Introduction

Heterocyclic compounds are indispensable structural units that are useful in medicinal chemistry and valuable as synthetic organic blocks. In various classes of natural heterocyclic compounds of biological interest, the coumarin moiety is commonly present (Wu et al., 2009). They display interesting biological and pharmacological profiles, such as the antibacterial (Al-Amiery et al., 2011a), cytotoxic (Al-Amiery et al., 2011a), antioxidant (Al-Amiery et al., 2011a), antifungal (Al-Amiery et al., 2012), anticoagulant (Manolov and Danchev, 1995), anti-inflammatory (Emmanuel-Giota et al., 2001), antitumor and anti-HIV (Harvey et al., 1988; Kostova et al., 2006) activities. In addition, these compounds have varied bioactivities and applications in cosmetics, pharmaceuticals, food, flavoring and agrochemicals (Borges et al., 2005; Kabalka et al., 2005; Morimoto et al., 2003; Srinivasan and Rampally, 2004).

Since the coumarin nucleus is associated with these diverse biological and pharmacological activities, several methods for the preparation of coumarin-based compounds have been reported (Gammonn et al., 2005; Messaoudi et al., 2010; Santana et al., 2006). Particularly, the use of 4-methylumbelliferone as a precursor represents a useful synthetic method for the preparation of such compounds (Ganesh et al., 2010; Kumar et al., 2011a; Ramesh et al., 2008).

Our research has been devoted to the development of a new class of heterocyclic systems which incorporate the coumarin moiety with the hope that they may be biologically active.

Herein, we report here the synthesis of ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate 2 and its use as a building block in the synthesis of some new coumarin-based derivatives 4–7. Their antimicrobial activities were evaluated, and the structure–activity relationship was also discussed.

Our approach to the target coumarin derivatives was firstly started by the synthesis of product 2 via condensation reaction of equimolar amount of 4-methylumbelliferone, ethyl chloroacetate and anhydrous K2CO3 in dry DMF (Al-Amiery et al., 2011b). Then, compound 2 was converted into the 2-(2-oxo-4-methyl-2H-chromen-7-yloxy) acetic acid 3 (Abd El-Fattah et al., 2011) which was esterified using a series of alcohols in the presence of iodine (Ramalinga et al., 2002; Jereb et al., 2009) to yield a new series of coumarin esters 4a–j (Scheme 1; Table 1).

Scheme 1
figure 1

Synthesis of new coumarin esters 4a–j

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Table 1 Synthesis of compounds 4a–j
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In the 1H-NMR spectrum of compound 2, a 3H triplet was observed at δ H 1.25 ppm (J = 7.2 Hz) due to the ester CH3 protons and a quartet at δ H 4.23 ppm (J = 7.2 Hz) due to the ester CH2 protons. The isolated CH2 protons were observed downfield as a singlet (2H) at δ H 4.63 ppm. The 13C-NMR spectrum analysis for compound 2, combined with the information from 1H-NMR experiment, can be considered enough to guide future synthetic work. The 13C NMR spectrum of this compound confirms the proposal structure by the observation of the signals at δ C 13.6 (C-4′), 61.1 (C-3′) and 167.4 (C-2′) ppm. The 13C-NMR spectrum analysis for compound 2, combined with the information from 1H-NMR experiments, can be considered enough to guide future synthetic work.

In the 1H-NMR spectrum of compound 3, the CH2 protons were observed a singlet (2H) at δ H 4.78 ppm and the CH3 of the coumarin moiety appeared at δ H 2.39 ppm. On the same spectrum, we noticed the appearance of a new signal at δ H 13.07 ppm corresponding to the OH group of the acetic acid fragment. The 13C-NMR spectrum of compound 3 shows in particular a signal at δ C 169.6 ppm attributable to the carbonyl of the acid group (C-2′).

The structure of these compounds (4a–j) was established on the basis of their analytical data. Thus, mass spectrum of 4a (as an example) gave a pseudo-molecular ion peak [M + H]+ at m/z 305.1381, which is consistent with the molecular formula (C17H21O5). In addition to the signals corresponding to the protons introduced by 4-methylumbelliferone, new signals related to the alcohol used were observed in the 1H-NMR spectrum. Examination at 300 MHz showed a multiplet at δ H 3.99 (2H, H-5′) and also a multiplet at δ H 0.87 (6H, H-6′, H-7′). The 13C-NMR spectrum confirmed the above spectral data by the observation of new signals at δC 11.2, 16.3, 25.9, 34.1 and 70.1 ppm relative to carbons of the alcohol used. The same spectrum showed a signal at δ C 168.1 ppm attributable to the carbonyl of the ester group (C-2′).

In the second step, we have subjected the key intermediate 2 to reaction with an aqueous solution of hydrazine in ethanol at reflux (Al-Amiery et al., 2011b). The so-formed hydrazide 5, which belongs to a class of intermediates, known to be highly reactive and used as precursors for the synthesis of new nitrogen’s compounds (Abdel-Aziz et al., 2007) was further converted into coumarin derivatives 6 and 7 by treatment with a series of aromatic aldehydes (Scheme 2; Table 2) and cyclic anhydrides, respectively (Scheme 3; Table 3).

