Thiosemicarbazones of Acetylpyrazines : Preparation and Their Hydrophobic Properties

Some substituted acetylpyrazine derivatives were prepared as the starting materials for the subsequent synthesis of thiosemicarbazones. General synthetic approach of all newly synthesized compounds is presented. All the thiosemicarbazone derivatives of acetylpyrazines were analyzed using the reversed phase high performance liquid chromatography (RP-HPLC) method for the lipophilicity measurement. The procedure was performed under isocratic conditions with methanol as an organic modifier in the mobile phase using end-capped non-polar C18 stationary RP column. In the present study the correlation between RP-HPLC retention parameter Log K (the logarithm of capacity factor K) and various calculated Log P values is shown. The relationships between the lipophilicity and the chemical structure of the studied compounds are discussed as well.


Introduction
Thiosemicarbazones are mostly prepared by the condensation of aldehydes or ketones with thiosemicarbazide.
Acetone thiosemicarbazone, which decomposes easily, can be used instead of the free thiosemicarbazide in some cases [1,2].Thiosemicarbazones substituted on the terminal nitrogen are usually obtained by the reaction of methyl hydrazinecarbodithioates with amines [3][4][5].Klayman and Lin [6,7] described the preparation of a variety of Nmono and N,N-disubstituted thiosemicarbazones by the displacement of the dimethylamino function of the corresponding N,N-dimethylthiosemicarbazones by a primary or secondary amine.The reaction was further improved by Scovill [8] who used N-methyl-N-phenylthiosemicarbazones as the starting compounds for transamination.Preparation methods, synthetic applications and biological importance of thiosemicarbazones have been reviewed recently [9,10].Examining biologically active derivatives of pyrazine, we have found that acetylpyrazine thiosemicarbazones exhibited promising biological effects [11,12].Since the potency of thiosemicarbazones often increases by the replacement of the terminal NH 2 by a substituted amino group, analogous N,N-dimethylthiosemicarbazones were prepared and tested for tested for antifungal, antimycobacterial and antiproliferative activity [13].
One of the major prerequisites for pharmacological screening and drug development is the prediction of absorption, e.g. the transport of a molecule through cellular membranes.The drugs most frequently cross the biological barriers by the passive transport, which strongly depends on the lipophilicity.Therefore hydrophobicity is one of the most important physical properties of biologically active compounds.This thermodynamic parameter describes the partitioning of a compound between an aqueous and an organic phase and is characterized by the partition (log P) coefficient.Classical methods for the determination of these constants are time consuming and not always sufficiently reliable.Therefore, reversed phase high performance liquid chromatography (RP-HPLC) methods have become popular and widely used for lipophilicity measurement.A general procedure is the measurement of the directly accessible retention time under isocratic conditions with varying amounts of an organic modifier in the mobile phase using end-capped non-polar C 18 stationary RP columns and calculating the capacity factor K. Log K, calculated from the capacity factor K, is used as the lipophilicity index converted to log P scale [14].

