# Regioselective Synthesis of Dispiro Pyrrolizidines as Potent Antimicrobial Agents for Human Pathogens

Authors: N. Sirisha; R. Raghunathan
Journal: ISRN Medicinal Chemistry
 (2013)

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## Abstract

Synthesis of a series of novel dispiro pyrrolizidines has been accomplished by 1,3-dipolar cycloaddition reaction of azomethine ylide generated from secondary amino acids and  diketones with  bischalcones. These compounds were evaluated for their antibacterial activity. Most of the synthetic compounds exhibited good antibacterial activity against microorganisms.


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## Body

## 1. Introduction

1,3-dipolar cycloaddition is one of the important tools for the construction of five membered heterocycles [1], and many important natural products have been synthesized by this method [2, 3]. Pyrrolidine-based natural products are very useful in preventing and treating rheumatoid arthritis, asthma, and allergies and also possess anti-influenza virus and anticonvulsant activities [4]. The azomethine ylide represents one of the most reactive and versatile classes of 1,3-dipoles and is readily trapped by a range of dipolarophiles forming substituted pyrrolidines [5]. Spiro compounds represent an important class of naturally occurring substances characterized by highly pronounced biological properties [6, 7].Chalcones belong to the broad class of compounds present in almost all vascular plants, not only in their terrestrial parts but also in roots, as well as in flakes and seeds. Chalcones are precursors of flavonoids and play a crucial role in their biosynthesis [8, 9]. Depending on the substitution pattern on the two aromatic rings, a wide range of pharmacological activities has been identified for various chalcones [10, 11]. These include among others cytotoxic, antiprotozoal [12], antibacterial, antifungal [13], and antitumor activities [14, 15]. More recently, there has been strong interest in the antimalarial activity of chalcones and bischalcones [16].As a part of the ongoing research program on the synthesis of complex novel spiro heterocycles [17], herein we report for the first time an expeditious protocol for the synthesis of novel dispiro pyrrolizidines through 1,3-dipolar cycloaddition reaction of azomethine ylide generated from various diketones and secondary amino acids with bischalcones derived from terephthalaldehyde and substituted acetophenones as dipolarophiles. The synthesized compounds were screened for antimicrobial activity, and the results are presented in this paper.


## 2. Materials and Methods

### 2.1. General Procedure for the Synthesis of Bischalcones3a–c

A solution of substituted acetophenone (2 equiv) and terephthalaldehyde (1 equiv) in methanolic solution of NaOH (60%) was stirred for 20 h at room temperature. The solution was poured into ice-cold water at pH 2 (pH adjusted by HCl). The solid separated was dissolved in CH2Cl2, washed with saturated solution of NaHCO3, and evaporated to dryness. The residue was purified by column chromatography using hexane, ethyl acetate mixture (9 : 1) as eluent. The compound was recrystallized in chloroform.(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-phenylprop-2-en-1-one) (
3a
). Pale yellow solid, yield 81%; m.p. 158°C; IR, 1653, 1610, 1541. 1H NMR (CDCl3, 300 MHz): δ 2.18 (s, 6H), 6.83 (d, J = 16 Hz, 2H), 7.24–7.51 (m, 8H), 7.75 (d, 4H), 8.02 (d, J = 16 Hz, 2H); 13C NMR (75 MHz); ppm 122.51, 128.08, 129.32, 131.14, 136.78, 138.45, 142.82, 192.37. MS (EI); m/z = 339.1. Anal. Calc. for C24H18O2:  C, 85.17; H. 5.3; found: C. 85.5; H 5.73.(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-p-tolylprop-2-en-1-one) (
3b
). Pale yellow solid, yield 75%; m.p. 178°C; IR, 1653, 1610, 1541. 1H NMR (CDCl3, 300 MHz): δ 6.9 (d, J = 15 Hz, 2H), 7.25–7.49 (m, 10H), 7.6 (d, 4H), 7.92 (d, J = 15 Hz, 2H); 13C NMR (75 MHz); ppm 31.22, 123.02, 128.51, 128.68, 132.94, 136.86, 138.05, 143.53, 194.4.  MS (EI); m/z = 367.4. C26H22O2: C, 85.22; H, 6.05; found: C. 85.41; H 5.94.(2E,2′E)-3,3′-(1,4-phenylene)bis(1-(2-hydroxyphenyl)prop-2-en-1-one) (
3c
). Pale yellow solid, yield 83%; m.p. 190°C; IR, 1653, 1610, 1541. 1H NMR (CDCl3, 300 MHz): δ 6.72 (d, J = 14 Hz, 2H), 7.15–7.32 (m, 8H), 7.5 (d, 4H), 7.89 (d, J = 14 Hz, 2H), 11.68 (s, 2H); 13C NMR (75 MHz); ppm 124.21, 127.34, 128.61, 129.52, 133–91, 138.11, 139.17, 144.92, 196.35. MS (EI); m/z = 371.4. Anal. Calc. for C24H18O4: C, 77.82; H, 4.90; found: C. 77.95; H 5.01.


