ISRN Medicinal Chemistry

Volume 2013 (2013), Article ID 619535, 11 pages

http://dx.doi.org/10.1155/2013/619535

Research Article

Synthesis and Evaluation of Substituted 4,4a-Dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one as Antimicrobial Agent

Amanpreet Kaur,¹ Vishal Sharma,¹ Abhishek Budhiraja,² Harpreet Kaur,³ Vivek Gupta,⁴ Rajni Kant,⁴ and Mohan Paul S. Ishar¹

¹Bio-Organic and Photochemistry Laboratory, Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab 143 005, India

²ISF College of Pharmacy, Moga, Punjab 143008, India

³Department of Microbiology, Guru Nanak Dev University, Amritsar, Punjab 143 005, India

⁴Post-Graduate Department of Physics, University of Jammu, Jammu-Tawi 180 006, India

Received 7 June 2013; Accepted 21 July 2013

Academic Editors: A. Contini, P. L. Kotian, and P. M. Sivakumar

Copyright © 2013 Amanpreet Kaur et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

A series of pyrano[4,3-b][1]benzopyranones (7a–t) were synthesized through hetero-Diels-Alder reaction of substituted 3-formylchromones (5) with enol ethers (6), characterized by IR, ¹H NMR, ¹³C NMR, and mass spectral techniques. All the compounds were evaluated for antimicrobial activity against various bacterial and fungal strains, found to possess significant inhibitory potential, particularly, compounds bearing electron withdrawing group -fluoro such as 7i and 7h. Compounds were also tested and displayed a significant inhibitory potential against methicillin-resistant Staphylococcus aureus (MRSA).

1. Introduction

Despite decades of extensive progress in treatment and prevention, infectious diseases remain a major cause of death and are responsible for worsening the living conditions of many millions of people around the world [1]. Additionally, resistance to known antibiotics is also a serious problem and presents a challenge for the medicinal chemists to develop new effective molecular entities against pathogenic microorganism resistant to available current treatments [2]. Chromones are an important class of heterocyclic molecules naturally occurring, and synthetic analogs are found to display a wide range of pharmacological activities such as antimicrobial, anticancer, neuroprotective, HIV-inhibitory, antifungal activities, and antioxidant [3–9]. Natural products such as aposhaerin A (1), isolated from Aposhaeria sp. possess remarkable antibacterial activity [10]. Recently, we have reported that 3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl-) chromen-4-ones (2) possess significant antibacterial activity against Shigella flexneri (Figure 1) [11].

Figure 1: Chromone based compounds as antimicrobial agents.

Similarly, pyran moiety is widely present in animal and plant kingdom; it exhibits diverse pharmacological activities such as antimicrobial, antiviral, antiproliferative, antitumor, antiinflammatory [12–16]. Pyrano[3,2-c]chromene derivatives (3a–c), bearing a 2-thiophenoxyquinoline nucleus, have been found to display excellent antibacterial activity against B. subtilis, E. coli, and P. aeruginosa, respectively, [17]. 2-Amino-3-cyano-6-(3,5-dibromo-4-methoxyphenyl)-4-arylpyrans (4) have been found to exhibit potent antimicrobial and antimycobacterial activity (Figure 2) [18].

Figure 2: Pyran based compounds as antimicrobial agents.

Taking cognizance of high antimicrobial activity of both chromone and pyran derivatives, it was decided to synthesize chromone fused pyrans and evaluate against various pathogenic bacterial and fungal strains.

2. Results and Discussion

2.1. Chemistry

Substituted pyrano[4,3-b][1]benzopyranones were synthesized by the hetero-Diels-Alder reaction of substituted 3-formylchromones (5a–j) with excess of enol ethers (6) in dichloromethane at room temperature [19–22]. All the purified products were characterized by rigorous spectroscopic techniques (IR, ¹H NMR, ¹³C NMR, and mass) and elemental analysis (Scheme 1, Table 1). Finally, the structure of compound 7k was confirmed by X-ray crystallography (Figure 3) [23].

Table 1: Reaction time (h) and yield (%) of various purified products.

Sr. number

Reaction time (h)
Products yield (%)

endo
exo

1
H
H
H
O-iBu
72
7a (77)
8a (traces)

2
H
H
CH₃
O-iBu
72
7b (70)
8b (traces)

3
Cl
H
Cl
O-iBu
36
7c (74)
8c (traces)

4
H
H
Cl
O-iBu
24
7d (72)
8d (traces)

5
H
Cl
F
O-iBu
36
7e (78)
8e (traces)

6
H
H
Br
O-iBu
24
7f (71)
8f (traces)

7
H
Br
H
O-iBu
48
7g (73)
8g (traces)

8
H
F
H
O-iBu
48
7h (70)
8h (traces)

9
H
H
F
O-iBu
72
7i (72)
8i (traces)

10
H
Cl
H
O-iBu
48
7j (73)
8j (traces)

11
H
H
H
OEt
168
7k (75)
8k (traces)

12
H
H
CH₃
OEt
96
7l (72)
8l (traces)

13
Cl
H
Cl
OEt
216
7m (73)
8m (traces)

14
H
H
Cl
OEt
192
7n (76)
8n (traces)

15
H
H
F
OEt
72
7o (72)
8o (traces)

16
H
H
Br
OEt
48
7p (74)
8p (traces)

17
H
Br
H
OEt
96
7q (75)
8q (traces)

18
H
F
H
OEt
120
7r (76)
8r (traces)

19
H
Cl
F
OEt
96
7s (74)
8s (traces)

20
H
Cl
H
OEt
72
7t (72)
8t (traces)

Scheme 1: Synthesis of substituted pyrano[4,3-b][1]benzopyranones.

Figure 3: ORTEP view of 7k.

¹H NMR spectrum of 7k displayed doublet of C9-H at 7.91 with  Hz, and C1-H showed up as a doublet at 7.51 with  Hz. Resonances of C3-H and C4a-H appeared as a multiplet at 5.16–5.10. C4-Ha resonance appeared at 2.53 as dd with  Hz and  Hz; C4-Hb showed up as a dt with  Hz and  Hz at 2.30. ¹³C NMR revealed a quaternary carbon resonance at 181.8 ppm attributed to carbonyl carbon (C10), which was further corroborated by a strong characteristic band at 1668 cm⁻¹ in the IR spectrum. Further, the mass spectrum of 7k (ESI) showed the highest ion peak at 247 (). The stereochemistry of product 7k (endo) was assigned on the basis of NMR spectral evidence. C4-Hb showed vicinal coupling constants of 6.9 and 2.4 Hz which can be attributed to axial-equitorial relationships with C4a-H and C3-H, whereas C4-Ha showed vicinal coupling of ~10.0 Hz with both C4a-H and C3-H indicating its diaxial relationship with both neighbouring protons, and this alludes to cis-relationship between C4a-H and C3-H; this trans-diaxial relationship was further confirmed by X-ray crystallographic structure determination of 7k (Figure 3) [20–22]. The corresponding exo-isomers (8a–t, traces) were detected by ¹H NMR of some column fractions, and the ratio of endo/exo was determined from NMR of crude reaction mixture (4 : 1 approximately). The endo and exo approaches leading to compounds 7 and 8 are shown in Figure 4.

