Synthesis of some 6-alkylureido-4-aryl-2(1H)-pyridones: further transformations and pharmacological activity

Lyubomir Dimitrov Raev; Ivo Christov Ivanov; Henri Angel Astroug; Wolfgang Frey; Silviya Georgieva Agontseva

doi:10.1515/znb-2015-0066

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Synthesis of some 6-alkylureido-4-aryl-2(1H)-pyridones: further transformations and pharmacological activity

Lyubomir Dimitrov Raev , Ivo Christov Ivanov , Henri Angel Astroug , Wolfgang Frey and Silviya Georgieva Agontseva

Published/Copyright: October 14, 2015

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From the journal Zeitschrift für Naturforschung B Volume 70 Issue 11

Abstract

The Michael addition of enaminoesters to coumarins (1) does not lead to the formation of simple adducts 3 but to the rearranged 4-aryl-2-pyridone 4a. Now, N-carbamoylation of the 6-amino-2-pyridone 4a with alkyl isocyanates and further transformation of the corresponding novel ureido-2-pyridone derivatives 6a–g into chromeno[3,4-c]pyridines 5d,g and O-acetyl derivatives 7a–g are reported. All newly synthesized compounds were characterized by means of ¹H/¹³C NMR, MS, IR spectra and elemental analysis. The structure of the ureide 6f and of the N-cyclohexyl-O-acetyl derivative 7g were additionally confirmed by crystal structure determinations. Acute toxicity after intraperitoneal administration, blood clotting time, analgesic activity and the effects on the hexobarbital sleeping time were tested on laboratory animals (compounds 4a, 6a, 6c, 6d and 6g).

Keywords: acute toxicity; analgesic activity; blood clotting time; chromeno[3,4-c]pyridines; crystal structure; enaminoesters; hexobarbital sleeping time; isocyanates; 2-pyridones; ureides

1 Introduction

In a series of earlier papers, we described the Michael addition of some enaminoesters to coumarin (1a, Scheme 1) and some of its 3-substituted (e.g. the coumarin-3-carboxylic acid 1b) and 3,6-disubstituted derivatives [1–5]. The initially formed adduct 3 spontaneously undergoes further intramolecular rearrangement to give the simplest of the resulting stable adducts, ethyl 2-amino-4-(2-hydroxyphenyl)-6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxylate (4a). Compound 4a could also be obtained from coumarin-3-carboxylic acid (1b) and ethyl 3,3-diaminoacrylate (2) accompanied by decarboxylation.

Scheme 1:

Synthetic route to the coumarin–enaminoester adduct 4a, its N-acetyl (4b), N,O-diacetyl (4c) and O-acetyl (4d) derivatives, cf. [1].

Carbamoylation of enamines with heterocumulenes, leading to either C- or N-adducts, has been widely used as the initial step in the synthesis of various heterocyclic compounds, mainly derivatives of uracil [6]. Thus, 3-aminopropenoic esters were added to organic isocyanates or isothiocyanates yielding N-adducts which readily cyclized to 6-substituted uracils [7] in the presence of a base. In a similar way, we previously developed the first general method for direct preparation of 3-monosubstituted 6-aminouracils [8] by the addition of the ene-diamine (ketene aminal) 2 to alkyl isocyanates. It seemed reasonable to try an analogous approach by carbamoylating the coumarin–enaminoester adduct, the 2-pyridone 4a, which contains the same ketene aminal moiety (Scheme 1). As we reported earlier [3], the adduct 4a, on the other hand, readily underwent simple recyclization on heating to give the substituted chromeno[3,4-c]pyridine 5a. Conditions have also been reported [1] for consecutive acetylation of the primary amino and the phenolic group to yield the corresponding acetyl derivatives 4b, c, d.

In the present paper, we wish to report N-carbamoylation of 4a with alkyl isocyanates and the results of some trials on further transformation of the 3-alkyl ureides 6a–g (Scheme 2) thus obtained. We also had to examine if new synthesis of 3-alkyl-5-arylpyrido[2,3-d]pyrimidine-2,4,7(1H,3H,8H)-triones of type 8 with promising pharmacological properties [9] was possible to be developed by cyclization of the resulting 3-alkylureides 6a–g (cf.Scheme 2).

Scheme 2:

N-Carbamoylation of 4a (→ 6a–g) and consequent acetylation (→ 7a–g). The structures 6f and 7g are confirmed by crystal structure determinations.

2 Results and discussion

2.1 Chemistry

Pursuing our interest in biologically active 2-pyridones [10–13], structurally related to 1,4-dihydropyridine calcium antagonists, we first studied the reaction of 4a with alkyl isocyanates in order to achieve selective C(2)-N-carbamoylation and to avoid competitive O-carbamoylation at the phenolic hydroxyl group (Scheme 2). The reaction was successfully performed in anhydrous acetonitrile at reflux to give the ureides 6a–g with good yields (76–97 %). The expected IR spectral bands could be observed for NH/OH-associated groups as well as for all three different carbonyl groups (ester, lactam, and urea) at about 1700, 1670 and 1620 cm⁻¹, respectively. All relevant proton groups show the expected signals in the ¹H NMR spectra. The ¹³C NMR data of two selected representatives 6a,d are schematically presented in Fig. 1. In addition, the structure of the ureides 6a–g was unambiguously confirmed by means of X-ray crystallographic analysis of 6f (Fig. 2). An attempt to bring 4a to reaction with methylisothiocyanate in acetonitrile failed, even after 40-h reflux, i.e. no product could be detected by means of thin-layer chromatography (TLC).

Fig. 1:

¹³C NMR chemical shifts (δ in ppm) of compounds 6a, d and 7d (in [D₆]DMSO).

Fig. 2:

Molecular structure of 6f in the crystal (displacement ellipsoids are drawn at the 50 % probability level; hydrogen atoms are omitted for clarity; only one alternative of the disordered atom C17 is shown).

The successive acetylation of the phenolic hydroxyl group was carried out in pyridine to selectively afford the corresponding O-acetyl derivatives 7a–g (cf. [1]) in quantitative yield. The molecular structure of the N-cyclohexyl-O-acetyl derivative 7g was confirmed by X-ray crystallographic analysis (Fig. 3).

Fig. 3:

Molecular structure of 7g in the crystal (displacement ellipsoids are drawn at the 50 % probability level; hydrogen atoms are omitted for clarity).

In order to prove the structure of the products 7a–g chemically, we also used an alternative synthetic route for 7d, starting from the O-acetyl derivative 4d and n-butylisocyanate. We obtained a product (7d) which was identical with that of the reaction of 6d with acetic anhydride–pyridine (Scheme 2).