### 2.2. General Procedure for Synthesis of Cycloadducts,6a–c and 8a–c

To a solution of acenaphthenequinone5/isatin 7 (2 eq), proline 4 (3 eq) in dry toluene (15 mL) bischalcones 3a–c (1eq) was added. The solution was refluxed until the completion of the reaction (5–7 h) as evidenced by TLC analysis. The solvent was removed in vacuo, and the crude product was subjected to column chromatography on silica gel (100–200 mesh) using petroleum ether/ethyl acetate (8 : 2) as eluent.(1R,1′R,2′R,7a′S)-2′-Benzoyl-1′-(4-((1S,1′S,2′S,7a′R)-2′-benzoyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizin]-2-one (
6a
). Yellow solid, yield: 66%. m.p: 172°C. IR (KBr): 1724, 1649 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.73–1.92 (m, 4H), 1.96 (m, 2H), 2.38 (m, 2H), 2.63 (m, 2H), 3.93 (m, 2H), 4.24 (m, 2H), 4.94 (d, 2H J = 11.4 Hz), 5.25 (m, 2H), 6.73–7.83 (m, 26H). 13C NMR (75 MHz); ppm 26.87, 30.39, 48.80, 53.09, 64.52, 72.13, 77.13. 121.73, 124.32, 125.12, 127.28, 127.39, 127.71, 127.87, 128.16, 128.52, 128.67, 130.30, 131.49, 132.19, 134.88, 137.07, 138.62, 141.75, 198.33, 206.41. MS (EI); m/z = 809.5 (M+). Anal. Calcd for: C56H44N2O4: C, 83.14; H, 5.48; N, 3.46; found; C, 83.23; H, 5.43; N, 3.50.(1R,1′R,2′R,7a′S)-2′-(4-Methylbenzoyl)-1′-(4-((1S,1′S,2′S,7a′R)-2′-(4-methylbenzoyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizin]-2-one (
6b
). Yellow solid, yield: 69%. m.p: 158°C.  IR (KBr): 1737, 1657 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.72–1.79 (m, 4H), 1.82–1.88 (m, 2H), 1.96 (s, 6H), 2.38 (m, 2H), 2.65 (m, 2H),   3.94 (m, 2H), 4.19–4.26 (m, 2H),  4.91 (d, 2H J = 11.10 Hz), 5.22 (m, 2H), 6.56–7.84 (m, 24H). 13C NMR (75 MHz); ppm 20.20, 25.77, 29.27, 47.80, 52.19, 63.18, 71.02, 76.44, 120.68, 123.35, 124.02, 126.56, 126.85, 127.41, 127.45, 129.32, 130.43, 130.51, 133.63, 133.95, 137.60, 140.79, 141.90, 196.86, 205.33. MS (EI); m/z = 837.3 (M+). Anal. Calcd for: C58H48N2O4: C, 83.23; H, 5.78; N, 3.35; found; C, 83.34; H, 5.69; N, 3.29.(1R,1′R,2′R,7a′S)-2′-(2-Hydroxybenzoyl)-1′-(4-((1S,1′S,2′S,7a′R)-2′-(2-hydroxybenzoyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizin]-2-one (
6c
). Yellow solid, yield: 71%. m.p: 134°C.  IR (KBr): 3038, 1727, 1642  cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.73–1.91 (m, 4H), 1.94–2.01 (m, 2H), 2.27 (m, 2H), 2.54 (m, 2H), 3.79 (m, 2H), 4.08 (m, 2H), 4.82 (d, J = 11.4 Hz, 2H), 5.19 (m, 2H), 6.59–7.83 (m, 24H). 12.17 (s, 2H). 13C NMR (75 MHz); ppm 26.09, 29.38, 48.10, 53.37, 63.98, 71.69, 76.22, 119.92, 120.34, 121.79, 121.98, 122.37, 123.78, 125.47, 126.76, 128.98, 129.15, 131.18, 132.75, 136.20, 140.81, 141.82, 197.18, 205.43  MS (EI); m/z = 841.2 (M+). Anal. Calcd for: C56H44N2O6: C, 79.98; H, 5.27; N, 3.33; O, 11.42; found; C, 80.09; H, 5.31; N, 3.24.(2′R,3R)-2′-Benzoyl-1′-(4-((2′S,3S)-2′-benzoyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (
8a
). Yellow solid, yield: 61%. m.p: 152°C. IR (KBr): 1712, 1644 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.49–1.57 (m, 4H), 1.70–1.77 (m, 2H), 1.89 (m, 2H), 2.01 (m, 2H), 2.19 (m, 2H), 2.54 (m, 2H), 3.55 (d, J = 11.10 Hz, 2H), 4.18 (m, 2H), 6.41–7.66 (m, 22H), 8.10 (s, 2H). 13C NMR (75 MHz); ppm 25.12, 26.39, 31.41, 42.03, 52.75, 62.19, 71.17, 120.93, 122.76, 125.53, 126.92, 127.52, 128.81, 129.08, 129.93, 131.61, 136.90, 140.08, 141.17, 181.33, 199.81. MS (EI); m/z = 739.2 (M+). Anal. Calcd for: C48H42N2O4: C, 78.03; H, 5.73; N, 7.58; found; C, 78.12; H, 5.81; N, 7.42.(2′R,3R)-2′-(4-Methylbenzoyl)-1′-(4-((2′S,3S)-2′-(4-methylbenzoyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (
8b
). Yellow solid, yield: 64%. m.p: 192°C. IR (KBr): 1712, 1656 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.65–1.72 (m, 4H), 1.78 (m, 2H), 1.96 (m, 2H), 2.01 (S, 6H), 2.48 (m, 2H), 3.81 (m, 2H), 4.04 (m, 2H),  4.85 (d, J = 11.10 Hz, 2H), 5.18 (m, 2H), 6.52–7.41 (m, 20H), 8.32 (s, 2H). 13C NMR (75 MHz); ppm 24.12, 27.45, 30.02, 36.91, 45.52, 54.57, 62.38, 71.17, 118.37, 125.51, 126.37, 128.23, 128.91, 130.15, 130.79, 132.24, 135.47, 137.76, 179.82, 201.31. MS (EI); m/z = 767.6 (M+). Anal. Calcd for: C50H46N4O4: C, 78.30; H, 6.05; N, 7.31; found; C, 78.37; H, 6.11; N, 7.27.(2′R,3R)-2′-(2-Hydroxybenzoyl)-1′-(4-((2′S,3S)-2′-(2-hydroxybenzoyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (
8c
). Yellow solid, yield: 68%. m.p: 146°C. IR (KBr): 3052, 1739, 1619 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.67–1.78 (m, 4H), 1.78 (m, 2H), 1.86–1.92 (m, 2H), 2.22 (m, 2H), 2.53 (m, 2H), 2.95 (m, 2H), 3.82 (m, 2H),  4.49 (d, J = 11.10 Hz, 2H), 5.67 (m, 2H), 6.59–7.62 (m, 20H), 8.07 (s, 2H). 12.08 (s, 2H), 13C NMR (75 MHz); ppm 26.48, 29.44, 37.48, 49.97, 54.35, 67.72, 70.97, 118.38, 120.07, 121.37, 121.84, 122.58, 123.39, 125.17, 127.15, 128.81, 130.07, 131.18, 133.83, 136.62, 136.91, 178.82, 202.11. MS (EI); m/z = 771.4 (M+). Anal. Calcd for: C48H42N4O6: C, 74.79; H, 5.49; N, 7.27; found; C, 74.85; H, 5.41; N, 7.22.