Scheme 2
figure 2

Synthesis of hydrazone 6a–f

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Table 2 Synthesis of compounds 6a–f
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Scheme 3
figure 3

Synthesis of novel coumarin derivatives 7a–c

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Table 3 Synthesis of compounds 7a–c
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The 1H-NMR spectrum of the intermediate 5 showed characteristic signals at δ H 5.02, 4.38 and 9.45 ppm corresponding to the methylene (C-1′), NH2 and NH groups, respectively. Its 13C-NMR spectrum reinforced this structure by the appearance of the signal of (C-2′) at δ C 169.6 ppm and the disappearance of the two signals at δ C 13.6 (C-4′) and 61.1 (C-3′) ppm attributed to the ethoxy carbons in compound 2.

Compound 6b was obtained as a white powder. Its ESHRMS gave pseudo-molecular ion peak [M + H]+ at m/z 327.0975, which is consistent with the molecular formula C17H14 N2O5. Thus, the 1H-NMR spectrum of 6b showed the absence of the NH2 protons at δ H 4.38 ppm, and the appearance of a new characteristic singlet at δ H 7.83 ppm, assignable to the proton –N = CH and the presence of the NHCO proton resonating at δ H 11.63 ppm.

Also the structure of compound 7 has been assigned from their analytical data. In fact the ESHRMS of compound 7a gave pseudo-molecular ion peak [M + H]+ at m/z 377.0777, which is consistent with the molecular formula C21H19O6N2. Furthermore, the 1H NMR spectrum of this compound was compatible with the proposed structure. In addition to the signals corresponding to most of the protons introduced by the hydrazide 5, we note the presence of a new multiplet (4H) at δ H 8.01 ppm relative to the aromatic protons of the phthalic anhydride. The 13C-NMR spectrum confirmed the above spectral data by the observation of new signals at δ C 129.3, 135.4, 154.5 and 164.9 ppm relative to carbons of the phthalic anhydride moiety.

Biological activity

Antimicrobial activity

The serious medical problem of bacterial and fungal resistance and the rapid rate of its development has led to increasing levels of resistance to classical antibiotics (Rice et al., 1999; Al-Amiery et al., 2009; Kadhum et al., 2011), and the discovery and development of effective antibacterial and antifungal drugs with novel mechanisms of action have become urgent tasks for infectious disease research programs (Kumar et al., 2011b). All the synthesized compounds were analyzed on what concerns their antimicrobial inhibition (Table 4). The antimicrobial inhibition was determined using the disc diffusion method described in the literature (Marmonier, 1987; Barry and Thornsberry, 1991).

Table 4 Antimicrobial activity of coumarin derivatives 4a–j, 6a–f and 7a–c
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According to the results given in Table 4, most compounds have displayed good antifungal activity against Botrytis cinerea and Fusarium oxysporum f. sp. lycopersici. Compounds 4a and 4g exhibited an important inhibitory activity against B. cinerea (IZ = 36 and 39 mm, respectively), while compound 4j was found to be the most active against F. oxysporum f. sp. lycopersici (IZ = 16 mm). The compounds 4a, 4i and 7a showed moderate activity against Aspergilus niger (IZ = 6–8 mm).

From the data of the antibacterial activity, few compounds (4f, 4h, 4i, 6e, 7a and 7c ) were found to be active against Pseudomonas sp. (Pa 499) and Bacillus sp (Bp 420) (IZ = 5–8 mm) (Table 4). However, compound 7b exhibited an interesting antibacterial activity against Pseudomonas sp. (IZ = 12 mm) which was comparable to that obtained with the standard reference (ampicillin) used.

Some results prove consistent with several studies showing that coumarins and some of their derivatives exhibited an important antimicrobial activity (Lin et al., 2012; Singh et al., 2010).

Moreover, it was indicated that the combination of the coumarin skeleton with some nitrogen-containing heterocyclic moieties could significantly increase their antimicrobial efficiency (Keri et al., 2009; Ronad et al., 2010).

The antimicrobial activity of the compounds 4a–j depends on the structure of the fragment introduced by the alcohol. Generally, the esterification does not seem beneficial for such antibacterial activity. Secondly, the behavior of the fungi used, such as B. cinerea, to compounds 4a–j appears complex, but in all cases the hydroxyl group or groups remained free in certain alcohols used (cases of 4b, 4c and 4d) did not appear an interesting factor that brings activity. The relatively high activity of compound 6d against F. oxysporum (IZ = 15 mm) could be explained by the presence of the free phenol function. The ortho-position of the latter allows the formation of a possible hydrogen bond with the free electron pair of the sp2 hybridized nitrogen atom of the hydrazone function, thus further stabilizing the molecule; this stability may be at the origin of the activity of this derivative. On the other hand, the para-position of this phenol function to the hydrazone in compound 6e and the presence of a methoxyl group certainly explain the attenuation of this activity against the same strain (IZ = 11 mm). The relatively high activity of compound 7b toward bacteria and fungi used except A. niger compared to its analogue 7a could be partly explained by the non-aromatic bicyclic structure of the moiety introduced by the anhydride used. The open structure of the fragment introduced by the succinic anhydride used in the preparation of compound 7c and the emergence of a carboxylic acid function were without significant effect on its antimicrobial activity.

