New (azulen-1-yldiazenyl)-heteroaromatic Compounds Containing 1,3,4-thiadiazole -3-yl Moieties

ALEXANDRU C. RAZUS1, LIVIU BIRZAN1*, MIHAELA CRISTEA1, VICTORITA TECUCEANU1, CONSTANTIN DRAGHICI1, ANAMARIA HANGANU1, MARIA MAGANU1, LUCIA PINTILIE2, ELEONORA MIHAELA UNGUREANU3 1Institute of Organic Chemistry C. D. Nenitzescu’ of RomanianAcademy, 202B Spl. Independentei, 060023, Bucharest, Romania 2National Institute of Chemical Pharmaceutical Research and Development Bucharest, 112 Vitan Av., 031299, Bucharest, Romania 3University Politehnica of Bucharest, Physical Chemistry and Electrochemistry, Department of Inorganic Chemistry, 1-7 Gh. Polizu, 011061, Bucharest, Romania

Numerous diazenes containing heterocycles have been prepared due to their easy availability and to their promising technical properties as dyes. Another important aspect is represented by the higher stability and lack of the toxicity of the compounds. Therefore, some of the obtained diazenes were studied for their biological properties as: anti-bacterial, anti-viral, anti-inflammatory or analgesic activities. An important heterocycle system largely used in diazenes structure was 1,3,4-thiadiazole, which can be readily obtained and is stable [1]. The building of numerous derivatives of this heterocycle with various arylazo substituents was reported [2,3] as well as their use as dyes [4][5][6][7][8][9][10] or in biological purposes [11,12] (antimicrobial and antifungal) [13], cytotoxicity against ascites carcinoma tumor cells [14]. Some cationic dyes were obtained by alkylation of the dyes containing strong electron donating groups (EDGs), like R 2 N on the thiadiazole ring [15]. The introduction of azulene-1-yl moiety as the second component of heteroaryl diazenes significantly extends the molecular electronic system. An important number of (azulen-1-yl)-azo derivatives containing heterocycles have been prepared by our group in the aim to study their synthesis, physical and chemical properties and to find possible technical applications. Thus were investigated azulene-1-azopyridines and azopyranylium salts [16], azulene-1-azopyridine 1'-oxides [17], azulene-1-azo-2thiazoles [18,19], azulene-1-azo-2-benzothiazol [20,21] and azulene-1-azo-1,2,5-oxadiazol [22]. On the other hand, the electron donor property of azulene-1-yl moiety can be used to afford push-pull systems especially when at the other end of nitrogen double bond there is an acceptor as a positive charged heterocycle [23]. The large use of diazenes containing 1,3,4-thiadiazole group together with the extended interest on azulene derivatives stimulated us to turn our attention to build diazenes with both these moieties and to examine some of their properties.