On the analogy with the published heterocyclization of other structurally similar ureide esters [6–8], we tried to perform the cyclization of 6a–g under the reported conditions, i.e. in the presence of sodium ethoxide at ambient temperature [7, 8]. However, instead of the expected pyrido[2,3-d]pyrimidine 8, we isolated the 2H-chromeno[3,4-c]pyridines 5d, g as products which are actually coumarin derivatives again (Scheme 2). Their structure was confirmed by means of spectral methods (IR, NMR and MS) and elemental analyses. Thus, three different carbonyl frequencies were observed at 1726, 1651 and 1622 cm⁻¹ in the IR spectrum of 5d, while its ¹H NMR spectrum showed a typical ABX spin system for the protons 5-H^AH^B and 4-H^X at δ = 2.61, 3.21 and 4.23 ppm, respectively, in addition to the expected D₂O-exchangeable signals due to the three NH groups at δ = 8.53, 11.01, and 11.73 ppm. In the positive ion CI-MS spectra, the base peaks matched the [M+1]⁺ ions and the corresponding isotope distribution confirmed the molecular formulae of 5d, g.

Many known approaches for the synthesis of [1]benzopyrano[3,4-c]pyridines or [1]benzopyrano[4,3-b]pyridines include coumarin derivatives as starting compounds or intermediates [14–19]; for a review see [20]. Evidently, the preparation of the 4-ureido-1,10b-dihydro-5H-[1]benzopyrano[3,4-c]pyridines 5d, g, as discussed above, is a new contribution to the synthesis of this heterocyclic class of compounds.

2.2 Pharmacology

The estimated acute toxicity LD₅₀ of the tested compounds after intraperitoneal administration to male line H mice is 4500 mg kg⁻¹ b.w. for compound 4a and more than 5000 mg kg⁻¹ b.w. for compounds 6a, 6c, 6d and 6g. The tested substances are practically non-toxic.

The results of the investigation of the effect of compound 4a on the blood clotting time compared to warfarin 24 h after administration are presented in Table 1. It was shown that 4a prolongs the clotting time of the control at a dose 1/10 of LD₅₀.

Table 1

Blood clotting time.

	Control (saline)	Warfarin	Compound 4a
Dose, mg kg⁻¹	–	150	450 (1/10 LD₅₀)	90 (1/50 LD₅₀)	45 (1/100 LD₅₀)
Clotting time, s	158	240^a	479^a	176.7	211
SD	±16.8	±35.9	±66.6	±58.7	±103.8

^ap < 0.001: level of significance compared to the controls.

The results of the investigation of the analgesic activity (hot-plate test in mice) of different doses of compound 4a 30 min after intraperitoneal administration are presented in Table 2. At doses 1/10, 1/50, and 1/100 of LD₅₀, the coumarin–enaminoester 4a exhibits analgesic activity.

Table 2

Analgesic activity (hot-plate test).

	Control (saline)	Compound 4a
Dose, mg kg⁻¹	–	450 (1/10 LD₅₀)	90 (1/50 LD₅₀)	45 (1/100 LD₅₀)
Time, s	14.2	21.7^a	26.3^a	28.8^b
SD	±1.5	±3.8	±6.0	±1.9

^ap < 0.001; ^bp < 0.05: level of significance compared to the controls.

The results of the investigation of the analgesic activity (acetic acid writhing test in mice) of different doses of compound 4a, administered intraperitoneally 60 min before the acetic acid injection, are presented in Table 3. The number of writhing was recorded during 30 min commencing 5 min after the acetic acid injection, as presented in Table 3. Metamizole (dose 25 mg kg⁻¹ equal to 1/10 of LD₅₀) was used as a positive control. The action of 4a is dose-dependent.

Table 3

Analgesic activity of 4a (acetic acid writhing test).

	Control (saline)	Metamizole	Compound 4a
Dose, mg kg⁻¹	–	25 (1/10 LD₅₀)	450 (1/10 LD₅₀)	90 (1/50 LD₅₀)	45 (1/100 LD₅₀)
Number of writhing	77.2	16.2	22.8^a	39.8^a	67.5^b
SD	±14.1	±11.5	±2.1	±8.5	±5.4

^ap < 0.001; ^bp < 0.05: level of significance compared to the controls.

In the same way, the analgesic effects of compounds 4a, 6a, 6c, 6d and 6g (all doses 1/10 of LD₅₀) are measured as given in Table 4. Compound 6g displays the highest analgesic effect at a dose 1/10 of LD₅₀.

Table 4

Analgesic activity of 4a, 6a, 6c, 6d and 6g (acetic acid writhing test).

Compound	Control (saline)	Metamizole	4a	6a	6c	6d	6g
Dose, mg kg⁻¹	–	25	450	500	500	500	500
Number of writhing	77.2	16.2	22.8^a	58.5^b	34.0	30.3^a	3.2^a
SD	±14.1	±11.5	±2.1	±6.0	±15.5	±14.4	±0.3

^ap < 0.001; ^bp < 0.05: level of significance compared to the controls.

The effects of compounds 4a, 6a, 6c, 6d and 6g (all doses 1/50 of LD₅₀) on the duration of sleep induced by hexobarbital (dose 80 mg kg⁻¹ b.w.) are presented in Table 5. All tested compounds were administered intraperitoneally 30 min before hexobarbital. It was found that all the substances (except 6g) administered at a dose 1/50 of LD₅₀ significantly prolong the hexobarbital sleep.

Table 5

Effects on hexobarbital sleeping time.

Compound	Hexobarbital (control)	4a	6a	6c	6d	6g
Dose, mg kg⁻¹	80	450	500	500	500	500
Sleeping time (min)	24.7	106.0^a	36.5^b	48.0^a	48.8^a	20.67
SD	±4.71	±7.67	±6.08	±6.63	±4.32	±3.44

^ap < 0.001; ^bp < 0.05: level of significance compared to the controls.

3 Conclusion

The addition reaction of the aminopyridone 4a to alkyl isocyanates was studied, and the conditions were specified under which the corresponding N-monocarbamoyl derivatives 6a–g could be obtained in good yields. Heterocyclization of some of the newly synthesized derivatives 6 by heating in inert solvents was also examined. Only the base-catalyzed re-lactonization leading to 4-ureidobenzopyrano[3,4-c]pyridines (5d, g) was possible but not the alternative heterocyclization to 5-arylpyrido[2,3-d]pyrimidine derivatives of type 8. It was found further that the N-carbamoyl derivatives 6a–g could be converted into the corresponding N,O-diacetyl derivatives 7a–g by treatment with acetic anhydride in anhydrous pyridine.