(a)

(b)

Figure 4: Modes of addition: endo and exo.

2.2. Antibacterial Activity

All the synthesized compounds (7a–t) were screened for their antibacterial potential in triplicate against two Gram-positive bacteria, Staphylococcus aureus (MTCC96), Bacillus subtilis (MTCC2451), and three gram-negative bacteria, Escherichia coli (MTCC 82), Pseudomonas aeruginosa (MTCC 2642), and Salmonella typhimurium (MTCC 1251), by using disc diffusion method [24]. The activity of compounds was determined in comparison to standard antibiotic discs of amoxicillin (5 μg) and ciprofloxacin (10 μg). Minimum inhibitory concentration (MIC) in μg/mL of compounds exhibiting activity (Table 2) was determined by using serial tube dilution method [25]. All tested compounds were found to exert prominent antibacterial activity against both gram-positive and gram-negative bacterial strains. Compound 7i showed comparable potent inhibitory activity with positive controls against various bacterial strains such as MIC 0.48 against both S. aureus and E. coli, whereas MIC 1.12 against both B. subtilis and P. aeruginosa. Compound 7d showed high inhibitory potential against gram-negative bacterial strain E. coli with MIC 1.56, followed by MIC 1.82 against both B. subtilis and S. aureus, and MIC 12.5 against P. aeruginosa and S. typhi. Compounds 7e and 7n showed good activity against B. subtilis with MIC 6.25, whereas compound 7j showed activity against E. coli and S. aureus with MIC 6.25 and 12.5, respectively. Compounds 7m and 7q showed significant activity against S. aureus and B. subtilis with MIC 6.25 and 12.5, respectively. Compounds 7n and 7o showed promising inhibitory activity against both S. aureus and E. coli, whereas compounds 7j and 7q showed inhibitory potential against P. aeruginosa with MIC 12.5. The literature reports reveal that these types of tricyclic compounds have been isolated from a strain of Chaetomium funicola, which act as potent broad-spectrum metallo-β-lactamase inhibitors [26]. 3-Formylchromones use as a starting reactant in the synthesis of these pyrano[4,3-b][1]benzopyranones has also shown good antibacterial activity against various bacterial strains [27, 28].

Table 2: MIC (µg/mL) of compounds 7a–t against different bacterial strains.

Comp. number
B. subtilis
S. aureus
E. coli
P. aeruginosa
S. typhi

7a
—
—
12.5
25
—

7b
—
12.5
—
—
—

7c
12.5
12.5
25
—
—

7d
1.82
1.82
1.56
12.5
12.5

7e
6.25
12.5
3.12
12.5
—

7f
12.5
25
25
—
—

7g
15.5
—
—
12.5
—

7h
12.5
32.5
—
—
—

7i
1.12
0.48
0.48
1.12
25

7j
25
12.5
6.25
12.5
—

7k
—
15.5
13.3
—
25

7l
25
12.5
25
—
—

7m
12.5
6.25
25
25
—

7n
6.25
12.5
12.5
25
—

7o
25
12.5
12.5
—
50

7p
—
15.5
—
30.5
—

7q
12.5
6.25
25
12.5
25

7r
—
25.5
—
—
—

7s
—
25
—
—
—

7t
12.5
—
30.5
—
—

Amoxicillin
0.5
0.5
0.12
1.0
0.9

Ciprofloxacin
0.75
1.2
0.9
1.8
—

Active compounds were further evaluated against bacterial resistant strains such as methicillin resistant staphylococcus aureus (MRSA), a clinically isolate obtained from PGIMER, Chandigarh, and Klebsiella pneumoniae (MTCC 530) by using disk-diffusion assay. Compounds were found to be active against MRSA and completely inactive against Klebsiella pneumoniae. Minimum inhibitory concentration (MIC) in mg/mL of compounds exhibiting activity (Table 3) was determined by using serial tube dilution method [25]. All the compounds were found to be active against resistant bacterial strain MRSA, whereas compound 7j showed a maximum activity in comparison to other compounds.

Table 3: MIC (mg/mL) of compounds against methicillin-resistant Staphylococcus aureus (MRSA).

Comp. number
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7p
7t

MRSA
0.7
0.5
0.7
0.5
0.5
0.7
0.7
0.5
0.6
0.1
0.5
0.7
0.4
0.5
0.6
0.5

2.3. Antifungal Activity

All synthesized compounds 7a–t were tested against five reference fungal strains: Aspergillus niger (MTCC 1344), Saccharomyces cerevisiae (MTCC 172), Candida albicans (MTCC 3018), Cryptococcus gastricus (MTCC 1715), and Microsporum gypseum (MTCC 4490) by using disc diffusion method [24]. Moreover, the compounds were found to exert prominent antifungal activity against various fungal strains, specially, against A. niger, S. cerevisiae, and C. albicans (Table 4). Compound 7h showed significant inhibitory activity with MIC 2.4, whereas, compounds 7d, 7g, 7j, and 7m exhibit good inhibitory potential against A. niger with . Compound 7j posseses maximum inhibitory potential against S. cerevisiae with MIC 2.9, whereas compounds 7b, 7h, 7i, and 7t displayed good inhibitory potential with . Compounds 7f, 7n, and 7r showed promising activity against C. albicans with . Compound 7q is found to display high antifungal activity as compared to standard drug with MIC 20.5 against C. gastricus.

Table 4: MIC (µg/mL) of compounds 7a–t against different fungal strains.

Compound no.
A. niger
S. cerevisiae
C. albicans
C. gastricus
M. gypseum

7a
55.4
31.5
>100
>100
88.4

7b
30
14.1
65.3
>100
>100

7c
25.2
18.6
32.5
>100
>100

7d
12.4
>100
70
48.5
90.2

7e
19.5
30.5
16.5
75.4
>100

7f
28.2
18.2
11.8
80.4
94

7g
13.8
32.1
55.5
>100
55.4

7h
2.4
11.7
16.6
>100
48.5

7i
16.6
10.5
17.8
58.4
>100

7j
11.2
2.9
61.2
>100
88.5

7k
29.9
71.4
54.5
>100
>100

7l
73.5
56.4
72.4
>100
>100

7m
13.2
40.5
76
>100
53.4

7n
34
>100
14.7
32.5
>100

7o
18.5
21.4
20.5
>100
93

7p
35
20
>100
43.5
>100

7q
21.0
32.1
>100
20.5
31.5

7r
15.3
>100
12.4
>100
53

7s
45.4
20
>100
63.5
>100

7t
21.0
12.1
>100
40.5
>100

Fluconazole
1.9
1.9
3.9
31.2
1.9

Compounds bearing electron withdrawing groups such as -fluoro and -chloro at chromone ring were found to display high activity against both bacterial and fungal strains, whereas substitution with electron donating group led to a decrease in activity. According to Craig’s plot, these -fluoro, -chloro, and -bromo groups are lipophilic in nature, having high π-values; from the literature, it was found that lipophilicity is essential for the compound permeability across the microbes cell membrane [27]. Therefore, compounds having these lipophilic groups exhibit valuable inhibitory potential; similarly, compounds having bulkier or lipophilic group such as Oi-Bu– at position 3 of the fused pyran ring were found to be more active than –OEt against various pathogenic bacterial strains. Disubstitution with electron withdrawing groups such as -fluoro and -chloro on chromone ring showed moderate inhibitory activity against both bacterial and fungal strains.