Experimental part Materials and instrumentation
Melting points (uncorrected) were measured with Kofler apparatus (Reichert Austria). Elemental analyses were performed using Perkin Elmer CHN 240B. UV-Vis spectra were obtained using Varian Cary 100 spectrophotometer (λ values are given in nm and the molar extinction, ε, in M -1 cm -1 ). For 1 H-and 13 C-NMR: Bruker Avance DRX4 ( 1 H: 400 MHz, 13 C: 100.62 MHz) and Gemini 300 ( 1 H: 300 MHz, 13 C: 75.47 MHz) spectrometers were used, with TMS as internal standard in CDCl 3 ; several signals were assigned on the basis of COSY, HETCOR and HMBC experiments. Mass spectra were obtained with Varian 1200L Triple Quadrupole LC/MS/MS spectrometer by direct injection in ESI. For the column chromatography, silica gel 60 or alumina [II-III Brockmann grade, 70e230 mesh ASTM] were used. The DCM was distilled over CaH 2 and the ether was preserved on sodium. Acetonitrile (Rathburn, HPLC grade), tetra-n-butylammonium perchlorate (TBAP) and tetra-n-butylammonium fluoroborate (TBABF4) from Fluka were used as received like solvent and supporting electrolytes, respectively. All potentials were referred to the potential of ferrocene/ferrocenium (Fc/Fc + ), which was 0.424 V vs Ag+/AgCl(std). Anodic and cathodic DPV curves were recorded individually, starting from the stationary potential. Concentration of diazenes was 10 -3 Min acetonitrile containing Et 4 NClO 4 (0.1 M), Pt electrode (i.d. 1.6 mm), scan rate = 100 mVs -1 . The starting 5substituted 1,3,4-thiadiazoles-2-amines, 1Q, where Q is described in scheme 1 were commercially available. The known amine 1(Nf) with 1-naphthyl in position 5 [24] was obtained on the route described in scheme 1 from the corresponding carboxylic acids and thiosemicarbazide (1-(aminomethyl)thiourea), 2, in the presence of dehydrating agent. The compounds nomenclature was obtained by the CambridgeSoft package of structure-to-name algorithm included with ChemBioDraw Ultra 11.0.
Diazotization and coupling of 2-amino-5-substituted-1,3,4thiadizole, 1(Q) Synthesis of compounds 5(Ph) -7(Ph) To a stirred mixture of phosphoric acid 85% (0.4 mL) and nitric acid 63% (0.4 mL), cooled to 0 o C, 2-amino-5phenyl-1,3,4-thiadiazole, 1(Ph) (177 mg, 1 mmol) was added at such a rate as to avoid overheating. The diazonium salt, orange colored, was obtained by the addition of solid sodium nitrite (69 mg, 1 mmol). After stirring at 0 o C for 10 minutes the viscous mixture was poured into a well stirred solution of azulene, 4a-c, (1 mmol) and a large excess of sodium acetate (5.5 g, 67 mmol) in methanol (20 mL) also cooled to 0 o C. The coupling reaction takes place in 30-40 minutes the color of reaction mixture changing from blue to red. After reaching room temperature DCM (50 mL) and water (50 mL) were added. The organic layer was separated and washed two times with water and then dried on sodium sulfate. The solvent was evaporated and the residue was chromatographed on silica gel obtaining diazene 5(Ph) -7(Ph) in yields shown in Table 1. Generally, at the chromatography of resulted mixture in this reaction, as well as in the following synthesis, the first collected fraction contained unreacted azulene.

Synthesis of compounds 5(Th) and 5(Fu)
To a stirred solution of phosphoric acid 85% (0.75 mL), cooled to 0 o C, 2-aminothiadiazole, 1(Th) or 1(Fu) (1 mmol), was added at such a rate as to avoid overheating. The diazonium salt was generated by the addition of solid sodium nitrite (69 mg, 1 mmol). After manually stirring at 0 o C for 10 minutes, the viscous mixture was poured under strong magnetically stirring into the solution of azulene, 4a, (128 mg, 1 mmol) and sodium acetate (5.6 g, 68 mmol) in methanol (20 mL) also cooled to 0 o C. The coupling reaction takes place in 30-40 minutes in which the reaction mixture turns from blue to red. Then it was let to reach the room temperature and DCM (50 mL) and water (50 mL) are added. The organic layer was separated and washed two times with water and then dried on sodium sulfate. The solvent was evaporated and the residue chromatographed on silica gel using DCM for azulene elution and a mixture DCM-AcOEt (with ester gradient from 5 to 20%) for next fractions. The third, brown band, represented the desired product (the other bands contained unknown mixtures in very small amounts). The yields in products 5(Th) and 5(Fu) are shown in table 1.

Synthesis of compounds 5(t-Bu)
To a stirred solution of 2-amino-5-tert-butyl-1,3,4tiadizole, 1(t-Bu) (157 mg, 1 mmol) in dichloroacetic acid (1.2 mL), cooled to 0 o C, solid sodium nitrite (138 mg, 2 mmol) was added. The mixture was kept for 5-10 min at this temperature and added to a solution of azulene (128 mg, 1 mmol) and sodium acetate (3.0 g, 36 mmol) in methanol (20 mL) cooled 0 o C and strongly stirred. After stirring for 30-40 min at 0 o C the work up was similar with that previously described but alumina was used for chromatography. Azulene was recovered from the column followed by diazene 5(t-Bu) as a brown band in the yield shown in table 1.

Synthesis of compounds 5(H)
Working in the same reaction conditions as above, the chromatography of reaction mixture gave 73% recovered azulene followed by (E)-2-(azulen-1-yldiazenyl)-1,3,4thiadiazole, 5(H), 23%. The color of the solution turns from blue to violet. After 1 hour of stirring the solution was diluted with water and the product was extracted in DCM. The organic phase was washed with water, dried on sodium sulfate, filtered and the solvent was evaporated. The residue was chromatographed on silica gel column and DCM was used for the elution of unreacted azulene (93 mg, 75%) and DCM-EtOAc (ester gradient from 5 to 100%) for the next fractions. The desired product, 5(S(O)Me), resulted in 60 mg, yield 20%.