Results and Discussion
The carboxamide moieties in the starting material 1a and substituted pyrazine-2-carboxamides 1b-1h were dehydrated to the corresponding nitrile groups (compounds 2a and 2b-2h) by means of phosphoryl chloride.
Alkylation of the amide 1a or the nitrile 2a was performed using the mixture of a carboxylic acid, ammonium peroxydisulfate and silver nitrate as a source of the alkyl radical.Pyrazine-2-carbonitrile (2a) and its 5-alkylated analogues 2b-2h were then converted to the corresponding acetyl derivatives 3a-3h via the Grignard reaction [29,30].Acetylpyrazines 3a-3h with N,N-dimethylthiosemicarbazide yielded the final thiosemicarbazones 4a-4h [3,13].Analogous acetophenone N,N-dimethylthiosemicarbazone 5 was prepared for comparison in the same synthetic pathway.The general synthetic approach to all newly synthesized compounds is shown in Scheme 1.
According to 1 H NMR experiments all the final products are E-isomers.The thioxo moiety of all thiosemicarbazones was confirmed by means of IR spectroscopy [13].
Scheme 1. Synthesis and structures of the target substituted N,N-dimethylthiosemicarbazones 4a-4h and 5.
Hydrophobicities (log P / Clog P data) of the studied compounds were calculated using two commercially available programs and measured by means of RP-HPLC determination of capacity factors K with a subsequent calculation of log K.The results are shown in Table 1 and Figure 1.In the figure they are arranged in the ascending manner according to the experimental log K values.
N,N-Dimethylthiosemicarbazones reported here are much more hydrophobic (log K = 0.2335 -2.6160) than the thiosemicarbazones described previously (log K = 0.1437 -1.7246) [11,12].However, the higher lipophilicity did not automatically result in higher biological activity.Antifungal activity was only improved with the compound 4a.In all other cases it remained the same or was lowered.The same applies for antiproliferative activity where compound 4a was the most potent.Nonetheless, in the antiproliferative assay the potency of alkylated derivatives 4b-4h gradually increased with the number of atoms in the alkyl chain and the lipophilicity, and the high activity of 4a was rather surprising.Antimycobacterial activity of N,N-dimethylthiosemicarbazones was mostly lower compared to thiosemicarbazones, except for 4g and 4h.Compound 5 was inactive in all performed bioassays [13].These results clearly indicate that the lipophilicity is only one of the factors affecting biological activity of thiosemicarbazones.contrary to all the computed data.1-(5-Isobutylpyrazin-2-yl)ethan-1-one N,N-dimethylthiosemicarbazone 4e is slightly less lipophilic than its congeners 4d (R = butyl) and 4f (R = tert-butyl).This is in a good agreement with the results of our previous studies [12,22], as well as with the computed log P data.
Great differences between the experimental and calculated log P (ChemOffice) could be observed for the compound 5 (acetophenone N,N-dimethylthiosemicarbazone), see Figure 1.The non-heterocyclic derivative 5 is situated between 4c (R = isopropyl) and 4e (R = isobutyl) according to log K and shows relatively low lipophilicity.
According to the calculated log P (ChemOffice), its hydrophobicity should rather correspond to that of 4f (R = isobutyl) or 4g (R = pentyl).were measured in minutes.

Experimetal
The capacity factors K were calculated using the Millennium32 ® Chromatography Manager Software according to the formula K = (T R -T D ) / T D , where T R is the retention time of the solute, whereas T D denotes the dead time obtained via an unretained analyte.The log K values of the individual compounds, calculated from the capacity factor K, are shown in Table 1.

Lipophilicity calculations
Log P, i.e. the logarithm of the partition coefficient for n-octanol/water, was calculated using the programs CS ChemOffice Ultra ver.9.0 (CambridgeSoft, Cambridge, MA, U.S.A.

Figure 1 .
Figure 1.Comparison of the log P / Clog P data calculated using the two programs with the experimentally found log K values.The discussed compounds are ordered according to the log K values increase.
Lipophilicity HPLC determination (capacity factor K / calculated log K) The HPLC separation module Waters Alliance 2695 XE and Waters Photodiode Array Detector 2996 (Waters Corp., Milford, MA, U.S.A.) were used.The chromatographic column Symmetry ® C 18 5 µm, 4.6 × 250 mm, Part No. WAT054275, (Waters Corp., Milford, MA, U.S.A.) was used.The HPLC separation process was monitored by Millennium32 ® Chromatography Manager Software, Waters 2004 (Waters Corp., Milford, MA, U.S.A.).The mixture of MeOH p.a. (50.0%) and H 2 O-HPLC -Mili-Q Grade (50.0%) was used as a mobile phase.The total flow of the column was 0.9 ml/min, injection 30 µl, column temperature 25 °C and sample temperature 10 °C.The detection wavelength 210 nm was chosen.The KI methanolic solution was used for the dead time (T D ) determination.Retention times (T R ) ) and ACD/LogP ver.1.0 (Advanced Chemistry Development Inc., Toronto, Canada).Clog P values (the logarithm of n-octanol/water partition coefficient based on established chemical interactions) were generated by means of CS ChemOffice Ultra ver.9.0 (CambridgeSoft, Cambridge, MA, U.S.A.) software.The results are shown in

Table 1 .
1. Calculated lipophilicities (log P / Clog P) and determined log K of the studied compounds