3. Conclusion

Variously substituted pyrano[4,3-b][1]benzopyrans (7a–t) were synthesized through the hetero-Diels-Alder reaction [4 + 2] of substituted 3-formylchromones (5) with excess of enol ethers (6) in dichloromethane at room temperature. Compounds bearing electron withdrawing groups such as -fluoro and -chloro at chromone ring were found to display high activity against both bacterial and fungal strains such as compound 7i which showed excellent antibacterial activity and compound 7h which displayed promising antifungal activity. All active compounds were also evaluated against bacterial resistant strain MRSA and found to posseses good inhibitory potential, particularly, compound 7j. These “lead” compounds can be taken under consideration for further antimicrobial development and their mode of action.

4. Experimental

4.1. General

Starting materials and reagents were purchased from commercial suppliers and used after further purification (crystallization/distillation). Bruker (400 MHz), JEOL AL-300 FT (300 MHz), and NMR spectrometer were used to record ¹H NMR (300 MHz and 400 MHz) and ¹³C NMR (75 MHz and 100 MHz) spectra, and chemical shifts () are reported as downfield displacements from tetramethylsilane (TMS) used as an internal standard, and coupling constants () are reported in Hz. IR spectrum was recorded with Shimadzu FT-IR-8400S and Bruker spectrophotometers on KBr pellets. Mass spectrum, EI, and ESI methods were recorded on Shimadzu GCMS-QP-2000A and Bruker Daltonics Esquire 300 mass spectrometer, respectively. Elemental analyses were carried out on a Thermoelectron EA-112 elemental analyzer and are reported in percent abundance.

4.2. Synthesis of Substituted Pyrano[4,3-b][1]benzopyrans

Substituted pyrano[4,3-b][1]benzopyrans (7a–t) were synthesized by the [4 + 2] cycloaddition of substituted 3-formylchromones (5a–j, 300 mg) with excess of alkoxy-ethenes (6) in dichloromethane at room temperature [19–22]. The progress of the reaction was determined by thin layer chromatography (TLC). After completion of reaction, the residue obtained on removal of solvent under vacuum was purified by column chromatography, using neutral (pH~7) silica gel 60–120 mesh, (Loba Chemie, 30 g, packed in hexane), and eluted with 1%-2% ethyl acetate in hexane. All the purified products were characterized by rigorous spectroscopic techniques such as IR, ¹H and ¹³C NMR, and mass and elemental analysis. The spectroscopic data of purified compounds are as follows.

4.2.1. 3-Isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7a)

Light-yellow amorphous solid (231 mg, 77%), mp 135–144°C; IR (CHCl₃): 2960, 2875, 1668, 1610, and 1461 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.91 (d, 1H,  Hz, C₉H), 7.52 (s, 1H, C₁H), 7.45–7.01 (m, 2H, Ar-Hs), 6.90 (d, 1H,  Hz, C₆H), 5.18 (m, 2H, C4aH and C₃H), 3.75 (dd, 1H, .3 Hz and  Hz, –OCH₂), 3.33 (dd, 1H,  Hz and  Hz, –OCH₂), 2.57 (unresolved.dd, 1H,  Hz and  Hz, C₄Ha), 2.33 (dt, 1H,  Hz and  Hz, C₄Hb), 1.98–1.89 (m, 1H, –CH), 0.96 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 75 MHz): 180.1 (C=O), 160.4 (q-arom.), 151.1 (olefinic-CH), 137.4 (Ar-CH), 127.1 (Ar-CH), 121.6 (q-arom.), 117.3 (Ar-CH), 100.5 (C4a and C₃), 70.18 (–OCH₂), 33.2 (–CH), 28.1 (C₄), 18.9 (2 × –CH₃); mass (ESI) : 274 (M⁺), 275 (); analysis: calculated for (C₁₆H₁₈O₄), C 70.06 H 6.61% and found C 70.02 H 6.55%.

4.2.2. 3-Isobutoxy-8-methyl-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7b)

Yellowish amorphous solid (210 mg, 70%), mp 138–147°C; IR (CHCl₃): 2954, 2887, 1668, 1618, and 1465 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.70 (brs, 1H, C₉H), 7.50 (s, 1H, C₁H), 7.28–7.12 (m, 1H, C₇H), 6.85 (d, 1H,  Hz, C₆H), 5.16–5.10 (m, 2H, C4aH and C₃H), 3.77–3.72 (m, 1H, –OCH₂), 3.50–3.41 (m, 1H, –OCH₂), 2.54–2.51 (m, 1H, C₄Ha), 2.33–2.21 (m, 1H, C₄Hb), 1.95–1.90 (m, 1H, –CH), 2.30 (s, 3H, –CH₃) 0.95 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 75 MHz): 181.0 (C=O), 157.1 (q-arom.), 153.1 (olefinic-CH), 138.2 (Ar-CH), 129.1 (Ar-CH), 120.6 (q-arom.), 117.1 (Ar-CH), 100.2 (C4a and C₃), 70.18 (–OCH₂), 33.2 (–CH), 28.1 (C₄), 25.3 (–CH₃), and 18.9 (2 × –CH₃); mass (ESI) : 288 (M⁺), 289 (); analysis: calculated for (C₁₇H₂₀O₄), C 70.81 H 6.99% and found C 70.74 H 6.94%.

4.2.3. 6,8-Dichloro-3-isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7c)

Brownish amorphous solid (222 mg, 74%), mp 144–154°C; IR (CHCl₃): 2960, 2873, 1670, 1606, and 1456 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.79 (d, 1H,  Hz, C₉H), 7.56 (s, 1H,C₁H), 7.47 (d, 1H,  Hz, C₇H), 5.21 (m, 2H,  Hz and  Hz, C4aH and C₃H), 3.73 (dd, 1H,  Hz and  Hz, –OCH₂), 3.33 (dd, 1H,  Hz and  Hz, –OCH₂), 2.68 (ddd, 1H,  Hz and , 1.8 Hz, C₄Ha), 2.40 (dt, 1H,  Hz and  Hz, C₄Hb), 1.99–1.87 (m, 1H, –CH), 0.94 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 100 MHz): 180.2 (C=O), 153.0 (olefinic-CH), 134.7 (Ar-CH), 125.5 (Ar-CH), 124.0 (q-arom.), 110.2 (Ar-CH), 101.1 (C4a and C₃), 71.42 (–OCH₂), 33.2 (–CH), 28.4(C₄), 19.3(2 × –CH₃); mass (ESI) : 343 (M⁺); analysis: calculated for (C₁₆H₁₆Cl₂O₄), C 55.99 H 4.70% and found C 55.93 H 4.64%.

