Synthesis and molecular structure of novel 2-(alkylthio)-4-chloro-N-(4,5-dihydro-5-oxo-1H-1,2,4-triazol-3-yl)-5-methylbenzenesulfonamides with potential anticancer activity

Abstract A series of novel 4-chloro-N-(4,5-dihydro-5-oxo-1-R2-1H-1,2,4-triazol-3-yl)-5-methyl-2-(R1-methylthio)benzenesulfonamide derivatives have been synthesized as potential anticancer agents. The in vitro antitumor activity of some compounds was evaluated in the US National Cancer Institute (NCI) against the NCI-60 cell line panel. The most prominent compound showed remarkable activity against 13 human tumor cell lines representing lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast at low micromolar GI50 level in the range of 1.9–3.0 μM. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s00706-012-0849-7) contains supplementary material, which is available to authorized users.

It has been known that aryl/heteroarylsulfonamides may act as antitumor agents through a variety of mechanisms such as cell cycle perturbation in the G1 phase, disruption of microtubules, angiogenesis inhibition, and functional suppression of the transcriptional activator NF-Y. The most prominent mechanism was the inhibition of carbonic anhydrase isozymes [18][19][20][21][22]. Recently, a host of structurally novel arylsulfonamide derivatives have been reported to show substantial anticancer activities in vitro and/or in vivo [23][24][25][26]. We have reported the synthesis and anticancer activity of 2-mercaptobenzenesulfonamides and subsequently extended our study to analogues with various heterocyclic ring systems attached to the benzenesulfonamide scaffold [4-6, 8, 10, 15] (Fig. 1 structure A [4-6, 8, 15], B [8], C [10]).
In this article we investigated new sulfonamide derivatives containing a triazolone ring in their structure. Triazolones are described in the literature as biologically active compounds, including anti-inflammatories [27], Nk i antagonists [28], inhibitors of tumor necrosis factor-a-converting enzyme (TACE) [29], checkpoint kinase-1 inhibitor [30], anti-tumor agents [31][32][33][34], and molecular chaperone Hsp90 inhibitor, which is currently in clinical trials for a number of human cancers [35]. Taking into account the interesting properties of triazolones, we have synthesized novel compounds of general structure D (Fig. 1).
Starting 3-aminobenzodithiazine 1 could be readily converted to the corresponding dipotassium 2 and potassium salts 3 and 4, according to the reported procedure for preparation of N-(phenylsulfonyl)cyanamide potassium salts [36]. Novel potassium salts 5-10 were prepared by the reaction of 2 with the corresponding halomethyl electrophiles such as aryl/cycloalkyl/methyl chlorides in methanol or ethanol. Subsequent reaction of salts 3-10 with either hydrazine monohydrochloride, methylhydrazine, p-toluenesulfonyl hydrazide, or various 4-substituted phenylhydrazine hydrochlorides led to the formation of the desired 3-(R 2 -amino)-1-[4-chloro-5-methyl-2-(R 1 -methylthio)phenylsulfonyl]guanidine derivatives 11-25 as depicted in Scheme 1. It is pertinent to know, however, that aminoguanidine 15 was chosen for the synthesis in two different ways (route A and B in Scheme 1). This was supposed to explain some arising synthetic aspects: whether the usefulness of the potassium salt, i.e., 3 with tosyl hydrazide (route A), is higher than the reaction of aminoguanidine 11 with tosyl chloride (TsCl, route B), and whether the reaction proceeds on the N-terminal nitrogen atom of the sulfonylhydrazide moiety or on the second nitrogen atom neighboring the sulfonyl group. As it turned out, both methods products 15 were identical, with structures (IR, NMR) having a N'-substituted sulfonylhydrazide fragment and obtained in almost equal yields.
In the present study we utilized a new method for the synthesis of 1,2,4-triazol-5-ones in the reaction of the corresponding aminoguanudines 11-25 with an excess of p-toluenesulfonyl isocyanate (TsNCO, Scheme 1). The isocyanates are well known as carbonyl precursors [43] and electrophilic agents whose reactions with hydrazines lead to intramolecular cyclization to five-membered heterocyclic rings [44] or reagents in cycloaddition reactions with various compounds having C=N bonds [45].
Our experiments demonstrated that the proposed synthetic route was an efficient way to prepare the desired N-(4,5-dihydro-5-oxo-1H-1,2,4-triazol-3-yl)benzenesulfonamides 26-40 when an excess of three molar equivalents of tosyl isocyanate was applied in the reaction with the corresponding aminoguanidines 11-25 in anhydrous tetrahydrofuran (THF) for at least 9 h at reflux. It is noteworthy, however, when 2 equivalents of tosyl isocyanate were used, no cyclization product was observed and a complex mixture of products was formed, even after considerable extending of the reaction time.
The structure of the new compounds was confirmed by elemental analyses (C, H, N) and spectral (NMR, IR, MS) data presented in the experimental section. Moreover, X-ray analysis was undertaken to confirm proposed structures on the representative compound 31, which crystallized as pyridinium salt (further specified as 31Pyr, Figs. 2 and 3).

