Design and Synthesis of Novel Coumarin Derivatives with Biological Activity Evaluation

Introduction: Coumarins, whether of natural or synthetic origin, represent a large and diverse family of heterocyclic compounds characterized by a benzopyrone (1,2-benzopyrone or α-pyrone) core structure. These compounds are widely distributed in the plant kingdom and have attracted considerable attention due to their broad spectrum of biological and pharmacological activities. The most well-known coumarin, simply called “coumarin,” was first isolated from tonka bean (Dipteryx odorata Wild) and has been extensively investigated for its biochemical and therapeutic properties.¹ Another naturally occurring coumarin derivative, dicoumarol, was discovered in mouldy, wet sweet-clover hay and is recognized for its anticoagulant activity.²

Several other coumarins have recently drawn significant interest due to their pharmacological potential. Osthole, for example, found in Cnidium monnieri, exhibits a wide range of biological activities, including anti-inflammatory, anti-osteoporotic, and neuroprotective effects.³ Scoparone, isolated from Artemisia scoparia, has shown promising pharmacological properties such as immunosuppressive, vasorelaxant, and hepatoprotective effects (Figure 1).⁴

Today, coumarins are considered an important class of bioactive organic compounds with multiple applications in medicinal chemistry. They are used as bactericides¹–³, fungicides⁴, anti-inflammatory agents⁵, anticoagulants⁶, and antitumor agents⁷,⁸. Their versatile pharmacological properties make them attractive scaffolds for the design of novel therapeutic agents.

Motivated by these biological activities, we aimed to synthesize a series of new coumarin derivatives by incorporating different heterocyclic rings onto the coumarin scaffold. This structural modification is intended to enhance pharmacological potency and broaden the spectrum of biological activity. The resulting compounds are expected to serve as promising candidates for further investigation as multifunctional pharmacologically active molecules.

Biological Activity

Standard drugs, amoxicillin for bacteria and mycostatin for fungi, were used at a concentration of 1000 ppm as reference compounds. The biological activities of the synthesized compounds were evaluated using the filter paper disc method [11]. Each compound was dissolved in N,N-dimethylformamide to prepare a 1 mg/mL solution (1000 ppm). The inhibition zones around the 5 mm filter paper discs were measured in millimeters after an incubation period of 3 days—at 37 °C for Escherichia coli and at 28 °C for the other bacteria and fungi. N,N-dimethylformamide alone did not produce any inhibition.

The synthesized compounds were tested against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus), Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes), and fungi (Aspergillus niger, Penicillium italicum, Fusarium oxysporum). The experimental results are summarized in Tables 1 and 2.

Table 1. Antibacterial activity of the prepared compounds

Compd.	Organisms*
	1	2	3	4	5	6
3a	10	12	18	26	20	10
3b	10	18	15	24	18	12
5a	17	12	11	23	15	15
5b	8	13	10	24	12	14
6	12	14	18	27	15	15
7	11	16	14	18	13	12
8	19	15	12	21	29	16
Amoxicillin	11	13	21	28	36	10

• * Organisms :1- Pseudomonas aurignosa, 2-Bacillus subtilis, 3- Bacillus cereus, 4- Staphylococcus aureus , 5-Echerichia coli and 6- Enterobacter aerogenes.

Experimental

          Melting Points and Spectral Characterization
Melting points were determined using an Electrothermal capillary apparatus and are uncorrected. Infrared (IR) spectra were recorded on KBr disks in the 3100 cm⁻¹ region. Proton nuclear magnetic resonance (^1H-NMR) spectra were obtained on an ADVANCE-300 MHz spectrometer using DMSO-d₆ as the solvent, with chemical shifts reported in δ (ppm). Mass spectra were recorded on a Finnigan Mat SSQ-7000 mass spectrometer.

2-Amino-4-(4′-bromophenyl)-3-cyano-4H,5H-pyrano[3,2-c][1]benzopyran-5-one (3a)
A mixture of 4-hydroxycoumarin (1, 1.62 g, 0.01 mol) and α-cyano-p-bromocinnamonitrile (2a, 2.32 g, 0.01 mol) in ethanol (20 mL) containing piperidine (0.5 mL) was heated under reflux for 30 min. The solid product that precipitated during the reaction was filtered, dried, and recrystallized from dioxane to afford 3a (see Table 3).