4.2.4. 8-Chloro-3-isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7d)

Yellowish-brown amorphous solid (216 mg, 72%), mp 141–150°C; IR (CHCl₃): 2962, 2869, 1664, 1616, 1471 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.86 (d, 1H,  Hz, C₉H), 7.53 (s, 1H, C₁H), 7.36 (dd, 1H,  Hz and  Hz, C₇H), 6.87 (d, 1H,  Hz, C₆H), 5.14 (m, 2H,  Hz and  Hz, C4aH and C₃H), 3.74 (dd, 1H,  Hz and  Hz, –OCH₂), 3.32 (dd, 1H,  Hz and  Hz, –OCH₂), 2.56 (ddd, 1H,  Hz and , 2.1 Hz, C₄Ha), 2.30 (dt, 1H,  Hz and  Hz, C₄Hb), 1.96–1.87 (m, 1H, –CH), 0.94 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 75 MHz): 180.03 (C=O), 159.1 (q-arom.), 153.3 (olefinic-CH), 139.0 (Ar-CH), 127.4 (q-arom.), 126.7 (Ar-CH), 123.6 (q-arom.), 119.4 (Ar-CH), 110.9 (q-arom.), 100.9 (C4a and C₃), 70.6 (–OCH₂), 33.2 (–CH), 28.4 (C₄), 19.1 (2 × –CH₃); mass (ESI) : 308.5 (M⁺), 309 (); analysis: calculated for (C₁₆H₁₇ClO₄), C 62.24 H 5.55% and found C 62.18 H 5.48%.

4.2.5. 8-Fluoro-3-isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7e)

Yellowish-brown amorphous solid (234 mg, 78%), mp 137–146°C; IR (CHCl₃): 2960, 2885, 1668, 1610, 1458 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.58–7.52 (m, 1H, Ar-H), 7.53 (d, 1H,  Hz, C₁H), 7.17–6.87 (m, 2H, Ar-Hs), 5.13 (m, 2H,  Hz and  Hz, C4aH and C₃H), 3.74 (dd, 1H,  Hz and  Hz, –OCH₂), 3.32 (dd, 1H,  Hz and  Hz, –OCH₂), 2.55 (ddd, 1H,  Hz and , 2.1 Hz, C₄Ha), 2.30 (dt, 1H,  Hz and  Hz, C₄Hb), 1.96–1.87 (m, 1H, –CH), 0.94 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 100 MHz): 180.1 (C=O), 160.8 (q-arom.), 151.5 (olefinic-CH), 140.4 (q-arom.), 127.8 (Ar-CH), 122.5 (Ar-CH), 121.1 (q-arom.), 116.8 (Ar-CH), 111.0 (q-arom.), 100.8 (C4a and C₃), 70.7 (–OCH₂), 33.4 (–CH), 28.5 (C₄), 19.3 (2 × –CH₃); mass (ESI) : 292 (M⁺), 293 (); analysis: calculated for (C₁₆H₁₇FO₄), C 65.74 H 5.86% and found C 65.70 H 5.81%.

4.2.6. 8-Bromo-3-isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7f)

Yellowish-brown amorphous solid (213 mg, 71%), mp 143–153°C; IR (CHCl₃): 2960, 2867, 1664, 1616, 1457 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 8.04–8.01 (m, 1H, C₉H), 7.54 (s, 1H, C₁H), 7.28–7.24 (m, 1H, C₇H), 6.86–6.83 (m, 1H, C₆H), 5.20–5.13 (m, 2H, C4aH and C₃H), 3.76–3.63 (m, 1H, –OCH₂), 3.38–3.32 (m, 1H, –OCH₂), 2.59–2.53 (m, 1H, C₄Ha), 2.39–2.32 (m, 1H, C₄Hb), 1.98–1.90 (m, 1H, –CH), 0.98–0.91 (m, 6H, 2 × CH₃); ¹³C NMR (CDCl₃, 75 MHz): 179.2 (C=O), 159.7 (q-arom.), 152.5 (olefinic-CH), 136.4 (Ar-CH), 130.8 (Ar-CH), 123.1 (q-arom.), 118.7 (Ar-CH), 113.1 (q-arom.), 110.2 (q-arom.), 100.5 (C4a and C₃), 70.1 (–OCH₂), 32.4 (–CH), 25.1 (C₄), 18.7 (2 × –CH₃); mass (ESI) : 353 (M⁺); analysis: calculated for (C₁₆H₁₇BrO₄), C 54.41 H 4.85% and found C 54.34 H 4.80%.

4.2.7. 7-Bromo-3-isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7g)

Yellowish-brown amorphous solid (219 mg, 73%), mp 142–151°C; IR (CHCl₃): 2974, 2887, 1670, 1591, 1465 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.76 (d, 1H,  Hz, C₉H), 7.50 (d, 1H,  Hz, C₁H), 7.17 (dd, 1H,  Hz and  Hz, C₈H), 7.12 (d, 1H, .8 Hz, C₆H), 5.17 (m, 2H,  Hz and  Hz, C4aH and C₃H), 3.73 (dd, 1H,  Hz and  Hz, –OCH₂), 3.32 (dd, 1H,  Hz and  Hz, –OCH₂), 2.57 (ddd, 1H,  Hz and , 1.8 Hz, C₄Ha), 2.30 (dt, 1H,  Hz and  Hz, C₄Hb), 1.98–1.85 (m, 1H, –CH), 0.94 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 75 MHz): 179.8 (C=O), 158.6 (q-arom.), 153.1 (olefinic-CH), 135.3 (Ar-CH), 131.7 (Ar-CH), 122.1 (q-arom.), 118.3 (Ar-CH), 113.7 (q-arom.), 110.9 (q-arom.), 100.1 (C4a and C₃), 70.1 (–OCH₂), 32.7 (–CH), 25.4 (C₄), 18.2 (2 × –CH₃); mass (ESI) : 353 (M⁺), 355 (); analysis: calculated for (C₁₆H₁₇BrO₄), C 54.41 H 4.85% and found C 54.34 H 4.80%.

4.2.8. 7-Fluoro-3-isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7h)

Yellowish-brown amorphous solid (210 mg, 70%), mp 136–144°C; IR (CHCl₃): 2954, 2885, 1670, 1618, 1444 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.96–7.91(m, 1H, C₉H), 7.50 (s, 1H, C₁H), 6.78–6.59 (m, 2H, Ar-Hs), 5.16 (m, 2H,  Hz and  Hz, C4aH and C₃H), 3.73 (dd, 1H,  Hz and  Hz, –OCH₂), 3.31 (dd, 1H,  Hz and  Hz, –OCH₂), 2.55 (ddd, 1H,  Hz and and 2.1 Hz, C₄Ha), 2.31 (dt, 1H,  Hz and  Hz, C₄Hb), 1.98–1.87 (m, 1H, –CH), 0.94 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 100 MHz): 180.0 (C=O), 151.9 (olefinic-CH), 129.8 (q-arom.), 110.9 (Ar-CH), 110.0 (Ar-CH), 104.8 (Ar-CH), 100.8 (C4a and C₃), 71.06 (–OCH₂), 33.3 (–CH), 29.9 (C₄), 19.1 (2 × –CH₃); mass (ESI) : 292 (M⁺), 293 (); analysis: calculated for (C₁₆H₁₇FO₄), C 65.74 H 5.86% and found C 65.70 H 5.81%.

