Synthesis, investigation of the new derivatives of dihydropyrimidines and determination of their biological activity

Journal of Molecular Structure 1141 (2017) 39e43

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Synthesis, investigation of the new derivatives of dihydropyrimidines and determination of their biological activity A.M. Maharramov a, M.A. Ramazanov a, G.A. Guliyeva b, A.E. Huseynzada a, *, U.A. Hasanova a, N.G. Shikhaliyev a, G.M. Eyvazova a, S.F. Hajiyeva a, I.G. Mamedov a, M.M. Aghayev a a b

Baku State University, Z. Khalilov 23, Baku, AZ 1148, Azerbaijan Azerbaijan Republican Sanitary and Quarantine Inspection, Zargarpalan 121, Baku, AZ 1009, Azerbaijan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 December 2016 Received in revised form 19 March 2017 Accepted 21 March 2017 Available online 25 March 2017

We reported of synthesis and investigation of the new biologically active derivatives of dihydropyrimidines 2 and 3. The investigation of structures of compounds by various experiments of NMR spectroscopy revealed the splitting of the signals to doublets and multiplets that confirms the presence of diastereomers in solution of compound 2 and the presence of diastereomers and tautomers in solution of compound 3. The individual diastereomer of compound 3 has been isolated. Biological activity of the synthesized compounds was studied on various species of genus Aspergillus fungi. © 2017 Elsevier B.V. All rights reserved.

Keywords: Dihydropyrimidine Diastereomer Tautomer NMR spectroscopy IR spectroscopy Aspergillus fungi

1. Introduction Dihydropyrimidines, as a class of organic compounds with a broad spectrum of biological activity, are widely used in medicine. The biological investigations of these various molecules via molecular manipulation showed such activities as antifungal [1], antiproliferative [1], antiviral [1], antitumor [2e7], antiinflammatory [8e10], antihypertensive [11e15], anti-HIV [16], antiepileptic [17], anti-malarial [18], antibacterial [19e22], antitubercular [23], miscellaneous [24e26], potassium [27e29] and calcium channel antagonist [30]. Among the derivatives of dihydropyrimidines that found their application in medicine we can mention the following drugs: batzelladine A and B [16], (S)-monastrol [2e7], (S)-enastron [2e7], mon-97 [2e7], (R)-fluorastrol [3], terazosin [25] and etc … Moreover, dihydropyrimidines SQ 32926, SQ 32547 which are aza-analogues of nifedipine, are promising targets for using them as oral antihypertensive agents [1]. Dihydropyrimidines, studied in Ref. [17], have similar structure to

* Corresponding author. E-mail address: [email protected] (A.E. Huseynzada). http://dx.doi.org/10.1016/j.molstruc.2017.03.084 0022-2860/© 2017 Elsevier B.V. All rights reserved.

phenobarbital, thus have shown promising anti-epilepsy activity. Dihydropyrimidines are obtained by various methods [31] and Biginelli reaction is a convenient one, due to the fact that it is a onepot condensation reaction, and renewed exploration of the reaction conditions leads to obtaining of enantioenriched dihydropyrimidines [32]. Extraction of individual optical active compound is very important, especially if this compound is potent to be applied as a biologically active compound, because the presence of another enantiomer in drug leads to severe side effects [33e35]. Moreover, obtaining of individual enantiomer mostly is expensive in economic terms, because of using of special conditions and catalysts. According to above-mentioned, in some cases it is better to receive a mixture of diastereomers, which can be easily separated. Considering the importance of dihydropyrimidines, we carried out Biginelli reaction and received new derivatives of dihydropyrimidines. Furthermore, we obtained a mixture of diastereomers and extracted individual diastereomer. The structures of obtained compounds were investigated by various methods, such as nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and elemental analysis. The biological activity of synthesized compounds was also studied against various species of genus Aspergillus fungi.