Conclusion

In conclusion, the present study provides a novel and efficient synthetic route to the preparation of new coumarin esters, hydrazone and novel coumarin derivatives from 4-methylumbelliferone. The results showed that some of the synthetic compounds possessed good antibacterial and antifungal activities. Most of the compounds might be promising for any investigations to develop new effective antibacterial and antifungal drugs.

Experimental section

Chemistry

Melting points were determined on a Büchi 510 apparatus using capillary tubes. Mass spectra were obtained with ESI-TOF (LCT Premier XE, Waters) using the reflectron mode in the positive ion mode. Leucine-enkephalin peptide was employed as the LockSpray lockmass. 1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a Bruker AM-300 spectrometer, using CDCl3 and DMSO-d 6 as solvents and non-deuterated residual solvents as internal standards. Chemicals shifts (δ) are given in parts per million (ppm) and coupling constants (J) in Hertz. Commercial TLC plates (Silica gel 60, F254, SDS) were used to monitor the progress of the reaction. Column chromatography was performed with silica gel 60 (particle size 40–63 μm, SDS). The starting materials 2, 3 and 5 were prepared according to the literature (Ramesh et al., 2008; Al-Amiery et al., 2011).

Preparation of compounds 4a–j

A mixture of acid 3 (0.1 g, 0.4 mmol), alcohol (10 ml) and iodine (5 mg,) was boiled under reflux for 2 to 24 h. The progress of the reaction was monitored by TLC. The solvent (excess of alcohol) was evaporated in vacuo. The mixture was extracted twice with the appropriate solvent and then dried over Na2SO4. After removing the solvent under reduced pressure, the residue thus obtained was purified by column chromatography on silica gel eluted with a mixture PE/EtOAc (8:2) to give the desired carboxylic esters 4a–j.

Ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate (2)

Yellow solid, yield 72 %, mp 102 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 1.25 (t, 3H, J = 7,2 Hz, CH3), 2.33 (s, 3H, CH3), 4.23 (q, 2H, J = 7,2 Hz, H-3′), 4.63 (s, 2H, H-1′), 6.06 (s, 1H, H-3), 6.69 (d, 1H, J = 2,7 Hz, H-8), 6.83 (dd, 1H, J 1  = 8,7 Hz, J 2  = 2,7 Hz, H-6), 7.46 (d, 1H, J = 8,7 Hz, H-5). 13C-NMR (75 MHz, CDCl3): δ c 13.6 (CH3-4′), 18.0 (CH3-9), 61.1 (CH2-3′), 64.8 (CH2-1′), 101.2 (CH-8), 111.8 (CH-6 + C-3), 113.8 (C-4a), 125.3 (CH-5), 151.9 (C-8a), 154.4 (C-4), 160.1 (C-7 + C-2), 167.4 (C-2′).

2-(4-Methyl-2-oxo-2H-chromen-7-yloxy) acetic acid (3)

White solid, yield 67 %, mp 206 °C (EtOH).1H-NMR (300 MHz, DMSO-d 6 ): δ H 2.39 (s, 3H, CH3), 4.78 (s, 2H, H-2′), 6.21 (s, 1H, H-3), 6.98 (dd, 1H, J 1  = 8,4 Hz, J 2  = 2,4 Hz, H-8), 7.69 (d, 1H, J = 8,7 Hz, H-5), 13.07 (s, 1H, OH). 13C-NMR (75 MHz, DMSO-d 6 ): δ c 18.1 (CH3-9), 64.8 (CH2-1′), 101.5 (CH-8), 111.4 (CH-6), 112.2 (CH-3), 113.5 (C-4a), 126.5 (CH-5), 153.3 (C-8a), 154.5 (C-4), 160.7 (C-7 + C-2), 169.6 (C-2′).

2-Methylbutyl 2-(4-methyl-2-oxo-2H-chromen-7- yloxy) acetate (4a)

White solid, yield 53 %, mp 301 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 2.37 (s, 3H, CH3), 0.87 (m, 6H, H-6′ + H-7′), 1.13 (m, 1H, H-3′a), 1.35 (m, 1H, H-3′b), 1.70 (m, 1H, H-4′), 3.99 (m, 2H, H-5′), 4.69 (s, 2H, H-1′), 6.12 (s, 1H, H-3), 6.75 (d, 1H, J = 2.4 Hz, H-8), 6.83 (dd, 1H, J 1  = 8.7 Hz, J 2  = 2.7 Hz, H-6), 7.46 (d, 1H, J = 8.7 Hz, H-5).

13C-NMR (75 MHz CDCl3): δ c 11.2 (CH3-6′), 16.3 (CH3-7′), 18.7 (CH3-9), 25.9 (CH2-5′), 34.1 (CH-4′), 65.2 (CH2-1′), 70.1 (CH2-3′), 101.7 (CH-8), 112.4 (CH-6), 114.3 (CH-3), 125.8 (CH-5), 152.5 (C-8a), 155.0 (C-4), 160.5 (C-7), 161.1 (C-2), 168.2 (C-2′). ESI-HRMS m/z [M + H]+ calcd. for (C17H21O5)+:305.1389 found: 305.1381.