4.2.9. 8-Fluoro-7-chloro-3-isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7i)

Yellowish-brown amorphous solid (216 mg, 72%), mp 140–149°C; IR (CHCl₃): 2960, 2885, 1663, 1610, 1461 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.70 (d, 1H,  Hz, C₉H), 7.52 (s, 1H,C₁H), 6.89 (s, 1H, C₆H), 5.23 (m, 2H,  Hz and  Hz, C4aH and C₃H), 3.75 (dd, 1H,  Hz and  Hz, –OCH₂), 3.33 (dd, 1H,  Hz and  Hz, –OCH₂), 2.67 (dd, 1H,  Hz and , C₄Ha), 2.39 (dt, 1H,  Hz and  Hz, C₄Hb), 1.96–1.85 (m, 1H, –CH), 0.94 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 75 MHz): 180.1 (C=O), 159.7 (q-arom.), 151.1 (olefinic-CH), 129.1 (Ar-CH), 119.5 (q-arom.), 110.5 (Ar-CH), 104.3 (Ar-H), 100.2 (C4a), 71.4 (C₃), 65.5 (–OCH₂), 32.4 (C₄), 15.1 (–CH₃); mass (ESI) : 298.5 (M⁺), 299 (M⁺+1); analysis: calculated for (C₁₆H₁₇ClFO₄), C 58.81 H 4.94% and found C 58.75 H 4.87%.

4.2.10. 7-Chloro-3-isobutoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7j)

Yellowish-brown amorphous solid (219 mg, 73%), mp 139–147°C; IR (CHCl₃): 2937, 2887, 1670, 1614, 1423 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.89 (d, 1H,  Hz, C₉H), 7.51 (s, 1H,C₁H), 7.01 (dd, 1H,  Hz and  Hz, C₈H), 6.94 (d, 1H,  Hz, C₆H), 5.17 (m, 2H,  Hz and  Hz, C4aH and C₃H), 3.77 (dd, 1H,  Hz and  Hz, –OCH₂), 3.36 (dd, 1H,  Hz and  Hz, –OCH₂), 2.57 (ddd, 1H,  Hz and , 2.1 Hz, C₄Ha), 2.30 (dt, 1H,  Hz and  Hz, C₄Hb), 1.96–1.85 (m, 1H, –CH), 0.94 (d, 6H,  Hz, 2 × CH₃); ¹³C NMR (CDCl₃, 100 MHz): 180.2 (C=O), 161.1 (q-arom.), 152.1 (olefinic-CH), 141.03 (q-arom.), 128.5 (Ar-CH), 122.7 (Ar-CH), 121.2 (q-arom.), 117.9 (Ar-CH), 111.03 (q-arom.), 100.9 (C4a and C₃), 70.93 (–OCH₂), 33.2 (–CH), 29.61 (C₄), 19.1 (2 × –CH₃); mass (ESI) : 308.5 (M⁺), 309 (); analysis: calculated for (C₁₆H₁₇ClO₄), C 62.24 H 5.55% and found C 62.18 H 5.48%.

4.2.11. 3-Ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7k)

White amorphous solid (225 mg, 75%), mp 115–121°C; IR (CHCl₃): 2929, 2887, 1668, 1614, 1473 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.91 (d, 1H,  Hz, C₉H), 7.51 (d, 1H,  Hz, C₁H), 7.45–7.01 (m, 2H, Ar-Hs), 6.91 (d, 1H,  Hz, C₆H), 5.16–5.10 (m, 2H, C4aH and C₃H), 4.02 (dq, 1H,  Hz and  Hz, –OCH₂), 3.64 (dq, 1H,  Hz and  Hz, –OCH₂), 2.53 (dd, 1H,  Hz and  Hz, C₄Ha), 2.30 (dt, 1H,  Hz and  Hz, C₄Hb), 1.29 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 100 MHz): 181.8 (C=O), 160 (q-arom.), 151.6 (olefinic-CH), 135.2 (Ar-CH), 127.3 (Ar-CH), 122.7 (q-arom.), 122.0 (Ar-CH), 111.6 (Ar-CH), 100.5 (C4a), 70.4 (C₃), 65.6 (–OCH₂), 33.5 (C₄), 15.01 (–CH₃); mass (ESI) : 246 (M⁺), 247 (); analysis: calculated for (C₁₄H₁₄O₄), C 68.28 H 5.73% and found C 68.20 H 5.68%.

4.2.12. 3-Ethoxy-8-methyl-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7l)

Light-yellow amorphous solid (216 mg, 72%), mp 118–125°C; IR (CHCl₃): 2918, 2896, 1668, 1618, 1498 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.69 (d, 1H,  Hz, C₉H), 7.45 (d, 1H,  Hz, C₁H), 7.20 (dd, 1H,  Hz and 2.1 Hz, C₇H), 6.78 (d, 1H,  Hz, C₆H), 5.17 (m, 2H,  Hz and  Hz, C4aH and C₃H), 3.81 (dq, 1H,  Hz and  Hz, –OCH₂), 3.57 (dq, 1H,  Hz and  Hz, –OCH₂), 2.49 (ddd, 1H,  Hz and , 2.1 Hz, C₄Ha), 2.20 (dt, 1H,  Hz and  Hz, C₄Hb), 1.17 (t, 3H,  Hz, –CH₂CH₃), 0.05 (s, 3H, –CH₃); ¹³C NMR (CDCl₃, 75 MHz): 180.4 (C=O), 158.5 (q-arom.), 152.9 (olefinic-CH), 137.5 (Ar-CH), 129.6 (Ar-CH), 120.3 (q-arom.), 117.7 (Ar-CH), 100.1 (C4a), 70.05 (C₃), 65.1 (–OCH₂), 31.6 (C₄), 15.5 (–CH₃); mass (ESI) : 262 (M⁺), 261 (); analysis: calculated for (C₁₅H₁₇O₄), C 69.22 H 6.20% and found C 69.15 H 6.15%.

4.2.13. 6,8-Dichloro-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7m)

Brownish amorphous solid (219 mg, 73%), mp 125–131°C; IR (CHCl₃): 2975, 2898, 1674, 1610, 1456 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.79 (d, 1H,  Hz, C₉H), 7.57 (d, 1H,  Hz, C₁H), 7.49 (d, 1H,  Hz, C₇H), 5.22 (m, 2H,  Hz and , 1.8 Hz, C4aH and C₃H), 4.02 (dq, 1H,  Hz and  Hz, –OCH₂), 3.67 (dq, 1H,  Hz and  Hz, –OCH₂), 2.66 (ddd, 1H,  Hz and , 1.8 Hz, C₄Ha), 2.38 (dt, 1H,  Hz and  Hz, C₄Hb), 1.29 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 75 MHz): 180.1 (C=O), 153.5 (olefinic-CH), 133.2 (Ar-CH), 125.1 (Ar-CH), 124.5 (q-arom.), 110.6 (Ar-CH), 101.3 (C4a), 70.08 (C₃), 65.5 (–OCH₂), 31.1 (C₄), 15.6 (–CH₃); mass (ESI) : 315 (M⁺); analysis: calculated for (C₁₄H₁₂Cl₂O₄), C 53.36 H 3.84% and found C 53.30 H 3.79%.

