Substituted (сycloalkylcarbonylthioureido)aryl-(benzyl-)carboxylic(sulfonic) acids: synthesis, antimicrobial and growth-regulating activity

Acylisothiocyanates are a promising class of organic compounds that are present in the plant world and can be used in the synthesis of disubstituted thioureas and various heterocycles. These derivatives are characterized by growth-regulating, antibacterial, fungicidal, cytotoxicity, and other activities. Modification of acylisothiocyanates by fragments of substituted aminoarylcarboxylic (sulfo) acids is promising, as some of them (anthranilic, p-aminobenzoic acids) are precursors for the auxins and other natural compounds synthesis. Their combined activity is also an important aspect. Namely the simultaneous manifestation of both fungicidal and restrictive activity. Based on this, the synthesis of new substituted (cycloalkylcarbonylthioureido)aryl-(benzyl-)carboxylic (sulfonic) acids is relevant as promising regulators of plant growth with antibacterial activity.

Актуальные вопросы фармацевтической и медицинской науки и практики. 2021. Т. 14, № 1(35). С. 4-11 Kholodniak, V. V. Stavytskyi, S. I. Kovalenko Common plant growth regulators are auxins, gibberellins, cytokinins, brassinosteroids, etc. [1][2][3]. In addition to phytohormones, plants also produce secondary metabolic products, which also are growth regulators (flavonoids, amino acids, lipids, carboxylic acids, alkaloids, unsaturated lactones, terpenoids, etc.) [4]. However, the use of substances with direct hormonal activity is likely to give way to the use of chemical agents. The latter should modify the metabolism or transport of plant hormones [5]. This approach is advantageous because it increases yields, and synthetic growth regulators are more metabolically stable and cheaper than those with direct hormonal activity. Among the synthetic growth regulators, today are analogues of auxins and cytokinins; antiauxins and cytokinin antagonists; inhibitors of auxin transport and gibberellin biosynthesis; substances that emit ethylene or promote its biosynthesis in plants [3].
Aryl and aryloxyaliphatic acids, onium salts, heterocyclic compounds, etc. are of practical importance among synthetic plant growth regulators. Thus, 3-indolylbutyric, 3-indolyl-and N-naphthylacetic acids, chlormequat, mepiquat, 2,6-dimethylpyridine N-oxide, dimethyl sulfoxide, etc. are used to improve plant growth. Such compounds are used for growth retardation: onium compounds (chlormequat chloride, bromholin bromide, iodolin iodide, etc.), maleic and succinic acid hydrazides, 1,2,4-triazole derivatives (paclobutrazol, uniconazole, etc.), ethylene producers (dextrel hydrel, ectdrel and ether), dichloroisobutyrate (sodium dichloroisobutyrate), etc. It should be noted that these retardants are not universal, not all of them are able to inhibit all forms of growth (for example, rooting cuttings and distortion of coleoptiles), don't show a strong effect compared to phytohormones, are toxic, difficult to metabolize and accumulate in plants. Therefore, the development of new effective environmentally friendly plant growth regulators based on low molecular weight compounds is of great theoretical and practical interest. Their combined activity is also an important aspect. Namely the simultaneous manifestation of both fungicidal and restrictive activity.
Acylisothiocyanates are interesting objects in terms of antimicrobial and growth-regulating agents. Firstly, they are low-toxic compounds, widely represented in the plant world (plants of the cruciferous family) [6,7]. Secondly, the chemistry of cycloalkane carbonylisocyanates is diverse and can be used in the synthesis of functionalized acylthioureas and acylthiosemicarbazides [8][9][10][11][12], as well as various heterocycles [13] which are characterized by growth-regulating, antibacterial, fungicidal and cytotoxicity activity. Thirdly, the modification of acyl isothiocyanates by fragments of substituted aminoarylcarboxylic (sulfo) acids is promising, as some of them (anthranilic, p-aminobenzoic acids) are important precursors of the synthesis of auxins and other natural compounds [14,15].

Aim
Therefore, the aim of the study is to search of effective compounds with growth-regulating and antimicrobial activity among substituted (cycloalkylcarbonylthioureido)aryl-(benzyl-)carboxylic (sulfonic) acids.