The starting aminopyridone 4a and some of the resulting N-carbamoyl derivatives (6a, c, d, g) were submitted to initial pharmacological screening. The found toxic dose LD₅₀ of the tested compounds is more than 5000 mg kg⁻¹, i.e. they are practically non-toxic. Studies have shown that if applied in doses 1/10 of LD₅₀ they extend significantly the hexobarbital sleep and exhibit an analgesic effect, which is most pronounced by the ureide 6g.

4 Experimental section

4.1 General information

Melting points were determined on a Büchi B-510 (Switzerland) apparatus and were uncorrected. The IR spectra were recorded in nujol on a Shimadzu 8001 FTIR spectrometer (Shimadzu Corporation, Kyoto, Japan). ¹H and ¹³C NMR spectra were registered on a Bruker WP 100 MHz or a Bruker AMX 300/400 MHz spectrometer (Bruker Corporation, Billerica, MA, USA). Chemical shifts are expressed in δ (ppm) downfield from tetramethylsilane (TMS) as an internal reference. The signals at 400 MHz were assigned with the help of 2D correlation NMR spectra. Signals close to δ ≈ 2.5 ppm in [D₆]DMSO were partially overlapped by the solvent. Mass spectra were measured on a Varian MAT 711 spectrometer (Varian Inc., USA) at 70 eV (direct inlet). TLC monitoring was carried out on silica gel Merck GF₂₅₄ pre-coated aluminum sheets (Merck KGaA, Darmstadt, Germany), eluted with hexane–acetone 2:1 (v/v). Spots were visualized by treatment with I₂ (vapor) and under UV irradiation (λ = 254 nm).

4.2 Ethyl 2-amino-4-(2-hydroxyphenyl)-6-oxo-1,4,5,6-tetrahydro- 3-pyridinecarboxylate (4a) (improved procedure; cf. [1–5])

A mixture of 31.7 g (0.16 mol) of coumarin-3-carboxylic acid (1b) and 28.6 g (0.22 mol) of ethyl 3,3-diaminopropenoate (2) in 300 mL of anhydrous ethanol was refluxed under stirring for 5 h. The reaction mixture was concentrated in vacuum to one-third of the initial volume and was allowed to stay several hours at 5–10 °C. The separated crystals were filtered, washed with ethanol and recrystallized from ethanol. Yield: 27.6 g (66 %) of colorless platelets of 4a: m.p. 187–189 °C (dec.) ([3]: yield 44–57 %, m.p. 192–195 °C). ¹H NMR and ¹³C NMR spectral data are reported in [1].

4.3 General procedure for the reaction of ethyl 2-amino-6-oxo-4-(2-hydroxyphenyl)-1,4,5,6-tetrahydropyridine-3-carboxylate (4a) with alkyl isocyanates. Preparation of ethyl 4-(2-hydroxyphenyl)-2-(3-alkylureido)-6-oxo-1,4,5,6-tetrahydropyridine- 3-carboxylates (6a–g)

A solution of 10.0 mmol of the corresponding alkyl isocyanate dissolved in 5 mL of anhydrous acetonitrile was added dropwise under stirring to a solution of 1.38 g (5.0 mmol) of 4a in 100 mL of hot anhydrous acetonitrile for a period of 20 min. After reflux for several hours (TLC monitoring), the reaction mixture was cooled down to 0–5 °C and the separated crystals of 6a–g were filtered and washed with cold anhydrous acetonitrile. Recrystallization from ethanol yielded the corresponding pure product 6a–g.

4.3.1 Ethyl 4-(2-hydroxyphenyl)-2-(3-ethylureido)- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (6a)

Reflux: 4 h. This product was obtained in 97 % yield as a colorless solid with m.p. 200–202.5 °C, R_f = 0.28. – IR (nujol): ν = 3333, 3221, 1701, 1669, 1622, 1600 cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.38 (s, 1H, NH), 10.93 (s, 1H, NH), 9.56 (s, 1H, OH), 8.30 (br.t, NH), 6.67–7.01 (m, 4H_arom.), 4.39 (d, 1H, J = 3 Hz, 4-H^X), 3.98 (q, 2H, J = 7 Hz, OCH₂), 3.10 (q, 2H, J = 7 Hz, NCH₂), 2.82–3.05 (m, 1H, 5-H^B), ~2.56 (m, 1H, 5-H^A), 1.05 (t, 3H, J = 7 Hz, CH₃), 1.03 (t, 3H, J = 7 Hz, CH₃). – ¹³C NMR spectrum, see Fig. 1. – C₁₇H₂₁N₃O₅ (347.37): calcd. C 58.78 H 6.09 N 12.10; found C 58.74, H 6.09, N 12.10.

4.3.2 Ethyl 4-(2-hydroxyphenyl)-2-[3-(1-propyl)ureido]- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (6b)

Reflux: 6 h. This product was obtained in 90 % yield as a colorless solid with m.p. 206–209 °C, R_f = 0.30. – IR (nujol): ν = 3335, 3218, 1700, 1669, 1620, 1600 cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.38 (s, 1H, NH), 10.91 (s, 1H, NH), 9.52 (s, 1H, OH), 8.29 (br.t, NH), 6.66–7.00 (m, 4H_arom.), 4.39 (d, 1H, J = 3 Hz, 4-H^X), 3.99 (q, 2H, J = 6.4 Hz, OCH2), 3.00 (q, 2H, J = 5.6 Hz, NCH₂), 2.78–2.94 (m, 1H, 5-H^B), ~2.48 (m, 1H, 5-H^A), 1.34–1.48 (m, 2H, CH₂), 1.03 (t, 3H, J = 7 Hz, CH₃), 0.86 (t, 3H, J = 7 Hz, CH₃). – C₁₈H₂₃N₃O₅ (361.40): calcd. C 59.82, H 6.41, N 11.63; found C 59.82, H 6.43, N 11.60.

