Synthesis and biological activity of novel 3-heteroaryl-2H-pyrido[4,3-e][1,2,4]thiadiazine and 3-heteroaryl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxides

Abstract A series of novel 1,2,4-thiadiazine 1,1-dioxides were synthesized by condensation of 2-chlorobenzenesulfonamide and 4-chloropyridine-3-sulfonamide with heterocyclic methyl carbimidates obtained from heterocyclic carbonitriles and used at the time of their creation. Substituted amidines were isolated as the intermediates in the reaction with 2-chlorobenzenesulfonamide. Those intermediates were successfully cyclized to corresponding 1,2,4-thiadiazine 1,1-dioxides in pyridine with the addition of DBU. The newly synthesized compounds were evaluated for their tuberculostatic and anticancer activities. Eight compounds were able to inhibit the growth of some renal and non-small cell lung cancer cell lines. Graphical Abstract


Results and discussion
The aim of the study was to investigate the reactivity of heterocyclic methyl carbimidates towards sulfonamides that possess a chlorine atom as a substituent at the ortho position to the sulfonamide group. The use of such sulfonamides facilitates the cyclization of sulfonated amidines, formed in the first stage of the reaction, to 1,2,4-thiadiazine 1,1-dioxides. The literature describes methods for the synthesis of 1,2,4-thiadiazine 1,1-dioxides. The most common method is the reaction of 2-aminobenzenesulfonamides with carboxylic acids, their halides, or anhydrides [14,15]. Synthesis via the reaction of 2-aminosulfonamides with aldehydes is another method that has been used [16]. Other authors have reported the reaction of 2-halobenzenesulfonyl chlorides with amidines and aminopyridines in the presence of potassium carbonate [17]. The synthetic method in which substituted amidines react with TosNSO (N-sulfinyl-ptoluenesulfonamide) in acetic acid and hydrogen peroxide has also been described [18].
The method presented in this paper involved the use of heterocyclic methyl carbimidates as they are synthesized from the corresponding carbonitriles (Scheme 1). The carbimidates were reacted with 2-chlorobenzenesulfonamide and 4-chloropyridine-3-sulfonamide in methanol. We have previously described the diazabicyclo products of this reaction when it is carried out with a catalytic amount of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene). This gave a linear amidine [13]. When DBU was equimolar to the sulfonamide, the reaction with 2-chlorbenzenesulfonamide led to linear structures 1-5. However, when 4-chloropyridine-3sulfonamide was used, the corresponding 3-heteroarylsubstituted pyrido [4,3-e] [1,2,4]thiadiazine 1,1-dioxides 11-17 were the reaction products. Reducing the electron density on the carbon atoms of the pyridine ring at positions a and c increases their vulnerability to nucleophilic attack. A halogen at the c position of the pyridine ring is readily exchanged for a nucleophilic NH group. c-Halopyridines are even more reactive than a-isomers [19]. Therefore, products that were cyclized to 1,2,4-thiadiazine 1,1-dioxides were easily obtained.
Using an equimolar amount of DBU in the case of methyl 6-chloropyrazinecarbimidate led to the creation of a mixture of linear and thiadiazine (16) structures that were quite easy to separate. However, the chlorine atom was replaced with a methoxy group.
The 1 H NMR signals for the aromatic protons and NHgroup protons were observed at 12-13 ppm. To elucidate the possible tautomeric forms of the representative compounds 6 ( Fig. 1) and 11, we estimated the total energies of the isolated molecules shown in Table 1.
Calculations were performed using ab initio Hartree-Fock and DFT methods in the gas phase. From the data presented in Table 1, one can infer that the 2H tautomers of compounds 6 and 11 are more energetically favorable than the 4H tautomers by 42.94-93.19 kJ/mol according to ab initio RHF as well as the density functional B3LYP method with the 6-31G* basis set [20]. Moreover, the possible optimized structures for compound 6 indicated conditions favoring hydrogen-bond formation between the hydrogen at nitrogen atom N-2 and the nitrogen atom of the pyridine substituent at carbon C-3. In this way, a stable