Materials and methods
Cyclopropanecarbonyl chloride (1.1) was synthesized by known method [16]. Other starting materials and solvents were obtained from commercially available sources and were used without additional purification.
Melting points were determined in open capillary tubes in a 'Mettler Toledo МР 50" apparatus and were uncorrected. The elemental analyses (C, H, N, S) were performed using the ELEMENTAR vario EL cube analyzer (USA). Analyses were indicated by the symbols of the elements or functions within ±0.3 % of the theoretical values. IR spectra (4000-600 cm -1 ) were recorded on a Bruker ALPHA FT-IR spectrometer (Bruker Bioscience, Germany) using a module for measuring attenuated total reflection (ATR). 1 H NMR spectra (400 MHz) were recorded on a Varian-Mercury 400 spectrometer (Varian Inc., Palo Alto, CA, USA) with TMS as internal standard in DMSO-d 6 solution. LC-MS were recorded using chromatography/mass spectrometric system which consists of high-performance liquid chromatography Agilent 1100 Series (Agilent, Palo Alto, CA, USA) equipped with diode-matrix and mass-selective detector Agilent LC/MSD SL (atmospheric pressure chemical ionization -APCI).
Antimicrobial test. The sensitivity of the microorganisms to the synthesized compounds was evaluated according to the described methods [17]. The assay was conducted on Mueller-Hinton agar by two-fold serial dilution of the compound in 1 ml. After that, 0.1 ml of microbial seeding (106 cells/ml) was added. Minimal inhibit concentration of the compound was determined by the absence of visual growth in the test tube with a minimal concentration of the substance, minimal bactericide/ fungicide concentration was determined by the absence of growth on agar medium after inoculation of the microorganism from the transparent test-tubes. DMSO was used as a solvent, initial solution concentration was 1 mg/ml. For preliminary screening, the mentioned ahead standard test cultures were used: S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and C. albicans ATCC 885-653 standard test cultures. All test strains were received from bacteriological laboratory in Zaporizhzhia Regional Laboratory Center of State Sanitary and Epidemiological Service of Ukraine. Nitrofurazone and Ketoconazole were used as reference compounds with proved antibacterial/antifungal activity. Additional quality control of the culture media and solvents was conducted by commonly used methods [17].
Influence of synthesized compounds on growth rates. The effect of synthesized compounds on growth rates was assessed on wheat (variety Grom) at the laboratory of the State Enterprise "State Center for Certification and Examination of Agricultural Products" (Zaporizhzhia) by a known method [18]. The concentration of aqueous solutions of "Heteroauxin" (3-indolylacetic acid) and test compounds was 0.00002 %. To achieve that concentration a 0.2 g of substances was emulsified in 1 ml of Twin-80 and adjusted with water to 1 l. Then from the resulting solution, 1 ml was taken and again adjusted with water to 1 l. Irrigation with a solution of test substances was performed every other day, the rate of water consumption -40 ml per Petri dish. Control seeds were watered with water and an emulsifier. Grain germination on days 4 and 8, the total number and length of roots, total mass of aboveground part (considering the weight of the grain) were evaluated during liquidation of the experiment.