• IR (cm⁻¹): 3387–3316 (NH₂), 2191 (C≡N), 1710 (lactone C=O)
• ^1H-NMR (DMSO-d₆, δ ppm): 4.47 (s, 1H, pyran H-4), 7.2 (s, 2H, NH₂), 7.3–8.0 (m, 8H, ArH)
• MS (m/z): 394 (M⁺, 8.41%), 328 (23.87), 315 (3.85), 249 (100), 239 (40.92), 153 (4.08), 120 (20.86), 92 (26.33), 66 (15.15)

2-Amino-4-(4′-bromophenyl)-3-carboethoxy-4H,5H-pyrano[3,2-c][1]benzopyran-5-one (3b)
A solution of 4-hydroxycoumarin (1, 1.62 g, 0.01 mol) and α-carboethoxy-p-bromocinnamonitrile (2b, 2.79 g, 0.01 mol) in ethanol (20 mL) containing piperidine (0.5 mL) was refluxed for 8 h. The solvent was removed under reduced pressure, and the residue was triturated with methanol. The resulting yellow solid was filtered, dried, and recrystallized from benzene to yield 3b (see Table 3).

• IR (cm⁻¹): 3338–3280 (NH₂), 1730 (ester C=O), 1697 (lactone C=O)
• ^1H-NMR (δ ppm): 1.11 (t, 3H, CH₃), 3.99 (q, 2H, CH₂), 4.65 (s, 1H, pyran H-4), 7.18–7.9 (m, 10H, ArH + NH₂)
• MS (m/z): 441 (M⁺, 50%), 368 (28), 286 (100), 249 (32.86), 121 (39.65), 92 (4.74)

2-N-furoylamino-3-carboethoxy-4-(p-bromophenyl)-4H,5H-pyrano[3,2-c][1]benzopyran-5-one (5a)
A mixture of 3b (4.4 g, 0.01 mol), 2-furoyl chloride (1.3 g, 0.01 mol), and potassium carbonate (1.38 g, 0.01 mol) in dry acetone (20 mL) was refluxed for 4 h. After cooling, the reaction mixture was poured into water (100 mL). The precipitated solid was collected, dried, and recrystallized from ethanol to yield 5a (see Table 3).

• IR (cm⁻¹): 3414 (NH), 1740 (ester C=O), 1714 (lactone C=O), 1681 (amide C=O)
• ^1H-NMR (δ ppm): 1.11 (t, 3H, CH₃), 3.99 (q, 2H, CH₂), 4.80 (s, 1H, pyran H-4), 6.81–8.20 (m, 11H, ArH + furan), 11.18 (s, 1H, NH, disappeared after D₂O exchange)
• MS (m/z): 536 (M⁺, 1.99%), 535 (5.71), 380 (70.53), 327 (18.81), 329 (20.20), 286 (23.37), 249 (78.38), 121 (19.65), 95 (100)

2-N-Chloroacetylamino-3-carboethoxy-4-(p-bromophenyl)-4H,5H-pyrano[3,2-c][1]benzopyran-5-one (5b)
Compound 5b was synthesized from 3b (4.4 g, 0.01 mol) and chloroacetyl chloride (1.25 g, 0.01 mol) following the procedure described for 5a. Characterization data:

• IR (cm⁻¹): 3414 (NH), 1728 (ester C=O), 1713 (lactone C=O), 1666 (amide C=O)
• ¹H NMR (δ, ppm): 1.11 (t, 3H, CH₃), 3.99 (q, 2H, CH₂), 4.4 (s, 2H, CH₂), 7.20–7.90 (m, 8H, ArH)
• MS (m/z, %): 519 (M⁺, 13.79), 518 (3.51), 364 (35.12), 362 (100), 286 (59.53), 240 (52.49), 121 (59.56), 92 (18.31)

7-(p-Bromophenyl)-9-ethoxy-9,11-dihydropyridino[3′,2′-6,5]4H-pyrano[3,2-c][1]benzopyran-6,8,10-trione (6)
A mixture of 5b (5.1 g, 0.01 mol) and sodium metal (0.23 g, 0.01 mol) in absolute ethanol (15 mL) was refluxed for 2 h. After concentration and cooling, the reaction mixture was poured into ice-acetic acid and stirred for 1 h. The insoluble product was filtered, dried, and recrystallized from dilute ethanol to yield compound 6 (cf. Table 3). Characterization data:

• IR (cm⁻¹): 3419 (NH), 1716 (lactone C=O), 1691–1651 (2 C=O)
• ¹H NMR (δ, ppm): 1.12 (t, 3H, CH₃), 3.3 (br s, 1H, NH, disappears after D₂O exchange), 3.91 (q, 2H, CH₂), 4.69 (s, 1H, pyran H-4), 7.21–7.90 (m, 8H, ArH)
• MS (m/z, %): 482 (M⁺, 0), 443 (62.83), 368 (37.25), 286 (100), 240 (70.81), 121 (45.96), 92 (13.26), 75 (5.78)

7-(p-Bromophenyl)-9-[(p-bromophenyl)carbonyl]-8-hydroxy-pyrrolo[3′,2′-5,6]4H-pyrano[3,2-c][1]benzopyran-6-one (7)
Compound 7 was prepared by refluxing a solution of 3b (1.02 g, 0.0025 mol) in pyridine (20 mL) with 4-bromophenacyl bromide (5.08 g, 0.01 mol) for 1 h. The reaction mixture was poured into ice-HCl, and the separated solid was filtered and recrystallized from dimethylformamide. Characterization data:

• IR (cm⁻¹): 3471 (broad NH and OH), 1694 (lactone C=O), 1639 (C=O)
• ¹H NMR (δ, ppm): 4.7 (s, 1H, pyran H-4), 6.5 (s, 1H, C9-H), 7.8–9.0 (m, 13H, ArH + NH)
• MS (m/z, %): 593 (M⁺, 0), 459 (5.44), 276 (26.72), 274 (18.28), 185 (100), 184 (90.32), 154 (20.46), 120 (4.71), 89 (13.49), 79 (52.64)

2-Amino-4-(p-bromophenyl)-3-[N-phenylcarbamido]-dihydrobenzofurano[3,2-b]-4H-pyran (8)
A mixture of 3b (1.02 g, 0.0025 mol) and anthranilic acid (1.26 g, 0.01 mol) in pyridine (20 mL) was refluxed for 6 h. The reaction mixture was poured into ice-HCl, and the solid product was washed with water, dried, and recrystallized from benzene to yield compound 8 (cf. Table 3). Characterization data:

• IR (cm⁻¹): 3413 (NH₂), 3236 (NH), 1667 (amide C=O)
• ¹H NMR (δ, ppm): 7.01 (d, 1H, pyran H-4), 7.36 (d, 2H, furano H), 7.47–8.12 (m, 16H, ArH + NH₂ + NH)
• MS (m/z, %): 463 (M⁺, 0), 327 (100), 325 (94.03), 247 (26.08), 218 (18.55), 119 (36.21), 102 (51.69), 92 (24.83)

Table 2. Antifungal activity of the prepared compounds

Compd.	Organisms*
	A	B	C
3a	20	11	14
3b	18	15	15
5a	19	22	12
5b	21	20	18
6	19	12	10
7	12	13	10
8	11	18	13
Mycostatin	26	20	12

          *Organisms ( A) Fusarium Oxysporum,(B) Penicillium italicumand (C) Aspergillus niger

Table 3. Characterization data of the prepared compounds

Compd.	M.P. Co	Yield (%) Colour	Molecular Formula (M. wt.)	Analysis Calcd./Found
				C	H	N
3a	254	67 White	C19H11BrN2O3 (395.22)	57.74 57.81	2.81 2.70	7.09 6.92
3b	192	40 White	C21H16BrNO5 (442.27)	57.03 56.99	3.65 3.70	3.17 3.06
5a	140	40 White	C26H18BrNO7 (536.33)	58.22 58.10	3.38 3.43	2.61 2.36
5b	158	60 White	C23H17BrClNO6 (518.75)	53.25 53.56	3.30 3.45	2.70 2.70
6	196	45 Yellow	C23H16BrNO6 (482.28)	57.27 57.11	3.31 3.46	2.90 3.00
7	230	40 Yellow	C27H15Br2NO5 (593.22)	54.66 54.87	2.54 2.34	2.36 2.45
8	282	40 Yellow	C24H19BrN2O3 (463.33)	62.22 62.01	4.13 3.98	6.05 5.90

Results and Discussion

          The condensation of 4-hydroxycoumarin (4-hydroxy-2H-1-benzopyran-2-one, 1) with α-cyano-p-bromo-cinnamonitrile (2a) in ethanol, in the presence of a catalytic amount of piperidine, yielded a product that could potentially exist as two structural isomers: 3a or 4a (Scheme 1). Analysis of the ^1H-NMR spectra favored structure 3a over 4a, as indicated by a distinct signal at δ 4.5–5.2 ppm corresponding to a single proton attached to an sp^3-hybridized carbon. This chemical shift is consistent with signals reported for 4H-pyran derivatives [9]. In contrast, if 4a were formed, a 2H-pyran signal would be expected at a lower field. The formation of 3a is postulated to occur via nucleophilic addition of the coumarin C-3 carbon to the activated double bond of 2a, followed by cycloaddition of the resulting Michael adduct. Similarly, compound 3b was synthesized using α-carboethoxy-p-bromo-cinnamonitrile (2b, Scheme 1) under analogous reaction conditions.