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A.M. Maharramov et al. / Journal of Molecular Structure 1141 (2017) 39e43

2. Materials and methods All chemicals, applied in the synthesis, were of analytical grade and used as received. K2CO3, dimethyl sulfoxide (DMSO), 1,2dibromoethane, salicylaldehyde, CCl4, urea, acetylacetone, thiourea, ethanol, NH4Cl, acetic acid, benzaldehyde, acetone were purchased from Sigma-Aldrich (Taufkirchem, Germany). The control of the reactions progress and the determination of the synthesized compounds purity were done by TLC on Sorbfil plates, iodine vapors were used as a developer. Elemental analysis was performed on the analyzer Carlo Erba 1108. 2.1. NMR spectra The NMR experiments have been performed on a BRUKER FT NMR spectrometer AVANCE 300 (Bruker, Karlsruhe, Germany) (300 MHz for 1H and 75 MHz for 13C) with a BVT 3200 variable temperature unit in 5 mm sample tubes using Bruker Standard software (TopSpin 3.1). The 1H and 13C chemical shifts were referenced to internal tetramethylsilane (TMS); the experimental parameters for 1H are as follows: digital resolution ¼ 0.23 Hz, SWH (spectral width in Hz) ¼ 7530 Hz, TD (time domain) ¼ 32 K, SI  (Fourier transform size) ¼ 16 K, 90 pulse-length ¼ 10 ms, PL1 (power level for F1 channel) ¼ 3 dB, ns (number of scans) ¼ 1, ds (number of dummy scans) ¼ 0, d1 (relaxation delay) ¼ 1 s and for 13 C as follows: digital resolution ¼ 0.27 Hz, SWH ¼ 17985 Hz,  TD ¼ 64 K, SI ¼ 32 K, 90 pulse-length ¼ 9 ms, PL1 ¼ 1.5 dB, ns ¼ 100, ds ¼ 2, d1 ¼ 3 s. COSY: pulse program ¼ cosygpdf, digital resolution ¼ 1.97 Hz, SWH ¼ 2610, TD ¼ 1 K, SI ¼ 512, 90 pulse-length ¼ 10 ms, PL1 ¼ 3 dB, ns ¼ 4, ds ¼ 16, d1 ¼ 1 s. ROESY: pulse program ¼ roesyph, digital resolution ¼ 1.49 Hz, SWH ¼ 3063 Hz, TD ¼ 2 K, SI ¼ 512 K, 90 pulse-length ¼ 10 ms, PL1 ¼ 3 dB, ns ¼ 16, ds ¼ 4, d1 ¼ 2 s. The NMR-grade DMSO-d6 (99.7%, containing 0.3% H2O) was used for the solutions of 1, 2, 3, 4 and 5. TopSpin plot editor was used to edit the 2D NMR contour plots. 2.2. IR spectra FTIR spectra were recorded on a Varian 3600 FTIR spectrophotometer in KBr tablets. The spectrum was taken in the range of 4000e400 cm1 at room temperature. 2.2.1. Synthesis of 2.20 -(ethane-1.2-diylbis(oxy)) dibenzaldehyde (1) To a solution of 38.3 mmol of salicylaldehyde in 20 ml DMSO were added 19.8 mmol of 1,2-dibromoethane and 37.7 mmol of K2CO3. The reaction mixture was heated on a water bath for 3.5 h. Subsequently, it was cooled with ice. The precipitate was filtered, washed with distilled water, dried and afterwards washed with CCl4; yield is 80%, M. p. 116e118  C. 1 H NMR spectrum of compound 1: (DMSO-d6, d, ppm), 4.6 s (4H, 2OCH2), 7e7.8 m (8H, Ar), 10.3 s (2H, COH). 13 C NMR spectrum of compound 1: (DMSO-d6, d, ppm), 68 (2OCH2), 115 (2CH, Ar), 121 (2CH, Ar), 125 (2C, Ar), 128 (2CH, Ar), 138 (2CH, Ar), 161 (2C, Ar), 190 (2COH). Found, %: C 71.04; H 5.11. C16H14O4. Calculated, %: C 71.11; H 5.19. 2.2.2. Synthesis of 4.40 -(2.20 -(ethane-1.2-diylbis(oxy)bis(2.1phenylene))bis(5-ace-tyl-6-methyl-3.4-dihydropyrimidin-2(1H)one) (2) To a solution of 2 mmol of 2.2’-(ethane-1.2-diylbis(oxy)) dibenzaldehyde in 50 ml of ethanol were added 35 mmol of urea, 17 mmol of acetylacetone and 4 ml of ice acetic acid. The reaction mixture was heated on a water bath for 9 h. Subsequently, it was