4.3.3 Ethyl 4-(2-hydroxyphenyl)-2-[3-(2-propyl)ureido]- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (6c)

Reflux: 6 h. This product was obtained in 86 % yield as a colorless solid with m.p. 190–192 °C, R_f = 0.31. – IR (nujol): ν = 3328, 3227, 1700, 1663, 1620, 1603 cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.40 (s, 1H, NH), 10.85 (s, 1H, NH), 9.52 (s, 1H, OH), 8.27 (d, 1H, J = 2.5 Hz, NH), 6.66–7.00 (m, 4H_arom.), 4.38 (d, 1H, J = 3 Hz, 4-H^X), 3.99 (q, 2H, J = 6.2, OCH₂), 3.71–3.85 (m, 1H, CHMe₂), 2.79–3.04 (m, 1H, 5-H^B), ~2.48 (m, 1H, 5-H^A), 1.09 (d, 6H, J = 3.2 Hz, 2 × CH₃, isopropyl), 1.02 (t, 3H, J = 7 Hz, CH₃). – C₁₈H₂₃N₃O₅ (361.40): calcd. C 59.82, H 6.41, N 11.63; found C 59.88, H 6.42, N 11.79.

4.3.4 Ethyl 4-(2-hydroxyphenyl)-2-[3-(1-butyl)ureido]- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (6d)

Reflux: 3 h. This product was obtained in 76 % yield as a colorless solid with m.p. 195.5–197 °C, R_f = 0. 32. – IR (nujol): ν = 3305, 3250, 1705, 1668, 1622, 1600 cm⁻¹. – ¹H NMR (400 MHz, [D₆]DMSO): δ = 11.41 (s, 1H, NH), 10.92 (s, 1H, NH), 9.57 (s, 1H, OH), 8.31 (t, 1H, J = 5 Hz, NH), 6.69, 6.80, 6.86, and 7.02 (4 × m, 4 × 1H, 4H_arom.), 4.40 (d, 1H, J = 3.5 Hz, 4-H^X), 4.06 (m, 1H, OCH), 3.94 (m, 1H, OCH), 3.08 (dt, 2H, J = 5 Hz, J = 7 Hz, NCH₂), 2.90 (m, 1H, 5-H^B), 2.49 (m, 1H, 5-H^A), 1.42–1.43 (m, 2H, CH₂), 1.31–1.33 (m, 2H, CH₂), 1.05 (t, 3H, J = 7 Hz, CH₃), 0.89 (t, J = 7.3 Hz, CH₃). – ¹³C NMR spectrum, see Fig. 1. – C₁₉H₂₅N₃O₅ (375.42): calcd. C 60.79, H 6.71, N 11.19; found C 60.78, H 6.61, N 11.18.

4.3.5 Ethyl 4-(2-hydroxyphenyl)-2-[3-(t-butyl)ureido]- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (6e)

Reflux: 10 h. This product was obtained in 81 % yield as a colorless solid with m.p. 179–182 °C, R_f = 0.35. – IR (nujol): ν = 3314, 3256, 1703, 1671, 1624, 1602 cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.44 (s, 1H, NH), 10.73 (s, 1H, NH), 9.52 (s, 1H, OH), 8.09 (s, 1H, NH), 6.66–7.08 (m, 4H_arom.), 4.40 (d, 1H, J = 7 Hz, 4-H^X), 3.99 (q, 2H, J ≈ 7 Hz, OCH2), 2.83 (dd, 1H, J = 7 Hz, J = 16 Hz, 5-H^B), 2.29 (d, 1H, J = 16 Hz, 5-H^A), 1.28 (s, 9H, 3 × CH₃), 1.02 (t, 3H, J = 7 Hz, CH₃). – C₁₉H₂₅N₃O₅ (375.42): calcd. C 60.79, H 6.71, N 11.19; found C 60.80, H 6.66, N 11.16.

4.3.6 Ethyl 4-(2-hydroxyphenyl)-2-[3-(1-hexyl)ureido]- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (6f)

Reflux: 3.5 h. This product was obtained in 89 % yield as a colorless solid with m.p. 191–193 °C, R_f = 0.35. – IR (nujol): ν = 3303, 3231, 1701, 1667, 1620, 1601 cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.41 (s, 1H, NH), 10.92 (s, 1H, NH), 9.57 (s, 1H, OH), 8.3 (br.t, 1H, NH), 6.65–7.05 (m, 4H_arom.), 4.41 (d, 1H, J = 7 Hz, 4-H^X), 4.00 (m, 2H, OCH2), 3.9–4.1 (m, 2H, NCH₂), 2.82 (d, 1H, J = 7 Hz, 5-H^B), ~2.4 (m, overlapped by DMSO, 1H, 5-H^A), 1.12–1.60 (m, 8H, 4 × CH₂, hexyl), 1.03 (t, 3H, J = 7 Hz, CH₃), 0.88 (br.t, 3H, CH₃). – C₂₁H₂₉N₃O₅ (403.48): calcd. C 62.51, H 7.24, N 10.41; found C 62.50, H 7.24, N 10.39.

4.3.7 Ethyl 4-(2-hydroxyphenyl)-2-(3-cyclohexylureido)-6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (6g)

Reflux: 4.5 h. This product was obtained in 82 % yield as a colorless solid with m.p. 218–218.5 °C, R_f = 0.36. – IR (nujol): ν = 3302, 3235, 1700, 1669, 1620, 1603 cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.41 (s, 1H, NH), 10.86 (s, 1H, NH), 9.53 (s, 1H, OH), 8.26 (d, 1H, J = 4 Hz, NH), 6.66–7.00 (m, 4H_arom.), 4.42 (d, 1H, J = 7 Hz, 4-H^X), 3.9–4.1 (m, 1H, NCH), 3.99 (q, 2H, J = 7 Hz, OCH₂), 2.80–3.05 (m, 1H, 5-H^B), ~2.48 (m, overlapped by DMSO, 1H, 5-H^A), 1.7 and 1.2 (2 × m_c, 10H, 5 × CH₂, cyclohexyl), 1.03 (t, 3H, J = 7 Hz, CH₃). – C₂₁H₂₇N₃O₅ (401.46): calcd. C 62.83, H 6.78, N 10.47; found C 63.04, H 6.86, N 10.52.