Subsequently, 3b was treated with either 2-furoyl chloride or chloroacetyl chloride in dry acetone containing potassium carbonate to afford the corresponding N-substituted derivatives. Specifically, reaction with 2-furoyl chloride yielded the 2-N-furoylamino-3-carboethoxy-4-(p-bromophenyl)-4H,5H-pyrano[3,2-c][1]benzopyran-5-one derivative (5a), while treatment with chloroacetyl chloride produced the 2-N-chloroacetylamino-3-carboethoxy-4-(p-bromophenyl)-4H,5H-pyrano[3,2-c][1]benzopyran-5-one derivative (5b) (Scheme 2). Cyclization of compound 5b using sodium ethoxide afforded 7-(p-bromophenyl)-9-ethoxy-9,11-dihydropyridino[3′,2′:6,5]-4H-pyrano[3,2-c][1]benzopyran-6,8,10-trione (6) (Scheme 2).

Further functionalization of 3b was carried out by heating it with 4-bromophenacyl bromide in refluxing pyridine, which resulted in the formation of 7-(p-bromophenyl)-9-[(p-bromophenyl)carbonyl]-8-hydroxypyrrolo[3′,2′:5,6]-4H-pyrano[3,2-c][1]benzopyran-6-one (7) (Scheme 2). Treatment of 3b with anthranilic acid in boiling pyridine, on the other hand, led to the formation of an unexpected benzofuranopyran derivative. The structure of this product was assigned as 2-amino-4-(p-bromophenyl)-3-[N-phenylcarbamido]dihydrobenzofurano[3,2-b]-4H-pyran (8) (Scheme 2).

The proposed structure of compound 8 is supported by several spectral observations:
(i) The IR spectrum shows the absence of ester and δ-lactone carbonyl absorptions, while a characteristic amide C=O stretch appears at 1666 cm^-1.
(ii) The ^1H-NMR spectrum exhibits signals at δ 7.02 (d, 1H, pyran H-4), δ 7.34 (d, 2H, furan H), and δ 7.46–8.11 ppm (m, 16H, aromatic protons along with NH_2 and NH groups), consistent with the proposed benzofuranopyran framework.

These reactions collectively illustrate a versatile and efficient strategy for the synthesis of structurally diverse coumarin-based pyran, pyridino, and benzofuran derivatives with potential biological relevance.

Conclusion

          2-Amino-4-(p-bromophenyl)-3-cyano(carboethoxy)-4H,5H-pyrano[3,2-c][1]benzopyran-5-ones (3a,b) were employed as starting materials for the synthesis of novel coumarin derivatives featuring heterocyclic rings fused to the coumarin core.

Compound 3b was further reacted with either 2-furoyl chloride or chloroacetyl chloride to yield 2-N-furoylamino-3-carboethoxy-4-(p-bromophenyl)-4H,5H-pyrano[3,2-c][1]benzopyran-5-one (5a) and 2-N-chloroacetylamino-3-carboethoxy-4-(p-bromophenyl)-4H,5H-pyrano[3,2-c][1]benzopyran-5-one (5b), respectively. These intermediates subsequently underwent cyclization to form 7-(p-bromophenyl)-9-ethoxy-9,11-dihydro-pyridino[3’,2’-6,5]4H-pyrano[3,2-c][1]benzopyran-6,8,10-trione (6).

Additionally, compound 3b reacted with 4-bromophenacyl bromide and anthranilic acid to afford 7-(p-bromophenyl)-9-[(p-bromophenyl)carbonyl]-8-hydroxypyrrolo[3’,2’-5,6]4H-pyrano[3,2-c][1]benzopyran-6-one (7) and 2-amino-4-(p-bromophenyl)-3-[N-phenylcarbamido]-dihydrobenzofurano[3,2-b]-4H-pyran (8), respectively.

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