O) of the 6 th position of the purine ring or the 3 t position of the pyrimidine ring, in the case of cyd containing complexes, excluding the formation of a Pd-O bond through the carbonyl group or the coordination through the neighboring N1. A similar behavior was observed in the infrared spectra of trans-and cis-(nucl)2PdCl or mixed transor c__is-[(nucl)2Pd(nucl')2]Cl2, were nucl or nucl_ guanosine, inosine or cytidine [12,13]. In addition all spectra of the prepared complexes, exhibited a broad band at about 425 to 430 cm q, attributed to Pd-N stretching vibration, suggesting coordination through N7. The far-i.r, spectrum of the binary complex ci_s-(pen)PdCl2 exhibited two strong to medium intensity bands at 325 and 329 cm " assigned to Pd-CI stretching vibration, confirming the cis__= configuration ofthe chlorine atoms [16].
The characteristic i.r. bands for the complexes and the a The H8 resonance of penciclovir, in the H NMR spectra of all five prepared complexes showed a downfield shitt by 0.38 to 0.46 ppm, compared to the free ligand in D20, indicating a covalent interaction of the Pd(II) ion with the neighboring to HS, nitrogen atom of the purine ring (N7). Similar strong downfield shifts of the guanosine's H8 proton, were observed in the binary or ternary complexes of the ligand with Pd(II) [12,13]. It is noticeable that in the spectrum of cis-(pen)2PdClz ( in DCI IN) the H8 proton resonance shifts upfield, compared with the protonated at N7 form of penciclovir, pen (in DCI IN), by about 0.47 ppm, indicating that the IT causes higher electron deshielding in the magnetic environment of the H8 nucleus than the palladium ion. 250 MHz spectra, n.a not assigned.

Antiviral properties.
All prepared penciclovir complexes were markedly active against HSV-1 and HSV-2 strains but not against thymidine kinase-defieient (TK') HSV in E6SM cell cultures (  (TABLE V). They were also not active against human immunodeficieney virus type (IIIa) and type 2 (ROD) in CEM and MT-4 cell cultures (data not shown). None of the compounds proved markedly cytostatic against murine leukemia L1210, murine mammary carcinoma FM3A and human lymphocyte Molt4 and CEM cells (50% inhibitory concentration > 100 tM) (except for compound II that inhibited Molt4 and CEM cell proliferation at 30-34 tM) (TABLE VI). Clearly, the compounds I-V displayed a similar antiviral spectrum as the parent compound peneiclovir. However, they were not superior to penciclovir in inhibiting herpes virus-induced cytopathicity in cell culture. Also, the test compounds lost marked activity against a TK-defieient herpes simplex virus as also penciclovir did. In general, the most active compound was III that contained four pencielovir molecules for each Pd atom in the entire molecule.
In conclusion, the Pd containing penciclovir derivatives had a comparable antiviral specmun as penciclovir (i.e. herpes simplex virus type 1 and 2), but were not superior to the parent compound.
The cytostatic activity measurements were basically described [20]. Briefly, tumor cells were seeded at 250,000-300,000 cells/ml in 200 pl-wells of 96-wells microtiter plates and incubated for 2 days (L1210, FM3A) or 3 days (Molt4/C8, CEM) at 37C in a humidified CO2-controlled atmosphere. At the end of the incubation period, calls were counted with a Coulter counter and the IC0 (50% inhibitory concentration) determined as the compound concentration required to inhibit tumor cell proliferation by 50%.