Synthesis of compounds 5(S(O)Me) by oxidation
The diazene 5(SMe) 143 mg (0.5 mmol) and sodium periodate 428 mg (2 mmol) in methanol (26 mL) and water (16 mL) were refluxed for 3 h. Then the methanol was partially evaporated and DCM (50 mL) and water (50 mL) are added. After layers separation, the organic solution was washed again with water (50 mL) and dried on sodium sulfate. The solvent was evaporated and the residue was chromatographed on silica gel and eluted with a mixture DCM-AcOEt in increasing amounts, then AcOEt and AcOEt-MeOH (9:1 in %). The first collected fraction, brown colored, 81 mg (57%), was the starting material, 5(SMe) and the second reddish-brown fraction, 54 mg (36%), represented the desired product 5(S(O)Me).

Synthesis of compounds 5(S(O) 2 Me) by oxidation
The diazene 5(SMe) 143 mg (0.5 mmol) and sodium periodate 428 mg (2 mmol) in dioxane (8 mL) and water (5 mL) were refluxed for 5 h. Then the solvent partially evaporated and DCM (50 mL) and water (50 mL) were added. After layers separation, the organic solution was washed again with water (50 mL) and dried on sodium sulfate. The solvent was evaporated and the residue was chromatographed on silica gel and eluted with a mixture DCM-AcOEt in increasing amounts, then AcOEt and AcOEt-MeOH (9:1 in %). The first collected fraction, brown colored, 68 mg (48%) was 5(S(O) 2 Me) and the second, reddish-brown fraction, 68 mg (45%) was the compound

Results and discussions Synthesis
The proposed diazenes were achieved starting from the 5-substituted 1,3,4-thiadiazoles-2-amines, 1Q, where Q is described in Scheme 3. To obtain a fair yield of the diazonium salts at diazotization of amines 1Q, generally, nitrososulfuric acid was used at low temperatures mainly in acetic acid [25], or a mixture of propionic acid-acetic acid [6,26] (eq. 1 in Scheme 3). The diazotization of 1(SH) was realized with NaNO 2 in the water solution of HCl [27]. However it must be noted that the diazonium salts 3(Q) is not very stable as, for example, the pyridine or thiazole correspondents. The subsequent azo coupling of 3(Q) was described as arising in good yields in the presence of urea in the aim to nitroso cation elimination or, in the same purpose, adding different bases to buffered medium [6,[25][26][27].
The coupling with azulenes (eq. 2 in scheme 3), occurred with modest yields and low azulenes conversions as results from Table 1. Because the azulene is a strongly polarized aromatic hydrocarbon which has a relatively weak nucleophilicity, coupling with diazonium salts are at the limit of the azulene system reactivity. Therefore an efficient azo electrophilic substitution in position 1 takes place with parent azulene or with azulenes activated by the inductive effect of alkyl groups. Supplementary, the weak electrophiles as, for example 4-dimethylaminophenyldiazonium ion, are not able to substitute the proton in 1-position of azulene [28]. It seems that the diazonium salts generated from aminothiadiazoles, 1(Q), are also quite weak electrophiles (see the limit structures described in scheme 3 for salt 3(Q)) leading to low azulenes conversions and, therefore, to their damage prior to the coupling reaction. The particular reaction conditions and the yields for each diazotization-coupling sequence are shown in table 1.
More difficulties are encountered when the SH group is present as in starting compounds 1(SH) and in coupling products. Presumably, this group can be oxidized by the nitrosyl ion present in medium giving the reactive radical sulfur. Thus 1(SH), besides the providing of diazenes 5 or 7, can be substituted at sulfur atom giving the compounds 8. These last products, due to the amino group can be again diazotized and coupled with a new azulene. Therefore, the composition of the generated reaction mixture became very complex and a high amount of polymer accompanies the products (scheme 4). The attempts to separate Scheme 3. Diazotization and coupling reactions samples containing pure compounds have succeeded only starting from guaiazulene, 4c. Their higher reactivity accelerates the reaction with the diazonium group in position 1 together with the attack of sulfur radical avoiding to a good extent the polymerization. In this way were separated and characterized the normal coupling product 7(SH) alongside the product with guaiazulene moieties both at sulfur as well as at azo group, 9(R=3,7-Me 2 -5-iPr) (based only on the molar peak present in mass spectrum of reaction mixture can be also suggested the generation of amine 8(R=3,8-Me 2 -5-iPr)). Several products starting from azulene, 4a, as compounds 8(R=H) or 5(SH), were highlighted by mass spectrometry and 1 H-NMR spectra of the mixtures enriched in one of the component. Diazene 5(SMe) can be oxidized selectively at the sulfur atom in the presence of sodium periodate. While in methanol-water, at reflux the reaction stops at the stage of sulfoxide, 5(S(O)Me), in THF-water it gives a mixture of sulfone and sulfoxide. Trying to increase the conversions, the reaction becomes non selective and the system is destroyed.