4.2.14. 8-Chloro-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7n)

Yellowish amorphous solid (228 mg, 76%), mp 121–129°C; IR (CHCl₃): 2979, 2896, 1672, 1610, 1475 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.86 (d, 1H,  Hz, C₉H), 7.52 (s, 1H, C₁H), 7.36 (dd, 1H,  Hz and 2.7 Hz, C₇H), 6.86 (d, 1H,  Hz, C₆H), 5.18 (m, 2H,  Hz and  Hz, C4aH and C₃H), 4.04 (dq, 1H,  Hz and  Hz, –OCH₂), 3.68 (dq, 1H,  Hz and  Hz, –OCH₂), 2.55 (ddd, 1H,  Hz and , 2.1 Hz, C₄Ha), 2.29 (dt, 1H,  Hz and  Hz, C₄Hb), 1.29 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 100 MHz): 180.3 (C=O), 161.1 (q-arom.), 151.9 (olefinic-CH), 142.2 (q-arom.), 127.2 (Ar-CH), 121.7 (Ar-CH), 120.2 (Ar-CH), 117.1 (Ar-CH), 110.0 (q-arom.), 100.1 (C4a), 70.02 (C₃), 65.1 (–OCH₂), 31.9 (C₄), 15.2 (–CH₃); mass (ESI) : 280.5 (M⁺), 281 (); analysis: calculated for (C₁₄H₁₃ClO₄), C 59.90 H 4.67% and found C 59.82 H 4.62%.

4.2.15. 8-Flouoro-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7o)

Orange-brownish amorphous solid (216 mg, 72%), mp 120–127°C; IR (CHCl₃): 2992, 2904, 1662, 1598, 1483 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.67–7.61 (m, 1H, Ar-H), 7.33 (d, 1H,  Hz, C₁H), 7.22–6.96 (m, 2H, Ar-Hs), 5.26 (m, 2H,  Hz and  Hz, C4aH and C₃H), 4.04 (dq, 1H,  Hz and  Hz, –OCH₂), 3.65 (dq, 1H,  Hz and  Hz, –OCH₂), 2.54 (dt, 1H,  Hz and  Hz, C₄Ha), 2.31 (dist.dd, 1H,  Hz and  Hz, C₄Hb), 1.29 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 75 MHz): 180.1 (C=O), 159.8 (q-arom.), 151.4 (olefinic-CH), 129.3 (Ar-CH), 118.9 (q-arom.), 110.1 (Ar-CH), 104.2 (Ar-H), 100.7 (C4a), 71.07 (C₃), 65.1 (–OCH₂), 33.2 (C₄), 15.03 (–CH₃); mass (ESI) : 264 (M⁺), 265 (); analysis: calculated for (C₁₄H₁₃FO₄), C 63.63 H 4.96% and found C 63.58 H 4.91%.

4.2.16. 8-Bromo-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7p)

Orange-brownish amorphous solid (222 mg, 74%), mp 125–133°C; IR (CHCl₃): 2975, 2877, 1666, 1600, 1456 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 8.01 (d, 1H,  Hz, C₉H), 7.53 (s, 1H, C₁H), 7.49 (dd, 1H,  Hz and 2.4 Hz, C₇H), 6.82 (d, 1H,  Hz, C₆H), 5.16 (m, 2H,  Hz and  Hz, C4aH and C₃H), 4.04 (dq, 1H,  Hz and  Hz, –OCH₂), 3.65 (dq, 1H,  Hz and  Hz, –OCH₂), 2.55 (dt, 1H,  Hz and  Hz, C₄Ha), 2.29 (dist.dd, 1H,  Hz and  Hz, C₄Hb), 1.29 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 100 MHz): 179.3 (C=O), 159.5 (q-arom.), 152.1 (olefinic-CH), 137.7 (Ar-CH), 130.0 (Ar-CH), 124.2 (q-arom.), 119.7 (Ar-CH), 114.8 (q-arom.), 110.9 (q-arom.), 100.5 (C4a), 70.7 (C₃), 65.5 (–OCH₂), 33.5 (C₄), 15.1 (2 × –CH₃); mass (ESI) : 325 (M⁺); analysis: calculated for (C₁₄H₁₃BrO₄), C 51.71 H 4.03% and found C 51.65 H 3.97%.

4.2.17. 7-Bromo-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7q)

Yellowish-brown amorphous solid (225 mg, 75%), mp 123–129°C; IR (CHCl₃): 2985, 2896, 1683, 1616, 1419 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.76 (d, 1H,  Hz, C₉H), 7.50 (d, 1H,  Hz, C₁H), 7.31 (s, 1H, C₆H), 7.16 (d, 1H,  Hz, C₈H), 5.17 (m, 2H,  Hz and  Hz, and ), 4.01 (dq, 1H,  Hz and  Hz, –OCH₂), 3.64 (dq, 1H,  Hz and  Hz, –OCH₂), 2.54 (dist.dd, 1H,  Hz and  Hz, C₄Ha), 2.31 (dt, 1H,  Hz and  Hz, C₄Hb), 1.27 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 100 MHz): 180.3 (C=O), 161.8 (q-arom.), 152.8 (olefinic-CH), 129.5 (q-arom.), 128.5 (Ar-CH), 125.5 (Ar-CH), 121.0 (Ar-CH), 111.0 (q-arom.), 100.5 (C4a), 71.2 (C₃), 65.7 (–OCH₂), 33.3 (C₄), 15.03 (–CH₃); mass (ESI) : 325 (M⁺); analysis: calculated for (C₁₄H₁₃BrO₄), C 51.71 H 4.03% and found C 51.65 H 3.97%.

4.2.18. 7-Fluoro-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7r)

Orange-brownish amorphous solid (228 mg, 76%), mp 119–124°C; IR (CHCl₃): 2985, 2904, 1670, 1622, 1436 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.97–7.91 (m, 1H, Ar-H), 7.51 (d, 1H,  Hz, C₁H), 7.22–6.59 (m, 2H, Ar-Hs), 5.18 (m, 2H,  Hz and , 1.8 Hz, C4aH and C₃H), 4.02 (dq, 1H,  Hz and  Hz, –OCH₂), 3.65 (dq, 1H,  Hz and  Hz, –OCH₂), 2.55 (ddd, 1H,  Hz and , 1.8 Hz, C₄Ha), 2.31 (dt, 1H,  Hz and  Hz, C₄Hb), 1.29 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 100 MHz): 179.9 (C=O), 160.8 (q-arom.), 151.9 (olefinic-CH), 129.8 (Ar-CH), 119.4 (q-arom.), 110.2 (Ar-CH), 104.6 (Ar-H), 100.4 (C4a), 71.03 (C₃), 65.6 (–OCH₂), 33.3 (C₄), 15.01 (–CH₃); mass (ESI) : 264 (M⁺), 265 (); analysis: calculated for (C₁₄H₁₃FO₄), C 63.63 H 4.96% and found C 63.58 H 4.91%.