2-Hydroxyethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (4b)

White solid, yield 70 %, mp 308 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 2.33 (s, 3H, CH3), 3.82 (t, 2H, J 1  = 3.3 Hz, J 2  = 1.2 Hz, H-4′), 4.30 (t, 2H, J 1  = 3.3 Hz, J 2  = 1.3 Hz, H-3′), 4.68 (s, 2H, H-1′), 6.10 (s, 1H, H-3), 6.72 (d, 1H, J = 2.4 Hz, H-8), 6.87 (dd, 1H, J 1  = 8.7 Hz, J 2  = 2.4 Hz, H-6), 7.45 (d, 1H, J = 8.7 Hz, H-5). 13C-NMR (75 MHz, CDCl3): δ c 18.7 (CH3-9), 60.8 (CH2-4′), 65.2 (CH2-1′), 66.9 (CH2-3′), 101.7 (CH-8), 112.6 (CH-6), 114.5 (CH-3); 125.9 (CH-5), 152.5 (C-8a), 155.1 (C-4), 160.5 (C-7), 161.2 (C-2), 168.3 (C-2′). ESI-HRMS [M + H]+ calcd. for (C14H15O6)+: 279.0790 found: 279.0796.

2-Hydroxypropyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (4c)

White solid, yield 72 %, mp 219 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 1.22 (m, 3H, H-5′), 2.38 (s, 3H, CH3), 3.64 (m, 1H, H-4′), 4.12 (m, 2H, H-3′), 4.76 (s, 2H, H-1′), 6.14 (1H, s, H-3), 6.79 (d, 1H, J = 2.4 Hz, H-8), 6.87 (dd, 1H, J 1  = 8.7 Hz, J 2  = 2.4 Hz, H-6), 7.50 (d, 1H, J = 8.7 Hz, H-5). 13C-NMR (75 MHz, CDCl3): δ c 18.2 (CH3-9), 18.7 (CH3-5′), 64.7 (CH-4′), 69.3 (CH2-1′), 73.0 (CH2-3′), 101.3 (CH-8), 112.1 (CH-6), 114.0 (CH-3), 125.3 (CH-5), 151.9 (C-8a), 154.5 (C-4), 160.5 (C-7), 167.5 (C-2), 167.6 (C-2′). ESI-HRMS: m/z [M + H]+ calcd. for (C15H16O6)+: 292.0947 found: 292.0950.

2,3-Dihydroxypropyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (4d)

White solid, yield 22 %, mp 208 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 2.40 (s, 3H, CH3), 3.70 (dd, 2H, J 1  = 15.2 Hz, J 2  = 6 Hz, H-5′), 4.15 (dd, 2H, J = 2.1 Hz, J = 11.4 Hz, H-3′), 4.08 (m, 1H, H-4′), 4.78 (s, 2H, H-1′), 6.18 (s, 1H, H-3), 6.82 (d, 1H, J = 2.4 Hz,, H-8), 6.95 (dd, 1H, J = 8.7 Hz, J = 2.4 Hz,, H-6), 7.55 (d, 1H, J = 8.7 Hz,, H-5) 13C-NMR (75 MHz, CDCl3): δ c 20.2 (CH3-9), 62.4 (CH2-5′), 64.1 (CH-4′), 66.1 (CH2-3′), 70.1 (CH2-1′), 102.0 (CH-8), 110.9 (CH-6), 112.2 (CH-3), 115.3 (C-4a), 128.8 (CH-5), 152.0 (C-8a), 155.1 (C-4), 160.7 (C-7) 162.4 (C-2), 168.9 (C-2′). ESI-HRMS: m/z [M + H]+ calcd. for (C15H17O7)+: 309.0896 found: 309.0899.

Pentyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (4e)

White solid, yield 72 %, mp 202 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 1.18 (m, 8H, H-3′ + H-4′ + H-5′ + H-6′), 1.59 (m, 3H, CH3), 2.31 (s, 3H, CH3), 4.61 (s, 2H, H-1′), 6,05 (s, 1H, H-3), 6.69 (d, 1H, J = 2.4 Hz, H-8), 6.82 (dd, 1H, J 1  = 8.7 Hz, J 2  = 2.7 Hz, H-6), 7.44 (d, 1H, J = 8.7 Hz, H-5). 13C-NMR (75 MHz, CDCl3): δ c 18.7 (CH3-9), 18.6 (CH3-7′), 22.2 (CH2-6′), 29.4 (CH2-5′), 29.6 (CH2-4′), 65.7 (CH2-1′), 67.7 (CH2-3′), 101.7 (CH-8), 104.9 (CH-6), 114.3 (C-3), 125.7 (CH-5), 152.4 (C-8a), 155.1 (C-4), 160.6 (C-2), 160.9 (C-7), 168.1 (C-2′). ESI-HRMS: m/z [M + H]+ calcd. for (C17H21O5)+: 305.1311 found: 305.1318.

Isopropyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate (4f)

White solid, yield 78 %, mp 218 °C (EtOH).1H-NMR (300 MHz, CDCl3): δ H 1.20 (d, 6H, J = 6 Hz, H-4′ + H-5′), 2.31 (s, 3H, CH3), 4.55 (s, 2H, H-1′), 5.07 (hept, 1H, J = 6 Hz, H-3′), 6.07 (s, 1H, H-3), 6.71 (d, 1H, J = 2.7 Hz, H-8), 6.83 (dd, 1H, J 1  = 8.4 Hz, J 2  = 2.4 Hz, H-6), 7.42 (d, 1H, J = 8.4 Hz, H-5). 13C-NMR (75 MHz, CDCl3): δ c 21.2 (CH3-4′ + 5′), 18.1 (CH3-9), 65.1 (CH2-1′), 69.2 (CH-3′), 101.2 (CH), 112.0 (CH-6 + C-3), 113.8 (C-4a), 125.2 (CH-5), 151.8 (C-8a), 154.6 (C-4), 160.2 (C-2), 160.5 (C-7), 177.0 (C-2′). ESI-HRMS: m/z [M + H]+ calcd. for (C15H17O5)+: 277.0998 found: 277.0993.

Propyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (4 g)

White solid, yield 71 %, mp 212 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 0.91 (t, 3H, J = 7.5 Hz, CH3), 1.65 (m, 2H, H-4′), 2.36 (s, 3H, CH3), 4.15 (t, 2H, J = 6.6 Hz, H-3′), 4.67 (s, 2H, H-1′), 6.10 (s, 1H, H-3), 6.73 (d, 1H, J = 2.7 Hz, H-8), 6.88 (dd, 1H, J 1  = 8.4 Hz, J 2  = 2.4 Hz, H-6), 7.47 (d, 1H, J = 8.4 Hz, H-5).13C-NMR (75 MHz, CDCl3): δ c 21.8 (CH3-9), 18.6 (CH3-5′), 10.2 (CH2-4′), 65.2 (CH2-3′), 67.2 (CH2-1′), 101.7 (CH-8), 112.4 (CH-6), 114.3 (C-3), 125.2 (CH-5), 152.5 (C-8a), 154.9 (C-4), 160.6 (C-7), 161.1 (C-2), 168.1 (C-2′). ESI-HRMS: m/z [M + H]+ calcd. for (C15H17O5)+: 277.1076 found: 277.1073.

Isobutyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (4h)

White solid, yield 79 %, mp 228 °C (EtOH).1H-NMR (300 MHz, CDCl3): δ H 0.85 (d, 6H, J = 3.9 Hz, H-5′ + H-6′), 2.32 (s, 3H, CH3), 1.89 (hept, 1H, J = 6.9 Hz, H-4′), 3.92 (d, 2H, J = 6.6 Hz, H-3′), 4.63 (s, 2H, H-1′), 6.07 (s, 1H, H-3), 6.71 (d, 1H, J = 2.7 Hz, H-8), 6.83 (dd, 1H, J 1  = 8.4 Hz, J 2  = 2.4 Hz, H-6), 7.43 (d,1H, J = 8.4 Hz, H-5). 13C-NMR (75 MHz, CDCl3): δ c 27.7 (CH3-5′ + 6′), 18.6 (CH3-9), 65.3 (CH2-3′), 71.6 (CH2-1′), 101.7 (CH-8), 112,5 (CH-6), 114.4 (CH-3), 125.7 (CH-5), 152.3 (C-8a), 155.1 (C-4), 160.7 (C-7), 161.0 (C-2), 168.1 (C-2′).ESI-HRMS: m/z [M + H]+ calcd. for (C16H19O5)+: 291.1154 found: 291.1151.

Butyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (4i)

White solid, yield 65 %, mp 221 °C (EtOH). 1H-NMR (300 MHz CDCl3, 300 MHz): δ H 0.85 (t, 3H, J 1  = 7.5 Hz, J 2  = 7.2 Hz, CH3), 2.40 (s, 3H, CH3), 1.33 (m, 2H, H-5′), 1.65 (m, 2H, H-4′) 4.22 (t, 2H, J 1  = 6.9 Hz, J 2  = 6.6 Hz, H-3′), 4.69 (s, 2H, H-1′), 6.15 (s, 1H, H-3), 6.78 (d, 1H, J 1  = 2.7 Hz, H-8), 6.92 (dd, 1H, J 1  = 8.4 Hz, J 2  = 2.4 Hz, H-6), 7.51 (d, 1H, J = 8.4 Hz, H-5).13C-NMR (75 MHz, CDCl3): δ c 13.6 (CH3-6′), 18.7 (CH2-5′), 19.0 (CH3-9), 29.4 (CH2-4′), 65.3 (CH2-3′), 65.5 (CH2-1′), 101.7 (CH-8), 112.5 (CH-6), 114.4 (C-3), 125.8 (CH-5), 152.4 (C-8a), 155.1 (C-4), 160.6 (C-7), 161.1 (C-2), 168.1 (C-2′). ESI-HRMS: m/z [M + H]+ calcd. for (C16H19O5)+: 291.1154 found: 291.1159.