Semiempirical molecular orbital calculation
A computational study on (E)-2-(azulen-1-yldiazenyl)-1,3,4-thiadiazoles was undertaken based on density functional theory (DFT) using Spartan 14 software Wavefunction, Inc. Irvine CA USA on Intel(R) Core i5 at 3. 2 Ghz CPU PC. Calculations of molecular properties and topological descriptors has been carried out using software algorithm hybrid B3LYP model (the Becke's three parameter hybrid exchange functional with the Lee-Yang-Parr correlation functional) and polarization basis set 6-31G* vacuum, for equilibrium geometry at ground state. A series of molecular descriptors and properties of their optimized geometries (natural charges, molecular frontier orbitals energies, etc.) were calculated. Thermodynamic properties (dipole moments, polarizability, log P, solvation energies) have been also computed proving their lipophilic and strong polar character. Reduction and oxidation potentials have been correlated to their calculated energy levels for LUMO and HOMO orbitals.
The HOMO energies decrease when EWG-s are attached to the 5-position of the heterocyclic system making the compounds more stable. Contrary, aryl and donor groups increase these values what destabilize the molecules but the effect is modest. At the same time, LUMO energies  decrease when the EWG-s are present and increase in the presence of aryls or donor group. As a result, in the first case the molecules are less stable to reduction and in last one they are more resistant. Azulene alkylation increases both values of HOMO and LUMO energies, making the compounds more sensitive to oxidation and more stable to reduction. Polarizability is increased by substitution with EWGs on the heterocyclic moiety due to the intensification of the pull-push effect. The natural charges explain the polarization vector with the positive pole in the tropylium moiety and the negative pole toward the thiadiazole system. However, the negative charges are disseminated on more nitrogen atoms and azulenic C3 and therefore the system does not interact strongly with metal cations or electrophiles. For example it is methylated only in traces by MeI, while the corresponding thiadiazolamines react in high yields ( fig. 2 gives the mass spectrum of methylated diazene 5(SMe) separated in ver y small amount and incompletely characterized).
At the same time, the metal complexation at nitrogen atoms is disadvantaged and also the protonation is observed at very low pH units in ethanol 96% (pH = ~ 0 units). The most reactive site to soft electrophiles attack is at azulene C3 atom (scheme 6).
As expected, the alkyl groups substituted at azulene seven ring increases the molecule polarity and polarizability (table 3). The most polarizable molecule contains 1-naphthyl group at position 5 of thiadiazole moieties. Log P is the lowest for the patern compound and the highest for the naphthyl substituted thiadiazole derivative. It is obvious that while nonpolar substituents increase this parameter those polar decrease its value (table 3).
Mass and UV-Vis spectra All the diazenes 5 -7 have a similar behavior in the mass spectrometer and the figure 3 is an example. The molecular ion is present but it is not very stable. He generates the diazonium ion of azulene, also instable, which further split to azulene fragment after nitrogen elimination. It is interesting that the thiadiazole moiety or its diazonium salt are not present between the obtained fragments, which proves a selective split between the heterocycle and the azo group.
The replace of aryl group in 2-aryldiazenyl-5-phenyl-1,3,4-thiadiazoles, already described [29], with azulene chromophores as in diazenes 5 -7 is expected to modify the electronic spectra. Besides this, the study of solvatochromic behavior as well as that of acid-basic equilibrium of the last compounds can highlight some particular features for these compounds.
Both, the nature of substituents at 5-position in heterocycle as well as the presence of alkyl substituents at azulen-1-yl moiety influences the signal shift in visible. Thus, the substitution of 5-position by tBu does not alter the signal recorded for compound 5(H). The presence of aryl group in this position exerts a bathochromic shift more pronounced for the thienyl and furyl groups. The influence of aryl is due to the charge extension of the π-electronic Consideration on the acid-base properties of (azulen-1yldiazenyl) heteroaryls seems to be troublesome due to their extremely weak basicity (pK a values are around 0) which is situated at the limit for registration capacity of the common pH-meters. The absorption maxima of some 2-(azulen-1-yldiazenyl)-1,3,4-thiadiazoles in ethanol both in neutral and in strong acid medium are presented in table 5.  5). This can be explained by the intensification of the pushpull effect by heterocycle protonation (scheme 8). Because in basic medium the compounds 5 are yellow-brown or brick colored and in acidic medium becomes orange they can be used as pH indicator. The very low basicity of the studied compounds also hinders the complex generation with metals ions as these of Ni or Fe in ethanol; our attempt to obtain complexes remains unsuccessful. However, they formed complexes with BF 3 , a Lewis acid ( fig. 6), which was highlighted by UV-vis spectroscopy. In alcoholic solutions they are redviolet, ver y similar with the protonated species of compounds. As can be seen in figure 6-A the main visible bond belonging to compound 5(SMe) was bathochromic shifted from 494 nm to 561 nm by interaction with BF 3 -Et 2 O in ethanol 96%. Another salt possessing Lewis acid properties is SnCl 4 , which has a similar reactivity toward the ligand 5(SMe) ( fig.6-B).   We have previously mentioned that, usually, the oxidation of (azulen-1-yldiazenyl)-heteroaryls takes place at azulenyl moiety with the generation of the stabilized structure as radical cation whereas for the reduction the heteroaryl group is responsible (scheme 9) [22,30].
As expected, the electron rich substituents at heteroaryls moiety, Q, decrease the oxidation potential. Thus, the potential for compound 5(SMe) is lower by 84 mV as compared with 5(H). The inductive effect of tBu destabilizes the molecules toward oxidation but only with 60 eV. Contrary, the electron withdrawing group S(O)Me deeply increases this potential by 32 mV. The conjugation extension due to the presence of the Q = aryl group decreases the oxidation potential in order ((Nf) with 105 mV) < ((Th) with 125 mV). The inductive effect of alkyl groups at azulenyl moiety decrease significantly these potentials: the three Me groups in 4,6,8 positions decrease the oxidation potential with around 95 mV and the alkyls belonging to guaiazulenyl moiety decrease this potential even more, till 150 mV. As expected, the reduction potentials vary in reverse. The CV experiments revealed that all the processes are irreversible. However, as can be seen in figure 9, the first cathodic process is partially reversible.