4.4 Heterocyclization of the N-monoureides 6d,g to pyridocoumarins 5d, g

4.4.1 1-Butyl-3-(2,5-dioxo-1,3,5,10b-tetrahydro- 2H-chromeno[3,4-c]pyridin-4-yl)urea (5d)

A solution of sodium ethoxide, prepared from 0.12 g (5.0 mmol) of sodium and 5 mL of ethanol, was added dropwise under stirring to a suspension of 1.88 g (5.0 mmol) of 6d in 20 mL of anhydrous ethanol. The reaction mixture was allowed to stay at room temperature for 7 days and the separated sodium salt was collected by filtration. It was then mixed with some water and acidified with acetic acid to pH ≈ 5. The crystals formed were filtered, washed with water and recrystallized from ethanol to give pure 5d. Yield: 0.33 g (20 %) of colorless crystals with m.p. 258–260 °C. IR: ν = 2350–3350 (3287 br., NH assoc.), 1726 (C=O, lactone), 1651, 1622 (C=O) cm⁻¹. – ¹H NMR (400 MHz, [D₆]DMSO): δ = 11.73 (s, 1H, NH, D₂O-exchangeable), 11.01 (s, 1H, NH, D₂O-exchangeable), 8.53 (s, 1H, NH, D₂O-exchangeable), 7.04, 7.15, 7.28, 7.41 (4 × m_c, 4 × 1H, 4H_arom.), 4.23 (d, 1H, J = 14.3 Hz, 10b-H^X), 3.21 (d, 1H, J = 15.6 Hz, 1-H^B), 3.09 (s, 2H, N-CH₂), 2.61 (dd, 1H, J₁ = 14.3 Hz, J₂ = 14.5 Hz, 1-H^A), 1.42 (m, 2H, CH₂), 1.31 (m, 2H, CH₂), 0.89 (t, J = 3 Hz, 3H, CH₃). – ¹³C NMR (100 MHz, [D₆]DMSO): δ = 169.9 (C=O, lactam), 163.5 (C-4), 155.1 (C=O, lactone), 152.7 (C=O, urea), 150.2 (C-6a), 128.8 (C-8), 127.0 (C-10), 125.9 (C-9), 125.0 (C-10a), 116.5 (C-7), 75.3 (C-4a), 40.0 (NCH₂), 35.9 (C-1), 31.2 (CH₂), 29.2 (C-10b), 19.7 (CH₂), 13.9 (CH₃). – CI-MS (NH₃, 100 eV; positive): m/z (%) = 117 (15), 134 (6), 229 (14), 233 (3), 288 (1), 330 (100) [M+1]⁺; isotope distribution corresponds to [C₁₇H₂₀N₃O₄]⁺, 347 (13). – C₁₇H₁₉N₃O₄ (329.35): C 62.00, H 5.81, N 12.76; found C 61.79, H 5.74, N 12.55.

4.4.2 1-(2,5-Dioxo-1,3,5,10b-tetrahydro- 2H-chromeno[3,4-c]pyridin-4-yl)- 3-cyclohexylurea (5g)

A solution of sodium ethoxide, prepared from 0.12 g (5 mmol) of sodium and 5 mL of anhydrous ethanol, was added dropwise under stirring to a suspension of 2.0 g (5 mmol) of 6g in 10 mL of anhydrous ethanol. The reaction mixture was allowed to stay at 20–25 °C for 4 d. Then the solvent was removed in vacuo; the residue was mixed with some water and neutralized with acetic acid to pH ≈ 5. The precipitated product was filtered, washed with water and recrystallized from ethanol. Yield: 1.65 g (93 %) of colorless sparkling needles of 5g with m.p. 240–243 °C. – IR: ν = 2350–3350, 3339 (br., NH_assoc.), 1723 (C=O, lactone), 1657, 1622 (C=O) cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.75 (s, 1H, NH-lactam, D₂O-exchangeable), 10.92 (s, 1H, 4-NH), 8.46 (d, J = 6.9 Hz, 1H, NH-C₆H₁₁), 7.03–7.42 (m, 4H_arom.), 4.23 (dd, J₁ = 15 Hz, J₂ ≈ 6.7 Hz, 1H, 10b-H^X), 3.11 (d, J ≈ 5.3 Hz, 1H, 1-H^B), 2.78 (s, 1H, NCH), 2.4–2.6 (m, 2H, 1-H^A), 1.0–2.0 [broad m, 10H, CH(CH₂)₅]. – CI-MS (NH₃, 100 eV; positive): m/z (%) = 100 (4), 143 (18), 160 (2), 229 (17), 233 (3), 356 (100) [M+1]⁺; isotope distribution corresponds to [C₁₉H₂₂N₃O₄]⁺, 373 (9). – C₁₉H₂₁N₃O₄ (355.39): calcd. C 64.21, H 5.96, N 11.82; found C 63.98, H 5.94, N 11.86.

4.5 General procedure for the preparation of O-acetyl-3-monoalkylureides 7a–g

Acetic anhydride (0.56 g, 5.5 mmol) was added dropwise at ambient temperature (20–25 °C) under stirring to a solution of the corresponding 3-alkylureide 6a–g (5.0 mmol) in anhydrous pyridine (10 mL). Stirring was continued until exhaustion of the starting 3-alkylureide 6a–g (several days; TLC monitoring), the volatile components were removed in vacuo and the residue was recrystallized from ethanol to give pure 7a–g. More details are given below.

4.5.1 Ethyl 4-(2-acetoxyphenyl)-2-(3-ethylureido)- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (7a)

Stirring for 4 days. The recrystallized product was obtained in 92 % yield as colorless crystals with m.p. 199–201.5 °C, R_f = 0.36. – IR (nujol): ν = 3333, 3113, 1763, 1707, 1667, 1626 cm⁻¹. – ¹H NMR (100 MHz; [D₆]DMSO): δ = 11.50 (s, 1H, NH, D₂O-exchangeable), 10.84 (s, 1H, NH, D₂O-exchangeable), 8.30 (broad t, 1H, NH, D₂O-exchangeable), 7.03–7.20 (m, 4H_arom.), 4.28 (d, J = 7.3 Hz, 1H, 4-H^X), 3.95 (m, 2H, OCH₂), 2.87–3.16 (m, 3H, N-CH₂ and 5-H^B), 2.49 (m, 1H, 5-H^A), 2.3 (s, 3H, COCH₃), 1.05 (t, J = 7 Hz, 3H, COCH₂CH₃), 1.00 (t, J = 7 Hz, 3H, CH₃). – EI-MS (70 eV): m/z (%) = 389 (13) [M]⁺: isotope distribution corresponds to [C₁₉H₂₃N₃O₆]⁺, 347 (8) [M–CH₂CO]⁺, 318 (4) [M–C₂H₅NCO]⁺, 316 (6) [M–COOC₂H₅]⁺, 301 (8) [M–CH₂CO–C₂H₅OH]⁺, 300 (15), 275 (9) [M–C₂H₅NCO–COCH₃]⁺, 254 (6) [M– C₆H₄OCOCH₃]⁺, 245 (10) [M–C₂H₅NCO–COOC₂H₅]⁺, 230 (41) [M–C₂H₅NCO–COCH₃–OC₂H₅]⁺, 229 (100), 203 (19), 183 (15). – C₁₉H₂₃N₃O₆ (389.41): calcd. C 58.60, H 5.95, N 10.79; found C 58.46, H 6.03, N 10.60.