Cyclohexyl 2-(4-methyl-2-oxo-2H-chromen-7- yloxy)acetate (4j)

White solid, yield 69 %, mp 210 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 1.33 (m, 2H, H-6′), 1.42 (m, 2H, H-5′), 1.65 (m, 2H, H-4′), 1.89 (m, 2H, H-8′), 2.40 (s, 3H, CH3), 4.65 (s, 2H, H-1′), 4.90 (m, 1H, H-3′), 6.15 (s, 1H, H-3), 6.78 (d, 1H, J = 2.7 Hz, H-8), 6.93 (dd, 1H, J 1  = 8.7 Hz, J 2  = 2.7 Hz, H-6), 7.55 (d, 1H, J = 8.7 Hz, H-5). 13C-NMR (75 MHz, CDCl3): δ c 19.2 (CH3-9), 22.4 (CH2-8′), 23,4 (CH2-6′), 25.3 (CH2-5′), 31.0 (CH2-4′), 67.8 (CH2-1′), 72,2 (CH2-7′), 78.9 (CH-3′), 103.2 (CH-8), 112.0 (CH-6), 112.1 (C-4a), 116.5 (CH-3), 128.7 (CH-5), 155.3 (C-8a), 158.0 (C-4), 160.3 (C-7), 161.8 (C-2), 169.2 (C-2′). ESI-HRMS: m/z [M + H]+ calcd. for (C18H21O5)+: 316.1311 found: 316.1316.

2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (5)

White solid, yield 63 %, mp 212 °C (EtOH). 1H-NMR (300 MHz, DMSO-d 6 ): δ H 2.39 (s, 3H, CH3), 4.38 (s, 2H, NH2), 5.02 (s, 2H, H-1′), 6.23 (s, 1H, H-3), 7.0 (m, 2H, H-6 + H-8), 7.68 (d, 1H, H-5), 9.45 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d 6 ): δ c 18.1 (CH3-9), 64.9 (CH2-1′), 102.0 (CH-8), 111.6 (CH-6), 112.3 (CH-3), 113.5 (C-4a), 126.4 (CH-5), 153.5 (C-8a), 154.5 (C-4), 160.8 (C-7 + C-2), 169.9 (C-2′).

Preparation of compounds 6a–f: A mixture of compound 5

(4 mmol) and the appropriate aromatic aldehyde (1 eq) was refluxed in ethanol (20 mL) for 2 h. The excess of solvent was then removed under reduced pressure, the precipitate formed after cooling was collected by filtration and recrystallized from ethanol to give compounds 6a–f.

(E)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N’-(thiophen-2-ylmethylene)acetohydrazide (6a)

White solid, yield 66 %, mp 268 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 2.33 (s, 3H, CH3), 4.86 (s, 2H, H-2′), 6.22 (s, 1H, H-3), 6.65 (s, 1H, H-4′’), 6,94-7,09 (m, 3H, H-3″ + H-6 + H-8), 7.78 (d, 1H, J = 8,7 Hz, H-5), 7.92 (s, 1H, H-1″), 7.98 (s, 1H, H-5″), 11.63 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): δ c 18.1 (CH3-9), 66.7 (CH2-2′), 101.6 (CH-8), 113.4 (CH-3 + C-6), 113.7 (C-4a), 125.0 (CH-1″), 125.8 (CH-5″), 127.2 (CH-4″), 128.0 (CH-3″),, 145.4 (C-8a), 153.3 (C-4), 144.5 (C-2″), 160.6(C-7 + 2), 168.8 (C-1′). ESI-HRMS: m/z [M + H]+ calcd. for (C17H15 N2O4S)+: 342.0674, found: 342.0677.

(E)-N’-(furan-2-ylmethylene)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (6b)

White solid, yield 76 %, mp 248 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 2.39 (s, 3H, CH3), 4.80 (s, 2H, H-2′), 6.23 (s, 1H, H-3), 6.62 (s, 1H, H-4″), 6,92-7,06 (m, 3H, H-3″ + H-6 + H-8), 7.71 (d, 1H, J = 8,7 Hz, H-5), 7.83 (s, 1H, H-1″), 7.91 (s, 1H, H-5″), 11.63 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): δ c 18.1 (CH3-9), 65.8 (CH2-2′), 101.8 (CH-8), 113.5 (CH-3 + C-6), 114.2 (C-4a), 125.4 (CH-1″), 125.8 (CH-5″), 127.2 (CH-4″), 128.0 (CH-3″),, 145.4 (C-8a), 153.3 (C-4), 144.5 (C-2″), 160.6(C-7 + 2), 168.8 (C-1′). ESI-HRMS: m/z [M + H]+ calcd. for (C17H15 N2O5)+: 327.0981, found: 327.0975.

(E)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N’-(quinolin-6-ylmethylene)acetohydrazide (6c)

White solid, yield 84 %, mp 238 °C (EtOH). 1H-NMR (300 MHz, DMSO-d 6 ): δ H 2.40 (s, 3H, CH3), 5.41 (s, 2H, H-2′), 6.21 (s, 1H, H-3), 7.0 (m, 2H, H-6 + H-8), 7.69-9.0 (m, 8H, H-5 + H-1″ + H-3″ + H-4″ + H-5″ + H-6″ + H-7″ + H-8″), 12.03 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d 6 ): δ c 18.1 (CH3-9), 66.7 (CH2-2′), 101.6 (CH-8), 113.4 (CH-3 + C-6), 113.7 (C-4a), 119.7 (CH-8″), 120.2 (C-2″), 123.9 (CH-5), 124.5 (CH-5″), 126.4-126.5 (CH-7″ +3″a), 126.6 (CH-5″), 145.4 (C-8a), 153.3 (C-4), 154.5 (CH-1″), 160.6 (C-7 + 2), 164.2 (CH-6″), 168.8 (C-1′). ESI-HRMS: m/z [M + H]+ calcd. for (C22H18O4N3)+: 388.1297 found: 388.1301.

