New tricks of well-known aminoazoles in isocyanide-based multicomponent reactions and antibacterial activity of the compounds synthesized

The well-known aminoazoles, 3-amino-5-methylisoxazole and 5-amino-N-aryl-1H-pyrazole-4-carboxamides, were studied as an amine component in Ugi and Groebke–Blackburn–Bienaymé multicomponent reactions. The first example of an application of aminoazoles in an Ugi four-component reaction was discovered and novel features of a Groebke–Blackburn–Bienaymé cyclocondensation are established and discussed. The heterocycles obtained were evaluated for their antibacterial activity and several of them demonstrated a weak antimicrobial effect, but for most of the compounds a 30–50% increase in biomass of Gram-positive strains (mainly B. subtilis) compared to control was observed.


Introduction
An intensive progress in pharmaceutical and medicinal chemistry, as well as in the generation and improvement of medicinal technologies has led to defeating a wide scope of diseases.
Ugi-4CR has been applied in the synthesis of natural products, as bicyclomycin, furanomycin, penicillin etc. [53]. The high combinatorial potential of Ugi-4CR together with the ability to incorporate a variety of functionalities and modifications extend its application for the generation of organic compound libraries, following hit-to-lead optimization, choosing the hit structure and final marketed drug production [54][55][56][57][58]. Moreover, it has been acknowledged that the combination of two privileged scaffolds in a single molecule (e.g., the combination of a peptidomimetic structure with an azole fragment [59]) potentially creates more active, new entities with unusual bioproperties [20,60,61]. In addition, the application of polyfunctional reagents in Ugi-4CR opens ways to different post-cyclization reactions, thereby broadening the scope. Thus the Ugi-4CR involving substituted propiolic acids, can be followed by electrophilic ipso-iodocyclization [62] or transition-metal-initiated [63][64][65][66][67][68] and metal-free cyclizations [69,70].
Taking into account the above-mentioned facts, several aminoazoles, whose reactivity in isocyanide-based reactions had not Scheme 2: Reactivity of 5-amino-N-aryl-1H-pyrazole-4-carboxamide and 3-amino-5-methylisoxazole in GBB-3CR and  been studied yet, were examined as an amine component in Ugi-4CR and GBB-3CR. The generated compounds were screened for their biological activity towards Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.
This condensation was also carried out in TFE or MeOH with addition of HClO 4 (10 mol %), but a significant amount of Schiff base 5 (Table 1) was observed in this case. The reaction involving aldehydes 1f-h bearing strong electron-withdrawing groups under all the abovementioned conditions allowed isolating a mixture of imidazopyrazoles 4n-v with a large quantity of Schiff bases 5.
Obviously, this reaction requires a longer reaction time (min. 48 h) and a moderate temperature (not more than 85 °C) to avoid tarring. Thus, after 48 h of heating (oil bath) at 85 °C the starting materials 1f, 2b and 3a in EtOH/H 2 O with TFA (10 mol %), imidazopyrazole 4q was isolated in 56% yield. However, the mother liquor still contained unreacted Shiff base 5q (entry 7, Table 2 ). On the other hand, using DMF/HClO 4 (10 mol %, method B) allowed obtaining the target compound 4q in 88 % yield with no impurities (entry 9, Table 2).
It should be noted that the conditions of method B are also suitable for obtaining imidazopyrazoles 4a-m in comparatively high yields; however, the synthesis in EtOH/H 2 O medium is preferred from the point of view of green chemistry. Thereby, the optimal methodology for obtaining compounds 4a-m is the synthesis according to the method A (H 2 O/EtOH (1:1), TFA (10 mol %), rt, 24 h) while for compounds 4n-v method B (DMF, HClO 4 (10 mol %), rt, 48 h) proved to be superior (Table 1, entries [14][15][16][17][18][19][20][21][22]. We presume that such difference in the outcome of GBB-3CR depending on the substitution pattern in the carbonyl component is related with the ability of the intermediate Schiff bases to be protonated as well as with their solubility. In case of the presence of electron-withdrawing substituents in the aldehyde the corresponding Schiff bases 5 are less soluble and less basic. DMF increases solubility of imines 5, while the application of strong acid (HClO 4 ) promotes Shiff bases protonation.