cooled with ice. The precipitate was filtered, washed with distilled water and dried; yield is 90%, M. p. 347e350  C. 1 H NMR spectrum of compound 2: (DMSO-d6, d, ppm), 1.9 (6H, 2CH3), 2.3 (6H, 2CH3), 4.5 (4H, 2OCH2), 5.6 (2H, 2CH), 6.9e7.3 (12H, 2Arþ2NH), 9.1 (2H, 2NH). 13 C NMR spectrum of compound 2: (DMSO-d6, d, ppm), 19 (2CH3), 30 (2CH3), 49 (2OCH2), 67 (2CH), 108 (2C), 112 (2C), 121 (2CH, Ar), 128 (2CH, Ar), 129 (2CH, Ar), 131 (2CH, Ar), 149 (2C), 153 (2C, Ar), 157 (2CO), 195 (2CO). Found, %: C 64.81; H 5.71; N 10.71. C28H30N4O6. Calculated, %: C 64.86; H 5.79; N 10.81. FTIR spectrum is given in Fig. S23. The bands at 3383 cm1 and 3232 cm1 correspond to stretching vibrations of NH groups. The intense bands at 1697 cm1 and 1598 cm1 correspond to stretching vibrations of C]O groups. The peaks within the range 1487e1325 cm1 are corresponding to stretching vibrations of CeC in aromatic ring. The peaks within the range 1111e1062 cm1 are corresponding to deformation vibrations of CH groups in the plane of aromatic ring. The band at 1234 cm1 corresponds to stretching vibration of CAr-OCH2 group. 2.2.3. Synthesis of 1-[4-[2-[2-[2-(5-acetyl-1,2,3,4-tetrahydro-6methyl-2-thioxo-4-pyrimidinyl)phenoxy]ethoxy]phenyl]-1,2,3,4tetrahydro-6-methyl-2-thioxo-5-pyri-midinyl]ethanone (3) To a solution of 2 mmol of 2.2’-(ethane-1.2-diylbis(oxy)) dibenzaldehyde in 20 ml of ethanol were added 34 mmol of thiourea, 17 mmol of acetylacetone and 2 mmol of NH4Cl. The reaction mixture was heated on a water bath for 1.5 h. Subsequently, it was cooled with ice. The precipitate was filtered, washed with distilled water and dried; yield is 91%, M. p. 270e272  C. Extraction of individual diastereomer was implemented by dissolving in acetone, in which it doesn't dissolve. 1 Н NMR spectrum of compound 3: (DMSO-d6, d, ppm), 1.9 (6H, 2CH3), 2.2 (6H, 2CH3), 4.4 (4H, 2OCH2), 5.6 (2H, 2CH), 6.9e7.3 (10H, 2Ar), 9.3 (2H, 2NH), 10.2 (2H, 2NH), 13.4 (1H, SH). 13 C NMR spectrum of compound 3: (DMSO-d6, d, ppm), 18.3 (2CH3), 30 (2CH3), 49.8 (2OCH2), 66 (2CH), 109.3 (2C), 113 (2C), 121 (2CH, Ar), 128 (2CH, Ar), 129 (2CH, Ar), 131 (2CH, Ar), 145 (2C), 156 (2C, Ar), 174 (2CS), 195 (2CO). Found, %: C 61.01; H 5.37; N 10.08; S 11.52. C28H30N4S2O4. Calculated, %: C 61.09; H 5.45; N 10.18; S 11.63. FTIR spectrum is given in Fig. S24. The bands at 3399 cm1 and 3196 cm1 correspond to stretching vibrations of NH groups. The intense bands at 1677 cm1 correspond to stretching vibrations of C]O group and 1187 cm1 correspond to stretching vibrations of C]S group. The peaks within the range 1598e1285 cm1 are corresponding to stretching vibrations of CeC in aromatic ring. The peaks within the range 1125e1052 cm1 are corresponding to deformation vibrations of CH groups in the plane of aromatic ring. The band at 1237 cm1 corresponds to stretching vibration of CArOCH2 group. 2.2.4. Synthesis of 5-acetyl-6-methyl-4-phenyl-3.4dihydropyrimidin-2(1H)-one (4) and 1-(6-methyl-4-phenyl-2thioxo-1,2,3,4-tetrahydropyrimidin-5-il)ethanone (5) was implemented as described in literature [36,37] 1 H NMR spectrum of compound 4: (DMSO-d6, d, ppm), 2 s (3H, CH3), 2.3 s (3H, CH3), 5.2 s (1H, CH), 7e7.5 m (5H, Ar), 7.8 s (1H, NH), 9.1 s (1H, NH). 13 C NMR spectrum of compound 4: (DMSO-d6, d, ppm), 19 (CH3), 30 (CH3), 55 (CH), 110 (C), 127 (2CH, Ar), 128 (CH, Ar), 129 (2CH, Ar), 145 (C), 148 (C, Ar), 153 (CO), 194 (CO). Found, %: C 67.74; H 6.01; N 12.1. C13H14N2O2. Calculated, %: C 67.83; H 6.09; N 12.17. 1 Н NMR spectrum of compound 5: (DMSO-d6, d, ppm), 2.1 s (3H,