Biological activity
Two of the 1,2,4-thiadiazine 1,1-dioxides obtained (11,15) were evaluated for their in vitro tuberculostatic activity against the Mycobacterium tuberculosis H 37 Rv strain and two ''wild'' strains isolated from tuberculosis patients: one (Spec. 210) resistant to p-aminosalicylic acid (PAS), isonicotinic acid hydrazide (INH), ethambutol (ETB), and rifampicin (RFP), and another (Spec. 192) that was fully sensitive to the tuberculostatics administered (Table 2). Isoniazid (INH) was used as a reference drug. The tested compounds showed weak tuberculostatic activity, much lower than the reference INH (MIC 0.5-1.0 lg/cm 3 ). The MIC values obtained when the compounds were tested against three strains ranged from 25 to 100 lg/cm 3 for both compounds. Interestingly, both compounds were more active against the resistant 210 strain than the sensitive 192 one.
All of the newly synthesized compounds were also tested for antitumor activity. We previously described the synthesis of open sulfonamidine derivatives that are analogs of the 1,2,4-thiadiazine 1,1-dioxides presented here. We established that these compounds have notable antitumor activities [13]. It was interesting to see how the closure of the open structure to form the 1,2,4-thiadiazine 1,1-dioxide system affects this activity, especially considering that we have already reported the high affinity of the 1,2,4-thiadiazine 1,1-dioxide derivatives for isozyme CA IX (cancer-associated), an isoform of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) [21], and the significant antitumor activities of these derivatives [22].

Conclusion
A series of novel 1,2,4-thiadiazine 1,1-dioxides with different six-membered nitrogen heterocyclic systems at the C-3 position were successfully synthesized by the reaction of heterocyclic methyl carbimidates with 2-chlorobenzenesulfonamide and 4-chloropyridine-3-sulfonamide. Substituted amidines were isolated as the intermediates in the reaction with 2-chlorobenzenesulfonamide. Those intermediates were successfully cyclized to the corresponding 1,2, 4-thiadiazine 1,1-dioxides in pyridine with the addition of DBU. The syntheses of these new compounds were confirmed by analyzing their IR and NMR spectra as well as elemental analysis. The tuberculostatic and anticancer activities of the synthesized compounds were evaluated. The results showed that the synthesized 1,2,4-thiadiazine 1,1-dioxides exhibited rather poor tuberculostatic activities in vitro. Eight compounds (6,9,12,13,(15)(16)(17) were able to inhibit the growth of some cancer cell lines derived mainly from renal cancer and non-small cell lung cancer.

Experimental
All materials and solvents were of analytical reagent grade. The synthesis of sulfonylcarboximidamides 1-5 was described previously [13].

General method for the synthesis of 3-heteroaryl-2Hbenzo[e][1,2,4]thiadiazine 1-1-dioxides 6-10
The respective sulfonamide derivative 1-5 (5 mmol) was refluxed with 1.8 cm 3 DBU (12 mmol) in 3 cm 3 of pyridine for 2 h. The mixture was cooled down and 30 g of ice were added. The clear solution was acidified with glacial acetic acid. The precipitate was filtered off and purified by crystallization from a suitable solvent with activated carbon.  inhibited the growth of the tested tuberculosis strains in relation to the probe with no tested compound.

Anticancer activity
Compounds were tested at one concentration (10 lM). A mean graph midpoint (MG_MID) was calculated to give the average activity parameter over all cell lines. Cell lines that were insensitive in the screen were included in the calculate the MG_MID. Selectivity of a compound with respect to one or more cell lines of the screen was characterized by a high deviation of the particular cell line parameter from the MG-MID value. Details of the system and the information encoded by the activity pattern over all cell lines have been published [27][28][29].