Results
The synthesis of the original cyclopropanecarbonyl isothiocyanate was carried out according to a known synthetic approach, which included the interaction of cyclopropanecarbonyl chloride (1.1) with ammonium isothiocyanate (acetonitrile medium) ( Fig. 1) [12]. Cyclopropanecarbonyl isothiocyanate without isolation from the reaction medium (in situ method) regioselectively and easily has attached aminoaryl-(benzyl-)carboxylic acids, 4-aminobenzenesulfonic acid, and its amide. This was produced individual compounds 2.1-2.11 with satisfactory yields (48-74 %).
The structure and individuality of compounds 2 were proved using elemental analysis, chromato-mass, IR and 1 H NMR spectra.
Analysis of these chromato-mass spectra showed that compounds 2 had a characteristic quasimolecular ion [M+1], which corresponds to the calculated mass and confirms their structure and individuality.
The structure and individuality of compounds 2 were proved using elemental analysis, chromato-mass, IR, and 1 H NMR spectra. The structure and individuality of compounds 2 were confirmed by chromato-mass spectra, in which the quasimolecular ion [M+1] corresponded to the calculated mass. In the 1 H NMR spectra of compounds 2, singlet or broad singlet signals of protons of the -COOH group were registered at the 13.45-12.50 ppm, the -C(S) NH groups -at the 13.02-11.44 ppm, and the C(О)NH groups -at the 11.69-11.04 ppm. In compounds 2.2, 2.10, 2.11 signals of protons -COOH-group were absent, due to deuteroexchange with DMSO.
In the 1 H NMR spectra of compounds 2, there were proton signals of the cyclopropane fragment, which appear in a strong field as wide multiplets of sequentially arranged signals of axial and equatorial protons. Thus, for com- О. V. Kholodniak, V. V. Stavytskyi, S. I. Kovalenko pounds 2, the methine proton of the cyclopropane fragment was registered as a broad multiplet in the range of 2.53-2.05 ppm, and the methylene proton was observed as multiplets at the 1.00-0.70 ppm. Aromatic protons of compounds 2 in 1 H NMR spectra have "classical" multiplicity and chemical shifts, which were in accordance with the proposed structures [19].
Analysis of the IR spectra of compounds 2 showed the presence of wide bands of valence vibrations of the associated NH-groups in the range of 3995-3121 cm -1 , which indicated the presence of secondary amide and thioamide groups in the molecule. Compounds 2 were also characterized by vibrations of two υ CO groups (band "Amide I") at the 1693-1657 cm -1 and 1674-1602 cm -1 , mixed valence-strain vibrations of N-H and C-N bonds ("Amide II") at the 1591-1504 cm -1 . In addition, characteristic contours of low-intensity vibrations of υ C=C -bond of the aromatic ring at the 1486-1424 cm -1 , non-planar vibrations γ (=C-H) at the 850-666 cm -1 and intense bands of symmetric and antisymmetric vibrations of υ СH2 -groups at the 2994-2304 cm -1 (cyclopropane fragment) were present in the spectrum [20].
The conducted microbiological screening showed that compounds 2.1-2.11 inhibited the growth of S. aureus and P. aeruginosa at a concentration of 50.0 μg/ml and exhibited bactericidal activity at a concentration of 100 μg/ml ( Table 1). However, compounds 2.1-2.11 weren't effective against E. coli (MIC 100.0 μg/ml, MBC 200 μg/ml). It is important to note, that the antimicrobial activity of the studied compounds was significantly lower than the reference drug Nitrofurazone. Better results were obtained when the antifungal activity against C. albicans ( Table 1) was studied. For example, compounds 2.1-2.11 inhibited the growth of the C. albicans strain at the concentration of 25-100 μg/ml and exhibited fungicidal activity at the concentration of 50 μg/ml. Levels of antifungal activity of the compounds competed with the reference drug Ketoconazole (MIC 25 μg/ml, МFC 50 μg/ml).
Analysis of the results of the study of the effect of synthesized compounds on growth rates showed ( Table 2) that compounds 2.1-2.11, like IAAs, have virtually no effect on grain germination. However, auxin-stimulating activity (number of roots) of most tested compounds have exceeded the control, and compound 2.7 competes with IAAs. In addition, all compounds stimulated their growth (root length) exceeding the IAAs by 0.3-48.5 %. This figure was also confirmed by the total weight of the roots and, importantly, compounds 2.2, 2.4, and 2.7 also exceed IAAs.
Analysis of the structure-activity relationship didn't reveal a correct relationship between the studied compounds. The combination of aryl moiety with a carboxyl group in the molecule is the key factor in its manifestation. This provides both antibacterial and growth-promoting activity. Moreover, compounds 2.2, 2.4, and 2.7 with high growth activity contain fragments of 2-amino-and 4-aminobenzoic acids, which are precursors for the synthesis of auxins and other natural compounds.

Conclusions
A one-step method for the synthesis of the substituted (cyclopropanecarbonylthioureido)aryl-(benzyl-)carboxylic (sulfonic) acids was developed. The physical-chemical properties of the synthesized compounds were studied using a set of methods (IR, 1 H NMR spectroscopy, chromato-mass spectrometry, elemental analysis) and the peculiarities of the structure were discussed. The synthesized compounds showed moderate antimicrobial activity against S. aureus and P. aeruginosa (MIC 50 μg/ml, MBC 100 μg/ml) and significant antifungal activity against C. albicans (MIC 25-50 μg/ml, MFC 25-50 μg/ml). Compounds 2.2, 2.4, and 2.7 of combined activity (high antifungal and growth-stimulating) exceeding the natural growth stimulator (heteroauxin) were found.

Funding
The work was carried out on the budgetary theme of the Ministry of Health of Ukraine "Cycloalkylcarbonylisothiocyanates are effective precursors for the synthesis of substituted thioureas and the construction of heterocyclic systems", state registration № 0118U004261. The work was performed with the financial support of "Enamine Ltd" (Kyiv, Ukraine).