4.2.19. 8-Fluoro-7-chloro-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7s)

Orange-brownish amorphous solid (222 mg, 74%), mp 124–132°C; IR (CHCl₃): 2974, 2877, 1670, 1600, 1461 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.69 (s, 1H, C₉H), 7.52 (s, 1H, C₁H), 6.81 (s, 1H, C₆H), 5.21 (m, 2H,  Hz and  Hz, C4aH and C₃H), 4.01 (dq, 1H,  Hz and  Hz, –OCH₂), 3.66 (dq, 1H,  Hz and  Hz, –OCH₂), 2.60 (unresolved dd, 1H,  Hz and , C₄Ha), 2.36 (dt, 1H,  Hz and  Hz, C₄Hb), 1.29 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 75 MHz): 179.7 (C=O), 159.9 (q-arom.), 151.5 (olefinic-CH), 129.3 (Ar-CH), 119.2 (q-arom.), 110.1 (Ar-CH), 104.9 (Ar-H), 100.4 (C4a), 71.0 (C₃), 65.2 (–OCH₂), 33.1 (C₄), 15.6 (–CH₃); mass (ESI) : 298.5 (M⁺), 299 (); analysis: calculated for (C₁₄H₁₂ClFO₄), C 56.29 H 4.05% and found C 56.22 H 3.98%.

4.2.20. 7-Chloro-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7t)

Orange-brownish amorphous solid (216 mg, 72%), mp 121–129°C; IR (CHCl₃): 2360, 2331, 1670, 1616, 1458 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.85 (d, 1H,  Hz, C₉H), 7.52 (s, 1H, C₁H), 7.01 (dd, 1H,  Hz and 2.1 Hz, C₈H), 6.93 (d, 1H,  Hz, C₆H), 5.17 (m, 2H,  Hz and  Hz, C4aH and C₃H), 4.01 (dq, 1H,  Hz and  Hz, –OCH₂), 3.66 (dq, 1H,  Hz and  Hz, –OCH₂), 2.55 (ddd, 1H,  Hz and , 2.1 Hz, C₄Ha), 2.31 (dt, 1H,  Hz and  Hz, C₄Hb), 1.29 (t, 3H,  Hz, –CH₃); ¹³C NMR (CDCl₃, 100 MHz): 180.1 (C=O), 161.0 (q-arom.), 152.0 (olefinic-CH), 141.05 (q-arom.), 128.5 (Ar-CH), 122.7 (Ar-CH), 121.2 (Ar-CH), 117.9 (Ar-CH), 111.05 (q-arom.), 100.4 (C4a), 70.9 (C₃), 66.6 (–OCH₂), 33.3 (C₄), 15.01 (–CH₃); mass (ESI) : 280.5 (M⁺), 281 (); analysis: calculated for (C₁₄H₁₃ClO₄), C 59.90 H 4.67% and found C 59.82 H 4.62%.

4.3. X-Ray Data of Compound 7k

 CCDC No.: 885423  Crystal description: block  Crystal colour: white Crystal size: 0.30 × 0.20 × 0.20 mm Empirical formula: C₁₄H₁₄O₄ Formula weight: 246.25 Radiation, wavelength: Mo , 0.71073 Å  Unit cell dimensions: , , and  Crystal system: monoclinic  Space group: P2₁/c Unit cell volume: 1197.08(16) No. of molecules per unit cell, : 4 Temperature: 293(2)  Absorption coefficient: 0.100 mm⁻¹ : 520  Scan mode: scan   range for entire data collection:  Range of indices: to 13, to 25, and to 6  Reflections collected/unique: 39266/2343  Reflections observed (): 998 Structure determination: direct methods Refinement: full-matrix least-squares on  No. of parameters refined: 164 Final : 0.0798 wR (): 0.1831 Weight: , where  Goodness-of-fit: 0.997 : 0.002 Final residual electron density:  Measurement: X’calibur system—Oxford diffraction make, UK  Software for structure solution: SHELXS97 (Sheldrick, 2008) Software for refinement: SHELXL97 (Sheldrick, 2008) Software for molecular plotting: ORTEP-3 (Farrugia, 1997) and PLATON (Spek, 2009) Software for geometrical calculation: PLATON (Spek, 2009) and PARST (Nardelli, 1995).

4.4. Microbiological Evaluation

4.4.1. Antibacterial Activity

All the synthesized compounds (7a–t) were screened for their antibacterial potential in triplicate against two gram-positive bacteria, Staphylococcus aureus (MTCC96), Bacillus subtilis (MTCC2451), and three gram-negative bacteria, Escherichia coli (MTCC 82), Pseudomonas aeruginosa (MTCC 2642), and Salmonella typhimurium (MTCC 1251) by using disc diffusion method [24]. The activity of compounds was determined with comparison to standard antibiotic discs of amoxicillin (5 μg) and ciprofloxacin (10 μg). Prewarmed Mueller-Hinton agar plates were inoculated with 10⁶ CFU/mL of test bacteria. Each compound was dissolved in DMSO (1 mg/mL), and then 30 μL of each was pipetted onto sterile paper discs (6 mm diameter) placed on the surface of inoculated agar plates. Plates were incubated at 37°C for 24 h. Activity was expressed as the diameter of the inhibition zone (mm) produced by the compounds (Table 2). DMSO was used as negative control. MIC of compounds exhibiting considerable activity was evaluated by turbidimetry method [25]. The initial optical density (OD) of the medium was measured by spectrophotometer at 600 nm. The test strains were incubated in nutrient broth until the OD reached 0.4–0.6. Then, the different concentrations of compounds (0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 μg/mL) were tested for the inhibition of growth of these microbes, in separate tubes. The 10 mL tubes, each containing 5 mL nutrient broth and 1 mL of different concentrations of compounds, were incubated for 24 hrs with shaking at 180 rpm using a rotary shaker. Each tube corresponding to different concentrations was observed, and the concentration showing apparently no turbidity was considered to be the MIC of respective compound.

4.4.2. Antifungal Activity

All synthesized compounds 7a–t were tested against five reference fungal strains, Aspergillus niger (MTCC 1344), Saccharomyces cerevisiae (MTCC 172), Candida albicans (MTCC 3018), Cryptococcus gastricus (MTCC 1715), and Microsporum gypseum (MTCC 4490) by using disc diffusion method [20–22]. The antifungal activity of synthesized compounds was determined by observing the zone of inhibition in comparison to the standard antifungal discs (fluconazole and griseofulvin). Test compounds were dissolved in DMSO to make a stock solution of 1 mg/mL. The fresh subculture of strains in normal saline was added to the sterile assay medium (Sabouraud Dextrose agar with chloramphenicol) at 40–45°C and mixed well. The medium was poured into each of the petri dishes. Sterile discs of diameter 6 mm were placed on the medium. 20 μL of each test solution was added to the previously marked discs and the media were allowed to stand for 5 min. The petri dishes were kept aside for 1 h, and then incubated at 28°C for 48 h. Zone of inhibition was measured, and the average of the three readings was calculated; DMSO was also used as negative control. The MIC of active compounds (zone of inhibition) was determined by serial tube dilution method [25]. Different dilutions of test compounds (1.9 μg/mL–500 μg/mL) were made from stock solution, 1 mL nutrient broth was taken in each test tube, and 20 μL of standard strains was added to previously marked test tubes.

References

D. T. W. Chu, J. J. Plattner, and L. Katz, “New directions in antibacterial research,” Journal of Medicinal Chemistry, vol. 39, no. 20, pp. 3853–3874, 1996.