### 2.3.  General Procedure for Synthesis of Cycloadducts,10a–c and 11a–c

To a solution of acenaphthenequinone5/isatin 7 (2 eq), pipecolinic acid 9 (3 eq) in dry toluene (15 mL) bischalcones 3a–c (1eq) was added. The solution was refluxed until the completion of the reaction (6–8 h) as evidenced by TLC analysis. The solvent was removed in vacuo, and the crude product was subjected to column chromatography on silica gel (100–200 mesh) using petroleum ether/ethyl acetate (8 : 2) as eluent.(1′R,2′R,8a′S)-2′-Benzoyl-1′-(4-((1S,1′S,2′S,8a′R)-2′-benzoyl-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizin]-2-one (
10a
). Yellow solid, yield: 72%. m.p: 138°C. IR (KBr): 1721, 1658 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.19–1.31 (m, 5H), 1.35–1.42 (m, 2H), 1.78 (m, 3H), 2.22 (m, 2H), 2.36 (m, 2H), 3.68 (m, 2H), 4.03 (m, 2H),  4.45 (d, J = 9.6 Hz, 2H), 4.77 (m, 2H), 6.96–8.27 (m, 26H). 13C NMR (75 MHz); ppm 23.74, 25.74, 30.66, 45.86, 52.00, 61.49, 66.07, 76.11, 120.50, 121.67, 122.08, 123.43, 124.56, 127.11, 127.39, 127.67, 128.50, 128.61, 128.77, 129.37, 131.68, 131.82, 132.02, 132.64, 132.69, 133.54, 136.85, 137.64, 138.33, 142.40, 143.81, 144.79, 198.00, 210.04. MS (EI); m/z = 837.9 (M+). Anal. Calcd for: C58H48N2O4: C, 83.23; H, 5.78; N, 3.35; found; C, 83.34; H, 5.67; N, 3.28.(1′R,2′R,8a′S)-2′-(4-Methylbenzoyl)-1′-(4-((1S,1′S,2′S,8a′R)-2′-(4-methylbenzoyl)-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizin]-2-one (
10b
). Yellow solid, yield: 65%. m.p: 120°C.  IR (KBr): 1739, 1657 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.25–1.51 (m, 5H), 1.73 (m, 3H), 1.85 (m, 2H), 1.97 (s, 6H), 2.25 (m, 2H), 2.42 (m, 2H),   3.67 (m, 2H), 4.12 (m, 2H),  4.43 (d, J = 9.3 Hz, 2H), 4.52 (m, 2H), 6.53–8.28 (m, 24H). 13C NMR (75 MHz); ppm 21.68, 23.75, 25.74, 29.67, 45.88, 52.11, 61.30, 66.07, 77.50, 120.47, 122.08, 123.48, 124.50, 126.53, 127.47, 128.12, 128.66, 129.32, 132.67, 134.49, 137.66, 142.85, 143.56, 197.62, 205.61. MS (EI); m/z = 865.6 (M+). Anal. Calcd for: C60H52N2O4: C, 83.30; H, 6.06; N, 3.24; found; C, 83.37; H, 6.11; N, 3.16.(1′R,2′R,8a′S)-2′-(2-Hydroxybenzoyl)-1′-(4-((1S,1′S,2′S,8a′R)-2′-(2-hydroxybenzoyl)-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizin]-2-one (
10c