Molecular structure
Details on data collection, structure solution, and refinement are given in Table 1. Compound 31Pyr crystallizes in the monoclinic space group C2/c with (typical for this symmetry) eight molecules in the unit cell. The molecule, being a secondary benzenesulfonamide, is deprotonated at the N1 atom and in the crystal structure is present in the anionic form (Fig. 2). The proton is accepted by pyridine so a pyridinium ion acts as a counterion. Additionally the  The two ions are linked by a charge-assisted hydrogen bond of the (?)NHÁÁÁN(-) type; pyridinium N(5) is a donor, and sulfonamide N(1) is an acceptor. Bonds N(4)-H(4) interact with carbonyl oxygen atoms O5 from the triazolone moiety of the neighboring molecules forming intermolecular hydrogen bonds NHÁÁÁO. These interactions arranged in pairs can be described by the R 2 2 (8) motifs situated about local inversion centers (see Fig. 3). Detailed information on hydrogen bonds is given in Table 2. Packing of molecules in the solid state is reinforced also by p-p stacking interactions between adjacent aromatic rings C5-C10 whose centers of gravity (Cg or centroids) are distant at 3.8513(10) Å . The geometry of the interaction is more precisely characterized in Table 3.

Biological assay
Compounds 27, 28, and 30-39 were initially tested at a single dose (10 -5 M) in the full NCI-60 cell panel, and the results are shown in Table 4. The methodology of the in vitro cancer screen is described at the website http://www.dtp.nci.nih. gov/branches/btb/ivclsp.html.
The relatively highest sensitivity to the compounds described here was found for the cell lines of non-small cell lung cancer NCI-H522 cell line to compounds Table 4).
The following conclusions can be drawn from the structure-activity relationship study (   Symmetry code: (i) -x ? 1/2, -y ? 1/2, -z Table 3 Main p-p stacking interaction geometry in crystal structure of 31Pyr (7) Ring (1)    Further anticancer evaluation was performed at five-dose assay on the distinctive compound 36. The anticancer activity of the tested compound was reported for each cell line by the parameters GI 50 (molar concentration of the compounds that inhibit 50 % net cell growth), TGI (molar concentration of the compounds leading to total inhibition), and LC 50 (molar concentration of the compounds causing 50 % net cell death). The susceptibility of individual subpanels indicates the following order: prostate, colon, CNS, leukemia, ovarian, nonsmall cell lung, melanoma, renal, and breast cancer ( Table 5). As shown in Table 5, compound 36 exhibited remarkable activity at low GI 50 level \11.   A COMPARE [47] analysis at the NCI of compound 36 showed a moderate Pearson's correlation coefficient (PCC = 0.473-0.425) with agents disrupting microtubule formation such as maytansine and rhizoxin [48].

X-ray structure determination
Experimental diffraction data were collected on a KM4 CCD kappa-geometry diffractometer (Oxford diffraction), equipped with a Sapphire2 CCD detector. An enhanced X-ray Mo Ka radiation source with a graphite monochromator was used. Determination of the unit cell and diffraction data collection were carried out at 120 K in a stream of dry nitrogen (Oxford CryoSystems). All calculations (data reduction, structure solution, and refinement) were carried out using CrysAlisPro [49] package. The structure was solved by direct methods, and all nonhydrogen atoms were refined with anisotropic thermal parameters by full-matrix least squares procedure based on F 2 . Final refinements were carried out using the SHELX-97 package [50], run under control of WinGX program [51].
All hydrogen atoms were refined using isotropic model with U iso (H) values fixed to be 1.2 times U eq of C atoms for CH and CH 2 and 1.5 times U eq for CH 3 . Bond lengths C-H were fixed at 0.98 Å for methyl groups, and 0.95 Å for methylene and methine groups; distances N-H were set to 0.88 Å . Solvating water molecules generated an electron density peak of ca. 1.7 e Å -3 . Because the electron density maximum is placed at a special position (, y, ) localization of hydrogen atoms is additionally uncertain so we did not attempt to find H atoms. The occupation factor of oxygen atom O10 was refined freely to obtain 0.079. One incorrect reflection (-1 1 17)