B
A very good correlation between the experimental redox potentials and calculated energies of the frontier orbitals was observed in figure 10.

Conclusions
As a continuation of our studies regarding the preparation of azulen-1-yldiazenyl heteroaryl compounds several 2-(azulen-1-yldiazenyl)-1,3,4-thiadiazols were synthesized and characterized. Both the weak azulene nucleophilicity and the low reactivity of the diazonium salts arising from thiadiazolamines prevented the obtaining of high yields. Supplementary, the salts show a low selectivity, generating undesired compounds. Due to the oxidizing character toward the thiadiazole system of nitric acid, usually used for other diazotizations (e.g. for pyridines and thiazoles), this reaction was carried out in dichloroacetic acid. Starting from the compounds substituted at the diazonium position 5 of the thiadiazole moiety with Q = SH, some azulenic azulen-1-ylthioethers are generated.
All products were characterized and their spectra discussed. It must be noted that the protonated compounds splitting in mass spectrometer produces azulenediazonium ion and the unionized heterocycle. This peculiar decomposition could be the result of the low stability of diazonium salts arising from thiadiazole, which has also a higher oxidation capacity. The absorption maxima of the new compounds are close to those of the corresponding diazenes containing thiazole. They have brick-brown color in neutral medium and violet in strong acidic solutions. However, their nuances differ in neutral solvents with substituents: EDGs make them brownish and EWGs make them reddish-brown.
The redox potentials of products were also recorded noticing the influence of the substituents on the potentials. Linear correlations have been found for their oxidation and reduction potentials.