). Yellow solid, yield: 70%. m.p: 134°C.  IR (KBr): 3021, 1729, 1662 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.25–1.47 (m, 5H), 1.70 (m, 2H), 1.79 (m, 3H), 2.20 (m, 2H), 2.34 (m, 2H),   3.65 (m, 2H), 3.99 (m, 2H),  4.43 (d, J = 9.0 Hz, 2H), 4.58 (m, 2H), 5.92–8.29 (m, 24H), 11.85 (s, 2H). 13C NMR (75 MHz); ppm 23.70, 29.36, 29.70, 46.00, 51.39, 60.63, 66.42, 77.25, 117.55, 118.85, 122.08, 124.62, 127.74, 128.58, 129.76, 131.01, 131.98, 132.66, 133.44, 135.35, 145.85, 193.32, 206.12. MS (EI); m/z = 869.4 (M+). Anal. Calcd for: C58H48N2O6: C, 80.16; H, 5.57; N, 3.22; found; C, 80.27; H, 5.49; N, 3.19.(1′R,2′R,3R,8a′S)-2′-Benzoyl-1′-(4-((1′S,2′S,3S,8a′R)-2′-benzoyl-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizin]-2-one (
11a
). Yellow solid, yield: 65%. m.p: 198°C.  IR (KBr):  1718, 1639 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.18–1.36 (m, 4H), 1.42 (m, 3H), 1.81 (m, 4H), 2.26 (m, 2H), 2.34 (m, 2H),   3.37 (m, 2H), 3.75 (m, 2H),  4.33 (d, J = 9.9 Hz, 2H), 4.57 (m, 2H), 6.75–7.86 (m, 22H), 8.42 (s, 2H). 13C NMR (75 MHz); ppm 24.48, 26.38, 30.07, 45.48, 51.97, 59.37, 65.78, 71.37, 120.39, 121.44, 124.39, 125.78, 127.01, 127.67, 128.18, 128.97, 129.58, 132.78, 133.86, 137.15, 139.39, 141.15, 178.93, 197.39. MS (EI); m/z = 767.4 (M+). Anal. Calcd for: C50H46N4O4: C, 78.30; H, 6.05; N, 7.31; found; C, 78.43; H, 6.01; N, 7.26.(1′R,2′R,3R,8a′S)-2′-(4-Methylbenzoyl)-1′-(4-((1′S,2′S,3S,8a′R)-2′-(4-methylbenzoyl)-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizin]-2-one (
11b
). Yellow solid, yield: 67%. m.p: 194°C.  IR (KBr): 1721, 1646 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.18–1.38 (m, 3H), 1.51 (m, 3H), 1.88 (m, 2H), 1.92 (s, 6H), 2.09 (m, 2H), 2.32 (m, 2H),   3.22 (m, 2H), 3.61 (m, 2H), 4.09 (m, 2H),  4.40 (d, J = 9.9 Hz, 2H), 4.59 (m, 2H), 6.39–7.85 (m, 20H), 8.30 (s, 2H). 13C NMR (75 MHz); ppm 22.63, 25.90, 29.47, 30.91, 44.61, 50.44, 59.69, 64.33, 71.49, 107.77, 121.74, 123.36, 125.76, 126.70, 127.11, 127.11, 127.65, 128.34, 129.91, 133.96, 137.65, 139.65, 142.11, 179.86, 196.12. MS (EI); m/z = 795.5 (M+). Anal. Calcd for: C52H50N4O4: C, 78.56; H, 6.34; N, 7.05; found; C, 78.62; H, 6.28; N, 7.13.(1′R,2′R,3R,8a′S)-2′-(2-Hydroxybenzoyl)-1′-(4-((1′S,2′S,3S,8a′R)-2′-(2-hydroxybenzoyl)-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizin]-2-one (
11c
). Yellow solid, yield: 66%. m.p: 186°C.  IR (KBr): 3037, 1718, 1651 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.27–1.42 (m, 4H), 1.56 (m, 3H), 1.98 (m, 3H), 2.39 (m, 2H), 2.48 (m, 2H),  3.57 (m, 2H), 3.67 (m, 2H), 4.45 (d, J = 9.6 Hz, 2H), 4.76 (m, 2H), 6.57–7.89 (m, 20H), 8.39 (s, 2H), 11.91 (s, 2H). 13C NMR (75 MHz); ppm 23.60, 25.50, 30.52, 45.70, 51.36, 60.26, 65.19, 73.04, 118.62, 119.20, 119.79, 120.19, 123.13, 126.17, 127.30, 128.49, 129.24, 133.48, 136.42, 143.55, 144.82, 180.43, 202.91. MS (EI); m/z = 799.6 (M+). Anal. Calcd for: C50H46N4O6: C, 75.17; H, 5.80; N, 7.01; found; C, 75.24; H, 5.78; N, 7.08.