A.M. Maharramov et al. / Journal of Molecular Structure 1141 (2017) 39e43

deficient carbon of the aldehyde function and the formation of Nacyliminium ion intermediate [38e40]. Further, the cyclization occurs by attack of active methylene compound on this intermediate in a Michael fashion [38e40]. 1 H and 13C NMR spectra of compound 2 and 3 in DMSO-d6 solution are given in Fig. S1eS4. During interpretation of 1H and 13C NMR spectra of these compounds, we have detected the splitting of all signals to doublets (in case of compound 2) and multiplets (in case of compound 3). Splitting of all signals to doublets and multiplets in these compounds theoretically may be caused by the following reasons:

CH3), 2.3 s (3H, CH3), 5.3 s (1H, CH), 7.1e7.4 m (5H, Ar), 9.78 s (1H, NH), 10.3 s (1H, NH). 13 C NMR spectrum of compound 5: (DMSO-d6, d, ppm), 18.7 (CH3), 30.8 (CH3), 54.2 (CH), 110.9 (C), 127.2 (2CH, Ar), 128.1 (CH, Ar), 129 (2CH, Ar), 143.3 (C), 145 (C, Ar), 174.5 (CS), 195.2 (CO). Found, %: C 63.33; H 5.64; N 11.33; S 12.96. C13H14N2SO. Calculated, %: C 63.41; H 5.69; N 11.38; S 13. 2.3. Biological activity Efficiency of the investigated compounds 3, 5 and individual diastereomer of compound 3 (IDC3) was tested on genus Aspergillus fungi by the two traditional methods: disc-diffusion method by using thick nutrient medium and serial dilution method in liquid nutrient medium for the determination of the minimum inhibitory concentration (MIC) of the tested compounds. According to the disc-diffusion method, 1 ml of the diurnal suspension of the test culture (105 CFU/ml) was stratified on the surface of thick nutrient medium (meat-peptone agar, Potato Dextrose Agar). Inoculum was used during 15 min after preparation. Previously prepared discs with a certain concentrations were stratified on the surface of nutrient medium by the sterile tweezers. The weights of the samples were 0,01; 0,1; 0,2; 1; 10 and 12 mg, respectively, and were dissolved in 1 ml of DMSO. Dishes were incubated at 37  C during 24e48 h and in parallel at 28  C during 72e120 h. Similarly, record of the results was carried out, comparing with control dishes without investigated compound (solution) and with the known drug Amphotericin B. MIC of the investigated compounds was determined by the second method e serial dilution in liquid nutrient medium (meat-peptone broth, Sabouraud broth). Various samples (different series) were carried in a test-tube with a sterile broth of 4 ml volume and as a result biological systems with various concentrations were obtained. A day later, 0,1 ml of diurnal culture suspension was carried in them. After exposition during 24e72 h at 28  C the inoculation of 0,1 ml of suspension from test-tube on the surface of meat-peptone agar was made. Record of the results was made after 24 h incubation of dishes in thermostat at 37  C.

- The presence of keto-enol tautomerism, caused by transition of proton from amine group to the oxygen of carbonyl group (or presence a partial double bond between nitrogen and carbon) in case of compound 2, and the presence of tautomerism, caused by transition of proton from amine group to the sulphur of thiocarbonyl group (or presence a partial double bond between nitrogen and carbon) in case of compound 3; - Rotation around single bonds; - The presence of diastereomers in solution (there are two asymmetric carbon atoms in the structures of compounds 2 and 3). In order to determine which of the reasons caused the splitting of all signals to doublets and multiplets in the above-indicated compounds, various NMR investigations have been carried out. The NMR investigations of compound 2 have shown the absence of keto-enol tautomerism (the signal from the enol hydroxyl group hasn't been observed in 1Н NMR spectrum). Subsequently, no changes have been observed in the splitting of the signals in the spectra of compound 2 at the 20e95  C temperature intervals (Figs. S5 and S6), that proves the discrepancy of first and second reasons. Considering all facts, the splitting of the signals to doublets in the spectra of compound 2 is caused by the presence of two diastereomers in the solution of this compound. In order to confirm that the reason of splitting of signals to doublet is caused by the presence of two diastereomers in the solution of compound 2, we synthesized known in literature compound 4 which has only one asymmetric carbon atom (Scheme 2) and investigated its NMR spectra. The fact of absence of any splitting in the spectra of compound 4 (Figs. S17 and S18) also confirms that the splitting of the signals to doublets in the spectra of compound 2 is caused by the existing of two diastereomers in the solution of this compound. 1 H and 13C NMR spectra of compound 3 in DMSO-d6 solution are