(E)-N’-(2-hydroxybenzylidene)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (6d)

White solid, yield 82 %, mp 212 °C (EtOH). 1H-NMR (300 MHz, DMSO-d 6 ): δ H 2.19 (s, 3H, CH3), 5.28 (s, 2H, H-2′), 6.21 (s, 1H, H-3), 7.0 (m, 2H, H-6 + H-8), 7.05-8.22 (m, 5H, H-1″ + H-4″ + H-5″ + H-6″ +H-7″), 11.01 (s, 1H, OH), 11.85 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d 6 ): δ c 18.1 (CH3-9), 64.8 (CH2-2′), 101.5 (CH-8), 111,4 (CH-6), 112.2 (CH-3), 113.5 (C-4a), 120.2 (C-2″); 121.5 (CH-4″), 123.0 (CH-6″), 127.2 (CH-7″), 130.1 (CH-5″), 126.5 (CH-5), 153.3 (C-8a), 154.5 (C-4), 156.0 (CH-1″), 160,1 (C-3″), 163,5 (C-2 + 7), 168,1 (C-1′). ESI-HRMS: m/z [M + H]+ calcd. for (C19H17O5N2)+: 353.1059 found: 353.1063.

(E)-N’-(4-hydroxy-3-methoxybenzylidene)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (6e)

White solid, yield 78 %, mp 229 °C (EtOH). 1H-NMR (300 MHz, DMSO-d 6 ): δ H 2.19 (s, 3H, CH3), 3.85 (s, 3H, H-8″), 5.28 (s, 2H, H-2′), 6.21 (s, 1H, H-3), 7.02 (m, 2H, H-6 + H-8), 7.11-7.22 (m, 4H, H-1″ + H-3″ + H-5″ + H-7″), 11.01 (s, 1H, OH), 11.85 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d 6 ): δ c 18.1 (CH3-9), 56.2 (OCH3-8″), 66.5 (CH2-2′), 113.0 (C-5″), 126.5 (CH-5), 130.1 (CH), 131.4 (CH), 153,3 (C-8a), 154.5 (C-4), 156,1 (CH-1″); 160,6 (C-7); 160,1 (CH-7″); 160,0 (CH-3″); 163.5 (C-2 + 7), 168.1 (C-1′). ESI-HRMS: m/z [M + H]+ calcd. for (C20H19O6N2)+: 383.1165 found: 383.1169.

(E)-N′-(3,4-dimethoxybenzylidene)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (6f)

White solid, yield 81 mp 222 °C (EtOH). 1H-NMR (300 MHz, DMSO-d 6 ): δ H 2.19 (s, 3H, CH3), 3.85 (s, 3H, H-8″), 3.91 (s, 3H, H-9″), 5.28 (s, 2H, H-2′), 6.21 (d, 1H, J = 0.9 Hz, H-3), 7.02 (m, 2H, H-6 + H-8), 7.10-7.24 (m, 4H, H-1″ + H-3″ + H-6″ + H-7″), 11.85 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d 6 ): δ c 18.1 (CH3-9), 65.2 (OCH3-8″), 66.5 (CH2-2′), 163.5 (C-2), 160.8 (C-7), 160.0 (CH-1″), 131.4 (C-2″), 113.0 (CH-3″), 155.0 (C-4a), 152.0 (C-4″), 148.0 (C-5″), 130.1 (CH-6″), 121.0 (CH-7″), 126.5 (CH-5), 152.3 (C-8a), 154.5 (C-4), 156.0 (CH-1″), 168.5 (C-1′).ESI-HRMS: m/z [M + H]+ calcd. for (C21H23O6N2)+: 397.1321 found: 397.1325.

Preparation of compounds 7a–c

A mixture of hydrazide 5 (100 mg, 4 mmol), cyclic anhydride (1 eq) and 20 mL of ethanol was refluxed. The progress of the reaction was monitored by TLC. Once the reaction is completed, the precipitate was filtered and recrystallized from ethanol to obtain the desired products, 7a–c.

N-(1,3-dioxoisoindolin-2-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetamide (7a)

White solid, yield 26 %, mp 280 °C (EtOH). 1H-NMR (DMSO-d 6 , 300 MHz): δ H 2.42 (s, 3H, CH3), 5.0 (s, 2H, H-2′), 6.27 (s, 1H, H-3), 7.10 (m, 2H, H-6 + H-8), 7.76 (d, 1H, J = 8.4 Hz, H-5), 8.01 (m, 4H, H-5′ + H-6′ + H-7′ + H-8′), 11.10 (s, 1H, NH).

13C-NMR (75 MHz, DMSO-d 6 ): δ c 18.2 (CH3-9), 66.1 (CH2-2′), 111.6 (CH-8), 112.6 (CH-3 + C-6), 115.0 (C-4a), 123.9 (CH-5), 129.3 (CH-5′ + C-8′), 135.4 (CH-6′ + C-7′), 160.1-160.3 (C-2 + C-7), 166.9 (C-1′), 101.7 (CH-8), 153.4 (C-8a), 154.5 (C-4′a + C-9′a), 164.9 (C-9′ + C-4′). ESI-HRMS: m/z [M + H]+ calcd. For (C20H13O6N2)+: 377.0787 found: 377.0777.