B. Beovic, “Isolation of soil Streptomyces as source antibiotics active against antibiotic-resistant bacteria,” International Journal of Food Microbiology, vol. 112, pp. 280–287, 2006.

P. Valenti, A. Bisi, A. Rampa et al., “Synthesis and biological activity of some rigid analogues of flavone-8- acetic acid,” Bioorganic and Medicinal Chemistry, vol. 8, no. 1, pp. 239–246, 2000.

R. Larget, B. Lockhart, P. Renard, and M. Largeron, “A convenient extension of the Wessely-Moser rearrangement for the synthesis of substituted alkylaminoflavones as neuroprotective agents in vitro,” Bioorganic and Medicinal Chemistry Letters, vol. 10, no. 8, pp. 835–838, 2000.

A. Groweiss, J. H. Cardellins, and M. R. Boyd, “HIV-Inhibitory prenylated xanthones and flavones from Maclura tinctoria,” Journal of Natural Products, vol. 63, no. 11, pp. 1537–1539, 2000.

Y. Deng, J. P. Lee, M. Tianasoa-Ramamonjy et al., “New antimicrobial flavanones from Physena madagascariensis,” Journal of Natural Products, vol. 63, no. 8, pp. 1082–1089, 2000.

I. A. Khan, M. A. Avery, C. L. Burandt et al., “Antigiardial activity of isoflavones from Dalbergia frutescens bark,” Journal of Natural Products, vol. 63, no. 10, pp. 1414–1416, 2000.

K. Mori, G. Audran, and H. Monti, “The first synthesis of coniochaetones A and (±)-B: two benzopyranone derivatives,” Synlett, no. 3, pp. 259–260, 1998.

P.-G. Pietta, “Flavonoids as antioxidants,” Journal of Natural Products, vol. 63, no. 7, pp. 1035–1042, 2000.

A. Yenesew, M. Induli, S. Derese et al., “Anti-plasmodial flavonoids from the stem bark of Erythrina abyssinica,” Phytochemistry, vol. 65, no. 22, pp. 3029–3032, 2004.

T. Raj, R. K. Bhatia, R. K. Sharma, V. Gupta, D. Sharma, and M. P. S. Ishar, “Mechanism of unusual formation of 3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones and 4-oxo-4H-chromene-3-carbothioic acid N-phenylamides and their antimicrobial evaluation,” European Journal of Medicinal Chemistry, vol. 44, no. 8, pp. 3209–3216, 2009.

R. Sakhuja, S. S. Panda, L. Khanna, S. Khurana, and S. C. Jain, “Design and synthesis of spiro[indole-thiazolidine]spiro[indole-pyrans] as antimicrobial agents,” Bioorganic and Medicinal Chemistry Letters, vol. 21, no. 18, pp. 5465–5469, 2011.

A. M. El-Agrody, M. S. Abd El-Latif, N. A. El-Hady, A. H. Fakery, and A. H. Bedair, “Heteroaromatization with 4-hydroxycoumarin—part II: synthesis of some new pyrano[2,3-d]pyrimidines, [1,2,4]triazolo[1,5-c]pyrimidines and pyrimido[1,6-b]-[1,2,4]triazine derivatives,” Molecules, vol. 6, no. 6, pp. 519–527, 2001.

N. R. Taylor, A. Cleasby, O. Singh et al., “Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran-6-carboxamides and sialidase from influenza virus types A and B,” Journal of Medicinal Chemistry, vol. 41, no. 6, pp. 798–807, 1998.

K. Hiramoto, A. Nasuhara, K. Michikoshi, T. Kato, and K. Kikugawa, “DNA strand-breaking activity and mutagenicity of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), a Maillard reaction product of glucose and glycine,” Mutation Research, vol. 395, no. 1, pp. 47–56, 1997.

A. G. Martinez and L. J. Marco, “Friedländer reaction on 2-amino-3-cyano-4H-pyrans: synthesis of derivatives of 4H-pyran [2, 3-b] quinoline, new tacrine analogues,” Bioorganic & Medicinal Chemistry Letters, vol. 7, pp. 3165–3170, 1997.

S. Jain, P. K. Paliwal, G. N. Babu, and A. Bhatewara, “DABCO promoted one-pot synthesis of dihydropyrano(c)chromene and pyrano[2,3-d]pyrimidine derivatives and their biological activities,” Journal of Saudi Chemical Society, 2011.

D. H. Vyas, S. D. Tala, J. D. Akabri, M. F. Dhadukh, K. A. Joshi, and H. S. Joshi, “Synthesis and antimicrobial activity of some new cyanopyridine and cyanopyrans towards Mycobacterium tuberculosis and other microorganisms,” Indian Journal of Chemistry B, vol. 48, no. 6, pp. 833–839, 2009.

S. J. Coutts and T. W. Wallace, “Heterodiene cycloadditions: preparation and transformations of some substituted pyrano[4,3-b][1]benzopyrans,” Tetrahedron, vol. 50, no. 40, pp. 11755–11780, 1994.

C. K. Ghosh, N. Tewari, and A. Bhattacharyya, “Heterocyclic systems; 15¹. Formation and hydrolysis of the [4+2]-cycloadduct of 4-Oxo-4H-[1]benzopyran-3-carboxaldehyde with ethyl vinyl ether,” Synthesis, vol. 7, pp. 614–615, 1984.

A. Cook, I. W. Gunawardana, M. Huestis, et al., “Preparation of heterocyclic compounds as inhibitors of beta-secretase useful for the treatment of neurodegenerative diseases,” Patent International Application, WO 2012071458 A1 20120531, 2012

Z. N. Siddiqui and A. Zaman, “Pyrazoles from 3-formylchromone-ethyl vinyl ether adduct,” Journal of the Indian Chemical Society, vol. 76, pp. 368–369, 1999.

The crystal data of 7k had already been submitted to The Cambridge Crystallographic Data Centre (CCDC No. 885423)

D. Mitic culafic, B. Vukovic Gacic, J. Knezevic-Vukcevic, S. Stankovic, and D. Simic, “Comparative study on the antibacterial activity of volatiles from sage,” Archives of Biological Science, vol. 57, pp. 173–178, 2005.

J. Hindler, C. C. Mahon, and G. Manuselis, Eds., Textbook of Diagnostic Micro-Biology Special Antimicrobial Susceptibility Tests, 1995.

D. J. Payne, J. A. Hueso-Rodríguez, H. Boyd et al., “Identification of a series of tricyclic natural products as potent broad-spectrum inhibitors of metallo-β-lactamases,” Antimicrobial Agents and Chemotherapy, vol. 46, no. 6, pp. 1880–1886, 2002.

H. Haraguchi, K. Tanimoto, Y. Tamura, K. Mizutani, and T. Kinoshita, “Mode of antibacterial action of retrochalcones from Glycyrrhiza inflata,” Phytochemistry, vol. 48, no. 1, pp. 125–129, 1998.

M. Kawase, T. Tanaka, H. Kan, S. Tani, H. Nakashima, and H. Sakagami, “Biological activity of 3-formylchromones and related compound,” In Vivo, vol. 21, no. 5, pp. 829–834, 2007.