## 2.1. General Procedure for the Synthesis of Bischalcones3a–c

A solution of substituted acetophenone (2 equiv) and terephthalaldehyde (1 equiv) in methanolic solution of NaOH (60%) was stirred for 20 h at room temperature. The solution was poured into ice-cold water at pH 2 (pH adjusted by HCl). The solid separated was dissolved in CH2Cl2, washed with saturated solution of NaHCO3, and evaporated to dryness. The residue was purified by column chromatography using hexane, ethyl acetate mixture (9 : 1) as eluent. The compound was recrystallized in chloroform.(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-phenylprop-2-en-1-one) (
3a
). Pale yellow solid, yield 81%; m.p. 158°C; IR, 1653, 1610, 1541. 1H NMR (CDCl3, 300 MHz): δ 2.18 (s, 6H), 6.83 (d, J = 16 Hz, 2H), 7.24–7.51 (m, 8H), 7.75 (d, 4H), 8.02 (d, J = 16 Hz, 2H); 13C NMR (75 MHz); ppm 122.51, 128.08, 129.32, 131.14, 136.78, 138.45, 142.82, 192.37. MS (EI); m/z = 339.1. Anal. Calc. for C24H18O2:  C, 85.17; H. 5.3; found: C. 85.5; H 5.73.(2E,2′E)-3,3′-(1,4-Phenylene)bis(1-p-tolylprop-2-en-1-one) (
3b
). Pale yellow solid, yield 75%; m.p. 178°C; IR, 1653, 1610, 1541. 1H NMR (CDCl3, 300 MHz): δ 6.9 (d, J = 15 Hz, 2H), 7.25–7.49 (m, 10H), 7.6 (d, 4H), 7.92 (d, J = 15 Hz, 2H); 13C NMR (75 MHz); ppm 31.22, 123.02, 128.51, 128.68, 132.94, 136.86, 138.05, 143.53, 194.4.  MS (EI); m/z = 367.4. C26H22O2: C, 85.22; H, 6.05; found: C. 85.41; H 5.94.(2E,2′E)-3,3′-(1,4-phenylene)bis(1-(2-hydroxyphenyl)prop-2-en-1-one) (
3c
). Pale yellow solid, yield 83%; m.p. 190°C; IR, 1653, 1610, 1541. 1H NMR (CDCl3, 300 MHz): δ 6.72 (d, J = 14 Hz, 2H), 7.15–7.32 (m, 8H), 7.5 (d, 4H), 7.89 (d, J = 14 Hz, 2H), 11.68 (s, 2H); 13C NMR (75 MHz); ppm 124.21, 127.34, 128.61, 129.52, 133–91, 138.11, 139.17, 144.92, 196.35. MS (EI); m/z = 371.4. Anal. Calc. for C24H18O4: C, 77.82; H, 4.90; found: C. 77.95; H 5.01.