3. Results We obtained dialdehyde 1 by condensation of salicylaldehyde with 1,2-dibromoethane. On the basis of this dialdehyde we carried out a three-component Biginelli reaction and obtained new dihydropyrimidines 2 and 3 (Scheme 1). The mechanism of this reaction involved nucleophilic attack of urea (thiourea) on the electron

O

O

H + OH

O

Br Br

O +

O

H

O

O

+

H

O O

O

HN

O HN

N H

X

H 2N

NH2 X

X= O, S

1

X

41

N H

X= O - (2) X= S - (3) Scheme 1. Reactions of synthesis of compounds 1, 2, 3.

42

A.M. Maharramov et al. / Journal of Molecular Structure 1141 (2017) 39e43 Table 4 The biological activity of Amphotericin B.

+

+ O

O

H2N

O

NH2 HN

X

O

X= O - (4) X= S - (5)

Scheme 2. Reactions of synthesis of compounds 4 and 5.

13 14 12 15 11 10 HN3 4 5 2 1 6 N S H

16 16 O O

O

13 14 12 15 11 10

13 14 12 15 11 10

HN3 4 5 8 9 2 1 6 7 N S H

O 8 9 7

N3 4 5 2 6 1 N HS H

16 16 O O

O

13 14 12 15 11 10 O

N3 4 5 8 9 21 6 7 HS N H

8 9 7

Scheme 3. The tautomeric transition of the compound 3.

Table 1 The biological activity of the compound 5. Investigated compound

Compound 5

Inhibition zone of investigated compound, mm A. Flavus

A. Niger

12 15 19 25 39 45

9 13 19 23 29 37

Sample weight, mg

0,01 0,1 0,2 1 10 12

Table 2 The biological activity of the compound 3. Investigated compound

Compound 3

Inhibition zone of investigated compound, mm A. Flavus

A. Niger

8 11 13 17 25 33

14 18 23 29 41 49

Sample weight, mg

0,01 0,1 0,2 1 10 12

Table 3 The biological activity of the compound IDC3. Investigated compound

IDC3

Inhibition zone of investigated compound, mm A. Flavus

A. Niger

9 13 16 21 29 37

7 8 11 14 17 19

Inhibition zone of investigated compound against A.Niger, mm

Sample weight, mg

Amphotericin B

12

0.01

N H

X

X=O, S

H

Investigated compound

Sample weight, mg

0,01 0,1 0,2 1 10 12

given in Figs. S3 and S4. In comparison with the compound 2, the signal from the enthiol group has been observed at 13.4 ppm in 1Н NMR spectrum of compound 3. According to this data, we can conclude that the compound 3 exists in solution in the tautomeric form, caused by transition of proton from amine group to the sulphur of thiocarbonyl group. It is known that in this case the splitting of signals to doublets should be observed, but instead of doublets, multiplets have been observed in 1H and 13C NMR spectra of compound 3. In order to determine, which of the reasons leads to the splitting of the signals to multiplets, NMR experiments of compound 3 at 20, 60 and 80  С have been performed. 1H and 13C NMR spectra of compound 3 in DMSO-d6 solution at 20, 60 and 80  С are given in Figs. S7eS12. NMR investigations of compound 3 demonstrate simplification of the signals in the spectra till doublets, during increasing temperature from 20  С to 80  С, and the complete disappearance of the signal from the enthiol group. This is consistent with the fact that with rising of temperature the complete transition of tautomer, containing enthiol group, to the most stable tautomer, containing thiocarbonyl group, is observed. Thus, we can conclude that the splitting of signals to multiplets in the spectra of compound 3 is caused by the presence of tautomers and diastereomers in solution. In order to confirm that the reason of splitting of signals to multiplets is caused by the presence of tautomers and diastereomers in solution of compound 3, we synthesized known in literature compound 5, which has only one asymmetric carbon atom (Scheme 2) and investigated its NMR spectra. The fact of absence of any splitting in the spectra of compound 5 (Figs. S19 and S20) also confirms that the splitting of signals to multiplets in the spectra of compound 3 is caused by the presence of tautomers and diastereomers in solution. Furthermore, individual diastereomer of compound 3 has been isolated from the diastereomeric mixture and its individualism has been proved by 1H and 13C NMR spectra (Figs. S13 and S14). As seen from the 1H and 13C NMR spectra, splitting of some signals to doublets is observed instead of multiplets, and it is related to the tautomeric transition. Due to the presence of two types of amine groups in the structure of compound 3, the determination of amine group, which takes part in the tautomeric transition, is a matter of scientific interest. Comparison of 1Н NMR spectra of compound 3 at 20, 60 and 80  С allows us to conclude that the signal from amine group at 9.3 ppm participates in the tautomeric transition, due to the fact that with the rising of temperature the constriction of this signal is observed. In order to determine, which of the amine groups is conformed to the signal at 9.3 ppm, 2D COSY and ROESY NMR experiments have been performed (Figs. S15 and S16). As seen from the 2D COSY and ROESY NMR spectra, the signal from the amine group at 9.3 ppm correlates with the signal of CH group in the position 4. Hence, we can conclude that the amine group in the position 3 participates in the tautomeric transition, which can be demonstrated by the following way (Scheme 3). The biological activity of compounds 3, IDC3 and 5 were investigated against various species of genus Aspergillus fungi. Values of inhibition concentration of compounds 3, IDC3, 5 towards microorganisms were 0,01; 0,1; 0,2; 1; 10 and 12 mg/ml respectively. It was determined that in all cases, MICs of investigated