4.5.2 Ethyl 4-(2-acetoxyphenyl)-2-[3-(1-propyl)ureido]- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (7b)

Stirring for 24 h. The recrystallized product was obtained in 82 % yield as colorless crystals with m.p. 175.5–177 °C, R_f = 0.39. – IR (nujol): ν = 3341, 3127, 1763, 1705, 1667, 1626 cm⁻¹.-¹H NMR (100 MHz, [D₆]DMSO): δ = 11.51 (s, 1H, NH, D₂O-exchangeable), 10.85 (s, 1H, NH, D₂O-exchangeable), 8.32 (broad t, 1H, NH, D₂O-exchangeable), 7.03–7.24 (m, 4H_arom.), 4.28 (d, 1H, J = 7.1 Hz, 4-H^X), 3.85–3.99 (m, 2H, OCH₂), 2.92–3.20 (m, 3H, NCH₂ + 5-H^B), 2.49 (m_c, 1H, 5-H^A), 2.31 (s, 3H, COCH₃), 1.34–1.55 (m, 2H, CH₂, propyl), 1.00 (t, J = 7 Hz, 3H, CH₃, ester), 0.86 (t, J = 7 Hz, 3H, CH₃, propyl). – EI-MS (70 eV): m/z (%) = 403 (19) [M]⁺: isotope distribution corresponds to [C₂₀H₂₅N₃O₆]⁺; 361 (9) [M–CH₂CO]⁺, 330 (5) [M–COOC₂H₅]⁺, 315 (9) [M–CH₂CO–C₂H₅OH], 314 (20) [M–COCH₃–C₂H₅OH]⁺, 275 (12) [M–C₃H₇NCO–COCH₃]⁺, 245 (10) [M–C₃H₇NCO–COOC₂H₅]⁺, 230 (46) [M–C₃H₇NCO–COCH₃–OC₂H₅]⁺, 229 (100), 203 (20), 183 (18). – C₂₀H₂₅N₃O₆ (403.43): calcd. C 59.54, H 6.25, N 10.42; found C 59.43, H 6.18, N 10.30.

4.5.3 Ethyl 4-(2-acetoxyphenyl)-2-[3-(2-propyl)ureido]- 6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylate (7c)

Stirring for 24 h. The recrystallized product was obtained in 88.5 % yield as colorless crystals with m.p. 186–188 °C, R_f = 0.41. – IR (nujol): ν = 3302, 3100, 1755, 1701, 1669, 1618 cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.55 (s, 1H, NH, D₂O-exchangeable), 10.80 (s, 1H, NH, D₂O-exchangeable), 8.26 (d, 1H, J = 6.9 Hz, NH, D₂O-exchangeable), 7.11–7.25 (m, 4H_arom.), 4.29 (d, 1H, J = 7.4 Hz, 4-H^X), 3.73–4.07 (m, 3H, OCH₂ + NCH), 2.92–3.17 (m, 1H, 5-H^B), 2.48 (m_c, 1H, 5-H^A), 2.31 (s, 3H, COCH₃), 1.10 (d, 6H, J = 6.3 Hz, 2 × CH₃, isopropyl), 1.01 (t, 3H, J = 6,9 Hz, CH₃, ester). – EI-MS (70 eV): m/z (%) = 403 (21) [M]⁺: isotope distribution corresponds to [C₂₀H₂₅N₃O₆]⁺, 330 (6) [M–COOC₂H₅]⁺, 315 (7) [M–CH₂CO–C₂H₅OH]⁺, 314 (16) [M–COCH₃–C₂H₅OH]⁺, 275 (12) [M–C₃H₇NCO–COCH₃]⁺, 245 (13) [M–C₃H₇NCO–COOC₂H₅]⁺, 230 (49) [M–C₃H₇NCO–COCH₃–OC₂H₅]⁺, 229 (100), 203 (17), 183 (14). – C₂₀H₂₅N₃O₆ (403.43): calcd. C 59.54, H 6.25, N 10.42; found C 59.35, H 6.28, N 10.31.

4.5.4 Ethyl 4-(2-acetoxyphenyl)-2-[3-(1-butyl)ureido]- 6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxylate (7d)

Stirring for 4 days. This product was obtained from 6d in 93 % yield after recrystallization as colorless crystals with m.p. 168–170 °C. Alternative preparation from 4d (cf.Scheme 2): To a suspension of 159 mg (0.5 mmol) of 4d in 6 mL of anhydrous acetonitrile, a solution of 0.11 mL (1.0 mmol) of butylisocyanate in 1.0 mL of anhydrous acetonitrile was added dropwise under stirring. The mixture was allowed to stay for 12 days at 20–25 °C, and the separated crystals of 7d were filtered and washed with acetonitrile. Yield: 170 mg (81 %) of 7d, colorless crystals with m.p. 168–170 °C, R_f = 0.42. – IR (nujol): ν = 3364, 3134, 1754, 1673, 1655, 1615 cm⁻¹. – ¹H NMR (300 MHz, [D₆]DMSO): δ = 11.54 (s, 1H, NH), 10.86 (s, 1H, NH), 8.33 (br.s, 1H, NH), 7.07–7.30 (m, 4H_arom.), 4.31 (d, J = 7.4 Hz, 1H, 4-H^X), 3.85–4.08 (m, 2H, OCH₂), 2.98–3.18 (m, 3H, NCH₂ + 5-H^B), 2.33 (s, 3H, COCH₃), 2.24 (d, J = 16.2 Hz, 1H, 5-H^A), 1.21–1.55 (m, 4H, two CH₂), 1.03 (t, J = 7.0 Hz, 3H, CH₃, ester), 0.90 (t, J = 7.3 Hz, 3H, CH₃, butyl). – EI-MS (70 eV): m/z (%) = 417 (29), [M]⁺, 375 (8) [M–CH₂CO]⁺, 344 (7) [M–COOC₂H₅]⁺, 328 (19) [M–COCH₃–C₂H₅OH]⁺, 318 (8) [M–C₄H₉NCO]⁺, 275 (12) [M–C₄H₉NCO–COCH₃]⁺, 245 (13) [M–C₄H₉NCO–COOC₂H₅]⁺, 230 (48) [M–C₄H₉NCO–COCH₃–OC₂H₅]⁺, 229 (100), 203 (15), 183 (14). – ¹³C NMR spectrum of 7d, see Fig. 1. – C₂₁H₂₇N₃O₆ (417.46): calcd. C 60.42, H 6.52, N 10.07; found C 60.21, H 6.51, N 10.01.