Phenylpyruvic acid (1') was also applied as carbonyl component in GBB-3CR to obtain imidazopyrazoles having a carboxylic group. However, the process of decarboxylation took place in the reaction and 1-H-imidazopyrazole carboxamides 4w,x were isolated as the sole reaction products (Table 4).  Interestingly, in case of ethyl 2-isocyanoacetate (3b) the reaction proceeded equally well regardless of the substituent in the aldehyde. This can be connected with an increased reactivity of ethyl 2-isocyanoacetate (3b) compared to tert-butylisocyanide  (3a) due to sterical reasons thus leveling the influence of the solubility factor of imines 5.
As it has been already mentioned above, in contrast to 5-amino-N-aryl-1H-pyrazole-4-carboxamides 2, 3-amino-5-methylisoxazole (7) exhibited only properties of primary amines that allowed using it as an amine component in Ugi-4CR. Particularly, its reaction with aromatic aldehydes 1a-h, phenylpropiolic acid (8) and tert-butylisocyanide (3a) gave peptidomimetics 9 under stirring the starting reagents in MeOH at room temperature for 24 h. In the presence of strong electron-withdrawing substituents as nitro or cyano groups in para-position of the aldehyde, despite the variation of the reaction conditions, only imines 10 were isolated as the major products. In case of 4-cyanobenzaldehyde (1h) we managed to isolate Ugi product 9g in a low yield of 18% (Table 6). It should be noted that 5-amino-N-aryl-1H-pyrazole-4-carboxamides 2 were also introduced into Ugi-4CR with aromatic aldehydes 1, alkylisocyanides 3 and phenylpropiolic acid 8; however, the acid 8 acted as a catalyst favouring the formation of GBB-3CR-products 4. The attempts to carry out GBB-3CR involving 3-amino-5-methylisoxazole (7) according to the elaborated procedures (Table 1, methods A or B) as well as under other conditions were not successful.

Structure elucidation
The purity and structures of the heterocycles obtained were established by means of mass spectrometry (including HRMS), NMR spectroscopy and X-ray diffraction study.
The 1 H NMR spectra of imidazo[1,2-b]pyrazole-7-carboxamides 4 exhibit a broad signal for the NH group in the position 1 at ca. 11.8 ppm, a broad signal for the carboxamide NH at ≈9.5 ppm, a singlet for pyrazole CH in the position 6 at ≈8.2 ppm, a broad signal for the NH group near the position 3 at ≈5.1 ppm, a singlet for tert-butyl CH 3 groups at ≈1.0 ppm, resonances for the aromatic protons around 6.9-8.2 ppm as well as signals for other substituents. In case of imidazo[1,2-b]pyrazole-7-carboxamides 4w,x a broad signal for the tert-butyl NH group is shifted upfield to 4.2 ppm and an additional singlet for the benzyl CH 2 group is present at 3.9 ppm.
The 1 H NMR spectra of imidazo[1,2-b]pyrazole-7-carboxamides 6 exhibit a broad signal for the NH group in the position 1 at ≈11.8 ppm, a broad signal for the carboxamide NH group at ≈9.6 ppm, a singlet for the pyrazole CH in the position 6 at ≈8.2 ppm, a broad signal for the NH group near the position 3 at ≈5.7 ppm, a singlet for the CH 2 group in the acetate moiety at ≈4.2 ppm, peaks for the ethoxy group: a quartet for CH 2 group at ≈4.0 ppm and a triplet for the CH 3 group at ≈1.0 ppm, peaks for the aromatic protons around 6.7-7.7 ppm as well as signals for other substituents.
The 1 H NMR spectra of N-(1-arylethyl-2-(tert-butylamino)-2oxo)-N-(5-methylisoxazol-3-yl)-3-phenylpropiolamides 9 exhibit a broad signal for the amide NH group at ≈7.9 ppm, singlet for the isoxazole CH at ≈6.3 ppm, a singlet for the CH group in the position 1 at ≈6.0 ppm, a singlet for the isoxazole CH 3 group at ≈2.3 ppm, a singlet for the tert-butyl CH 3 groups at ≈1.2 ppm, peaks for the aromatic protons around 6.8-7.5 ppm as well as signals for other substituents.