N′-(2,9-dioxo ([2,2.1] hept-5-en)-2-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetohydrazide (7b)

White solid, yield 66 %, mp 270 °C (EtOH). 1H-NMR (300 MHz, CDCl3): δ H 1.71 (m, 2H, H-9′), 2.41 (s, 3H, CH3), 3.41 (m, 2H, H-4′a + H-8′a), 3.44 (m, 2H, H-8′ + H-5′), 4.81 (s, 2H, H-2′), 6.18 (s, 1H, H-3), 6.22 (s, 1H, H-6′), 6.31 (s, 1H, H-7′), 7.02 (m, 2H, H-6 + H-8), 7.80 (d, 1H, J = 8.7 Hz, H-5), 8.53 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δ c 18.7 (CH3-9), 22.1 (CH2-9′), 44.4 (C-4′a + C-8′a), 45.1 (CH-8′ + C-5′), 67.1 (CH2-2′), 111.6 (CH-8), 112.6 (CH-3 + C-6), 155.0 (C-4a), 123.9 (CH-5), 134.8 (CH-6′ + C-7′), 152.5 (C-8a), 160.1–160.6 (C-2 + C-7), 160,9 (C-4′), 161.1 (C-10′), 173.6 (C-1′). ESI-HRMS: m/z [M + H]+ calcd. for (C21H19O6N2)+: 395.1243 found: 395.1240.

4-(2-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)hydrazinyl)-4-oxobutanoic acid (7c)

White solid, yield 76 %, mp 266 °C (EtOH). 1H-NMR (300 MHz, DMSO-d 6 ) δ H 2.41 (s, 3H, CH3), 2.54 (m, 4H, H-6′ + H-7′), 4.81 (s, 2H, H-2′), 6.22 (s, 1H, H-3), 7.02 (m, 2H, H-6 + H-8), 7.80 (d, 1H, J = 8.7 Hz, H-5), 10.34 (s, 1H, NH), 10.53 (s, 1H, NH), 13 (s, 1H, OH). 13C-NMR (75 ppm, DMSO-d 6 ): δ c 18.2 (CH3-9), 28.0 (CH2-6′ + C-7′), 66.1 (CH2-2′), 111.0 (CH-8), 112.8 (CH-3 + C-6), 114.2 (C-4a), 155.1 (C-8a), 123.9 (CH-5), 160.1–160.6 (C-2 + C-7), 165.9 (C-1′), 170.1 (C-5′), 173.5 (C-8′). ESI-HRMS: m/z [M + H]+ calcd. for (C16H17O7N2)+: 349.0958 found: 349.0964.

Biological activities

Antimicrobial evaluation

All the synthesized compounds have been evaluated for their antibacterial and antifungal activities. For antibacterial test, three bacterial agents were selected as test microorganisms, Pseudomonas sp. (Pa 499), Burkholderia sp. (Bg 35) and Bacillus sp. (Bp 420). They were cultured at 25 °C on Nutrient Agar (NA) medium for 48 h before use.

Antifungal activity was performed against Aspergillus niger, Botrytis cinerea and Fusarium oxysporum f. sp. lycopersici. These fungi were obtained from the Laboratory of Phytopathology of the Regional Center of Research in Horticulture and Organic Agriculture (CRRHAB) of Chott-Mariem, Tunisia. They were cultured at 25 °C on potato dextrose agar (PDA) medium 1 week before use. The screening results were compared with those of ampicillin and carbendazim used as standard references for antibacterial for antifungal activities, respectively.

Antibacterial activity

The purified products were screened for their antibacterial activity by using the agar disc diffusion method (Marmonier, 1987). NA medium cooled at 45 °C was supplemented with a bacterial suspension (106 CFU/mL) and poured into Petri plates. After solidification, sterile Whatman paper discs (diameter 6 mm) were placed at the surface of the culture medium and 20 μL (1000 µg/mL) of the product dissolved in DMSO was dropped onto each disc. The negative control plates had no product added to the filter paper, whereas in the positive control plates, discs were impregnated with the same volume of ampicillin solution (5 mg/mL). The treated Petri dishes were incubated at 25 °C for 48 h. The antibacterial activity was evaluated by measuring the diameter of the inhibitory zones formed around the discs. The experiment was replicated twice.

Antifungal activity

Aspergillus niger, Botrytis cinerea and Fusarium oxysporum f. sp. lycopersici were used for the screening of antifungal activity of the products tested by using the disc diffusion method (Barry and Thornsberry, 1991). A conidial suspension of the tested fungi was prepared (104–105 CFU/mL) and added to PDA medium cooled at 45 °C and poured uniformly into Petri plates (diameter 90 mm). Sterilized paper discs (6 mm, Whatman No. 1 filter paper) were impregnated with 20 µL (1000 µg/mL) of the product dissolved in DMSO and placed on the culture plates, whereas the negative control plates had no product added to the filter paper. In the positive control plates, discs were imbibed with the same volume of a carbendazim suspension (0.5 mg/mL). The diameter of the inhibition zone (mm) around the disc was measured after incubation at 25 °C for 4 days and compared with control. The test was performed in triplicate.