4.5.5 Ethyl 4-(2-acetoxyphenyl)-2-(3-tert-butylureido)- 6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxylate (7e)

Stirring for 4 days. The recrystallized product was obtained in 87 % yield as a colorless solid with m.p. 166–168 °C, R_f = 0.43. – IR (nujol): ν = 3347, 3134, 1763, 1713, 1669, 1630 cm⁻¹. – ¹H NMR (100 MHz, [D₆]DMSO): δ = 11.65 (s, 1H, NH, D₂O-exchangeable), 10.71 (s, 1H, NH, D₂O-exchangeable), 8.17 (s, 1H, NH, D₂O-exchangeable), 7.15–7.23 (m, 4H_arom.), 4.32 (d, J = 7.1 Hz, 1H, 4-H^X), 3.83–4.01 (m, 2H, OCH₂), 2.93–3.18 (m_c, 3H, N-CH₂ + 5-H^B), 2.49 (m_c, 1H, 5-H^A), 2.31 (s, 3H, COCH₃), 1.28 [s, 9H, C(CH₃)₃], 1.00 (t, 3H, J = 7 Hz, CH₃). – EI-MS (70 eV): m/z (%) = 417 (21) [M]⁺: isotope distribution corresponds to [C₂₁H₂₇N₃O₆]⁺; 375 (4) [M–CH₂CO]⁺, 344 (4) [M–COOC₂H₅]⁺, 328 (8) [M–COCH₃–C₂H₅OH]⁺, 318 (10) [M–C₄H₉NCO]⁺, 275 (13) [M– C₄H₉NCO–COCH₃]⁺, 245 (17) [M–C₄H₉NCO–COOC₂H₅]⁺, 230 (52) [M–C₄H₉NCO–COCH₃–OC₂H₅]⁺, 229 (100), 203 (12), 183 (10). – C₂₁H₂₇N₃O₆ (417.46): C 60.42, H 6.52, N 10.07; found C 60.30, H 6.52, N 10.01.

4.5.6 Ethyl 4-(2-acetoxyphenyl)-2-[3-(1-hexyl)ureido]- 6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxylate (7f)

Stirring for 4 days. The recrystallized product was obtained in 89 % yield as a colorless solid with m.p. 163–166 °C, R_f = 0.41. – IR (nujol): ν = 3292, 3094, 1755, 1684, 1667, 1618 cm⁻¹. – ¹H NMR (300 MHz, [D₆]DMSO): δ = 11.54 (s, 1H, NH, D₂O-exchangeable), 10.85 (s, 1H, NH, D₂O-exchangeable), 8.32 (broad t, 1H, NH, D₂O-exchangeable), 7.09–7.29 (m, 4H_arom.), 4.30 (d, 1H, J = 7.5 Hz, 4-H^X), 3.85–4.07 (m, 2H, OCH₂), 3.01–3.15 (m_c, 3H, N-CH₂ + 5-H^B), 2.49 (d, J = 7.5 Hz, 1H, 5-H^A), 2.33 (s, 3H, COCH₃), 1.24–1.50 [m, 8H, (CH₂)₄], 1.03 (t, 3H, J = 7 Hz, CH₃, ester), 0.88 (t, J = 7 Hz, 3H, CH₃, hexyl). – EI-MS (70 eV): m/z (%) = 445 (10) [M]⁺: isotope distribution corresponds to [C₂₃H₃₁N₃O₆]⁺, 403 (5) [M–CH₂CO]⁺, 372 (4) [M–COOC₂H₅]⁺, 356 (12) [M–COCH₃–C₂H₅OH]⁺, 318 (6) [M–C₆H₁₃NCO]⁺, 245 (12) [M–C₆H₁₃NCO–COOC₂H₅]⁺, 230 (46) [M–C₆H₁₃NCO–COCH₃–OC₂H₅]⁺, 229 (100), 203 (17), 183 (13). – C₂₃H₃₁N₃O₆ (445.51): calcd. C 62.01, H 7.01, N 9.43; found C 61.89, H 7.01, N 9.35.

4.5.7 Ethyl 4-(2-acetoxyphenyl)-2-(3-cyclohexylureido)- 6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxylate (7g)

Stirring for 4 days. The recrystallized product was obtained in 88 % yield as a colorless solid with m.p. 197–199 °C, R_f = 0.44. The Oak Ridge Thermal Ellipsoid Plot (ORTEP) view of the molecule 7g with the atomic numbering is shown in Fig. 2. – IR (nujol): ν = 3335, 3141, 1769, 1707, 1672, 1628 cm⁻¹. – ¹H NMR (100 MHz; [D₆]DMSO): δ = 11.54 (s, 1H, NH, D₂O-exchangeable), 10.78 (s, 1H, NH, D₂O-exchangeable), 8.20 (d, J = 7 Hz, 1H, NH, D₂O-exchangeable), 7.08–7.22 (m, 4H_arom.), 4.28 (d, 1H, J = 7.3 Hz, 4-H^X), 3.93 (q, 2H, J₁ = J₂ = 3.9 Hz, OCH₂), 2.83–3.18 (m, 2H, N-CH + 5-H^B), ~2.45 (m, 1H, 5-H^A, overlapped by DMSO), 2.31 (s, 3H, COCH₃), 1.10–1.33 and 1.63–1.89 (m, 10H, five CH₂), 1.00 (t, J = 7 Hz, 3H, CH₃). – EI-MS (70 eV): m/z (%) = 443 (10) [M]⁺: isotope distribution corresponds to [C₂₃H₂₉N₃O₆]⁺, 401 (4) [M–CH₂CO]⁺, 370 (3) [M–COOC₂H₅]⁺, 354 (10) [M–COCH₃–C₂H₅OH]⁺, 318 (6) [M–C₆H₁₁NCO]⁺, 275 (11) [M–C₆H₁₁NCO–COCH₃]⁺, 245 (13) [M–C₆H₁₁NCO–COOC₂H₅]⁺, 230 (45) [M– C₆H₁₁NCO–COCH₃–OC₂H₅]⁺, 229 (100), 203 (15), 183 (12). – C₂₃H₂₉N₃O₆ (443.50): C 62.29 H 6.59 N 9.47; found C 62.13, H 6.59, N 9.30.

4.6 Crystal structure determinations

Suitable single crystals of 6f and 7g were obtained by simple recrystallization from anhydrous ethanol with charcoal decolorization and slowly cooling the hot solution. Data collection and data reduction were performed with Xscans, release 2.32 (Siemens, 1999). The structures were solved by Direct Methods (Shelxs-97 [21]) and refined with Shelxl-97 [22]. Molecular graphics were made with Xp (Shelxtl-Plus [23]).