As it was found earlier for 2-aminopyrimidines [107][108][109] GBB-3CR may lead to the formation of two positional isomers A and B (Figure 1). Experiment with D 2 O allowed to identify the signals of NH protons while the HSQC spectrum showed the correlations between the signals of protons and corresponding carbon atoms (in the position 6 and in tert-butyl group). The correlations between the signals of NH protons and corresponding carbon atoms (through two and three bonds, Figure 2) allowed final distinguishing the shifts of three NH groups signals in 1 H NMR spectra. However, the final assignment of the structure A for heterocycles 4 was made with the help of X-ray analysis (Figure 3). In the case of compounds 9 the presence of NOE between the signals of the methyl group and the CH group in the isoxazole moiety allowed to distinguish closely located signals of two CH groups (Figure 4). Ultimately, the structure of compounds 9 was proven by an X-ray analysis of compound 9e ( Figure 5).

Antibacterial activity
The antibacterial activity of compounds 4, 6 and 9 (Table 7) was studied (see Experimental part in Supporting Information File 1 for details) against reference bacterial cultures: Bacillus subtilis (strain 1211), Staphylococcus aureus (strain 2231) (Gram-positive) and Escherichia coli (strain 1257), Pseudomonas aeruginosa (strain 1111) (Gram-negative).  As it follows from the results obtained several of the substances studied inhibit the growth of test-microorganisms demonstrating a weak antimicrobial effect (Table 7). Generally, the compounds were found to be less active than nitroxoline (being the reference substance).
The antimicrobial effect of the heterocycles studied is different depending on each bacterical strain; however, some rules can be seen. Only a few substances inhibited the growth of Gram-negative bacteria (strains of E. coli and P. aeruginosa) in an effective way. Particularly, compounds 4a and 6g inhibited the growth of E. coli in concentration 125 mg/L. The bacteriostatic activity against P. aeruginosa of compounds 4v and 6e was fixed only in the highest concentration 500 mg/L. The Grampositive bacterium S. aureus showed the resistance to almost all the compounds tested in the given concentration range. The strain of B. subtilis was found to be sensitive to compounds 4i  and 6d, but the bacteriostatic activity was fixed only in the highest concentration 500 mg/L. The information about the influence of the compounds on bacteria is important from the point of view of choosing the further strategy for the investiga-tions of their biological action. An absence or a low level of antibacterial activity of the heterocycles synthesized is a good prerequisite for carrying out the research on the other promising types of activity, e.g., anticancer, antidiabetic, etc., because in this case a negative influence on a microflora of the organism is decreased [110].
The other interesting feature of most of the compounds was the 30-50% increase in biomass of Gram-positive strains (mainly B. subtilis) compared to control. As it follows from a brief literature overview there are a lot of applications of metabolites (recombinant insulin [111], polyhydroxyalkanoates [112,113] etc. [114][115][116][117][118][119]) produced by the strains studied. Therefore, the found probiotic effect of the heterocycles has a very promising area for the further application while scaling up the production of biomass with the aim of shortening the time and saving resources [120,121]. Although this is a subject for a future detailed study the results of antibacterial activity allowed outlining the positive tendency.
The optimal reaction conditions were different depending on the substituent in the carbonyl component and the structure of the isocyanide. Thus, GBB-3CR involving tert-butylisocyanide in the case of aldehydes with electron-donating substituents was carried out in an EtOH/H 2 O mixture with TFA (10 mol %) at rt for 24 h and in DMF/HClO 4 (10 mol %) at rt for 48 h in case of electron-withdrawing groups. When replacing tert-butylisocyanide with ethyl 2-isocyanoacetate the similar imidazo[1,2b]pyrazole-7-carboxamides were isolated from the treatment in TFE/HClO 4 (10 mol %) at rt for 24 h. Ugi-4CR involving tert-butylisocyanide proceeded under standard conditions in MeOH.
The broad antibacterial activity of the obtained compounds was studied as well. Several of the substances inhibited the growth of test microorganisms demonstrating a weak antimicrobial effect. For most of the stuctures a 30-50% increase in biomass of Gram-positive strains (mainly B. subtilis) compared to control was observed. After a detailed study this effect may be used to stimulate the growth of producers of biologically active compounds.