A.M. Maharramov et al. / Journal of Molecular Structure 1141 (2017) 39e43

compounds were 0,01 mg/ml. Investigated compounds showed different activities and results are given in Table 1e3. The highest fungal toxicity against A. Niger was revealed on compounds 5 and 3. Record of the results was carried out, comparing with control dishes without investigated compound (solution) and with the known drug Amphotericin B. It was determined that DMSO doesn't influence on various species of genus Aspergillus fungi. The result of the Amphotericin B effect is given in Table 4. Comparing results, we can conclude that synthesized compounds demonstrate good antifungal activity against A. Niger. Taking into consideration the structure-activity correlation it could be supposed that pronounced antifungal activity of investigated compounds is due to the presence of NH-C(S)-NH moiety in the molecule [41,42]. Compounds 5 and IDC3 demonstrate pronounced antifungal properties against A. Flavus. It was found that both methods were informative during the investigation of influence of synthesized compounds with various concentrations on antifungal activity against A. Niger and A. Flavus. By using serial dilution method was found that samples 3, IDC3, 5 did not demonstrate any inhibition activity on test cultures of A. Ochraceus and possible reasons of this experimental fact require further investigations. 4. Conclusion The compounds 2 and 3, which are the new derivatives of dihydropyrimidines, were synthesized and their structures were investigated by spectroscopic methods. Application of various NMR experiments proved the presence of diastereomers in the solution of compound 2 and the presence of tautomers and diastereomers in the solution of compound 3. Individual diastereomer of compound 3 has been isolated from the diastereomeric mixture by applying the selective dissolving of it in acetone. The individualism of diastereomer has been confirmed by the NMR spectroscopy. The mechanism of the tautomeric transition of the compound 3 has also been clarified and revealed that only the amine group in the position 3 participates in the tautomeric transition. Considering that proposed substances 3, IDC3 and 5, being the derivatives of dihydropyrimidines, can have an ability to act as antifungal drugs, they were tested for the biological activity against various species of genus Aspergillus fungi. Obtained promising results showed that synthesized compounds indeed represent an interest as potential biological active compounds with fungicidal activity that in future, by applying innovative technologies, can lead to the creation of new effective antifungal drugs. The isolation of individual diastereomer and comparing of its biological activity with that of diastereomeric mixture is also very important for studying the structure-activity dependence of chiral drugs. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.molstruc.2017.03. 084. These data include MOL files and InChiKeys of the most important compounds described in this article. References [1] Suresh, S. Jagir, Sandhu, Past, Present and Future of the Biginelli Reaction: a Critical Perspective, ARKIVOC, 2012, pp. 66e133 (i). [2] E. Klein, S. DeBonis, B. Thiede, D.A. Skoufias, F. Kozielskib, L. Lebeaua, Bioorg. Med. Chem. 15 (2007) 6474. [3] H.Y.K. Kaan, V. Ulaganathan, O. Rath, H. Prokopcov, D. Dallinger, C.O. Kappe, F.J. Kozielski, Med. Chem. 53 (2010) 5676.

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