Important crystal structure data are summarized in Table 6. Atomic coordinates, bond lengths (Å) and angles (deg), anisotropic displacement parameters, hydrogen coordinates, torsion angles (deg) have been deposited at Cambridge Crystallographic Data Centre.

Table 6

Crystal data, data collection and structure refinement details for 6f and 7g.

	6f	7g
Chemical formula	C₂₁H₂₉N₃O₅	C₂₃H₂₉N₃O₆
M_r	403.47	443.49
Temperature, K	293	293
Crystal system	Triclinic	Triclinic
Space group	P1̅	P1̅
a, Å	8.0779 (7)	8.3097 (4)
b, Å	9.8541 (9)	10.3876 (7)
c, Å	14.4837 (16)	14.4613 (8)
α, deg	87.487 (8)	73.328 (6)
β, deg	89.678 (9)	86.191 (5)
γ, deg	68.654 (7)	76.718 (6)
V, Å³	1072.72 (18)	1163.81 (12)
Z	2	2
Radiation; λ, Å; monochromator	CuK_α; 1.54178; graphite
μ(CuK_α), mm⁻¹	0.74	0.76
Crystal size, mm³	0.30 × 0.07 × 0.07	0.50 × 0.20 × 0.10
Data collection
Diffractometer	Siemens P4
Absorption correction	None
(sin θλ)_max, Å⁻¹	0.599	0.601
Refl. total/unique/R_int	4631/3737/0.025	4689/3889/0.022
Refinement
H atom treatment	H atoms located by a difference fourier map and refined constrained using standard riding models
Ref. param./restraints	274/0	290/0
R (F)/wR(F²) (all data)	0.1228/0.2851	0.1178/0.3058
GoF (F²)	1.066	1.065
Δρ_max/min, e Å⁻³	0.39/−0.27	0.35/−0.41

CCDC 908171 (6f) and CCDC 908172 (7g) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge viawww.ccdc.cam.ac.uk/data_request/cif.

4.7 Pharmacology

4.7.1 Laboratory animals

Male mice line H (body weight 20–25 g) were used. The mice were housed in plexiglass cages (six per cage) in a 12/12 light/dark cycle, under standard laboratory conditions (ambient temperature 20 ± 2 °C and humidity 72 ± 4 %) with free access to water and standard pelleted rat food 53-3, produced according to ISO 9001:2008. Animals were purchased from the National Breeding Center, Sofia, Bulgaria. A minimum of 7 days of acclimatization was allowed before the commencement of the study and their health was monitored regularly by a veterinary physician. Vivarium (certificate of registration of farm No. 0072/01.08.2007) was inspected by the Bulgarian Drug Agency in order to check the husbandry conditions (No. A-11-1081/03.11.2011). All performed procedures were approved by the Institutional Animal Care Committee (KENIMUS) and the principles stated in the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123) (Council of Europe, 1991) were strictly followed throughout the experiment [24].

4.7.2 Acute toxicity

The acute toxicity was established after intraperitoneal administration (i.p. LD₅₀) of the studied compounds. Compounds were dissolved in saline (0.9 % NaCl) with one drop of Tween 80. LD₅₀ was evaluated for five different doses (1000, 1500, 2000, 4000 and 5000 mg kg⁻¹), each on six animals, and calculated by the Litchfield–Wilcoxon method, using a personal computer.

4.7.3 Blood clotting time

The influence of the investigated compounds on blood clotting time was determined by the method of Morawitz. The investigation was performed on 30 male white mice (six animals in a group). The compounds were administered intraperitoneally in doses of 1/10, 1/50 and 1/100 of LD₅₀. Twenty-four hours after the compound administration the clotting time was assessed after small incision of sublingual vein and measuring the clotting time of the second drop blood on clean glass [25].

4.7.4 Analgesic activity

Hot-plate test in mice [26]. The effects of compounds on the reaction times of mice placed on a hot plate thermostatically maintained at 55 °C were determined. The time at which mice displayed a nociceptive response, licking the hind paws, was recorded and the animal was removed from the hotplate at this time. Apparatus: Cold Hot Plate Analgesia Meter Apparatus for mice and rats (Part # PE34), IITC Life Science (Woodland Hills, CA, USA) [27].

Acetic acid writhing test in mice [28]. Mice (b.w. 20–30 g) were selected 1 day prior to each test and were divided into groups of six mice each. One group served as control (saline 0.1 mL/10 g) intraperitoneally. The second group was given metamizole (25 mg kg⁻¹ = 1/10 LD₅₀) by the same route, as a reference drug. The remaining groups were treated with the respective compounds. All animals received 0.1 mL kg⁻¹ (i.p.) of 3 % acetic acid 30 min after treatment. The number of writhing was recorded during 30 min commencing 5 min after the acetic acid injection. A writhe is indicated by abdominal constriction and stretching of at least one hind limb [29].

4.7.5 Hexobarbital-induced sleeping time

The effects of the compounds administered intraperitoneally on the duration of sleep induced by hexobarbital (dose 80 mg kg⁻¹ b.w.). Compounds were administered intraperitoneally 30 min before hexobarbital in doses equal to 1/50 of LD₅₀.

4.7.6 Statistical analysis

Data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using Student’s t-test. The significance of difference was considered to include values of p < 0.05 or p < 0.001.

Dedicated to Professor Gerhard Maas on the occasion of his 65^th birthday.

Corresponding author: Ivo Christov Ivanov, Faculty of Pharmacy, Medical University of Sofia, Dunav 2, BG-1000 Sofia, Bulgaria, Fax: +35-929879874, E-mail: ivanov43@gmail.com

Acknowledgments

This project was funded by the State Research Funds of the Medical University of Sofia. We wish to thank the Management of the Faculty of Pharmacy for providing the research facilities. We gratefully acknowledge the Microanalytical Laboratory and the Laboratory of Mass Spectrometry of the Institute of Organic Chemistry, University of Stuttgart (Head Dr. Joachim Opitz).

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Received: 2015-4-4

Accepted: 2015-6-7

Published Online: 2015-10-14

Published in Print: 2015-11-1

Articles in the same Issue

https://doi.org/10.1515/znb-2015-0066

Keywords for this article

acute toxicity; analgesic activity; blood clotting time; chromeno[3,4-c]pyridines; crystal structure; enaminoesters; hexobarbital sleeping time; isocyanates; 2-pyridones; ureides