## 2.2. General Procedure for Synthesis of Cycloadducts,6a–c and 8a–c

To a solution of acenaphthenequinone5/isatin 7 (2 eq), proline 4 (3 eq) in dry toluene (15 mL) bischalcones 3a–c (1eq) was added. The solution was refluxed until the completion of the reaction (5–7 h) as evidenced by TLC analysis. The solvent was removed in vacuo, and the crude product was subjected to column chromatography on silica gel (100–200 mesh) using petroleum ether/ethyl acetate (8 : 2) as eluent.(1R,1′R,2′R,7a′S)-2′-Benzoyl-1′-(4-((1S,1′S,2′S,7a′R)-2′-benzoyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizin]-2-one (
6a
). Yellow solid, yield: 66%. m.p: 172°C. IR (KBr): 1724, 1649 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.73–1.92 (m, 4H), 1.96 (m, 2H), 2.38 (m, 2H), 2.63 (m, 2H), 3.93 (m, 2H), 4.24 (m, 2H), 4.94 (d, 2H J = 11.4 Hz), 5.25 (m, 2H), 6.73–7.83 (m, 26H). 13C NMR (75 MHz); ppm 26.87, 30.39, 48.80, 53.09, 64.52, 72.13, 77.13. 121.73, 124.32, 125.12, 127.28, 127.39, 127.71, 127.87, 128.16, 128.52, 128.67, 130.30, 131.49, 132.19, 134.88, 137.07, 138.62, 141.75, 198.33, 206.41. MS (EI); m/z = 809.5 (M+). Anal. Calcd for: C56H44N2O4: C, 83.14; H, 5.48; N, 3.46; found; C, 83.23; H, 5.43; N, 3.50.(1R,1′R,2′R,7a′S)-2′-(4-Methylbenzoyl)-1′-(4-((1S,1′S,2′S,7a′R)-2′-(4-methylbenzoyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizin]-2-one (
6b
). Yellow solid, yield: 69%. m.p: 158°C.  IR (KBr): 1737, 1657 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.72–1.79 (m, 4H), 1.82–1.88 (m, 2H), 1.96 (s, 6H), 2.38 (m, 2H), 2.65 (m, 2H),   3.94 (m, 2H), 4.19–4.26 (m, 2H),  4.91 (d, 2H J = 11.10 Hz), 5.22 (m, 2H), 6.56–7.84 (m, 24H). 13C NMR (75 MHz); ppm 20.20, 25.77, 29.27, 47.80, 52.19, 63.18, 71.02, 76.44, 120.68, 123.35, 124.02, 126.56, 126.85, 127.41, 127.45, 129.32, 130.43, 130.51, 133.63, 133.95, 137.60, 140.79, 141.90, 196.86, 205.33. MS (EI); m/z = 837.3 (M+). Anal. Calcd for: C58H48N2O4: C, 83.23; H, 5.78; N, 3.35; found; C, 83.34; H, 5.69; N, 3.29.(1R,1′R,2′R,7a′S)-2′-(2-Hydroxybenzoyl)-1′-(4-((1S,1′S,2′S,7a′R)-2′-(2-hydroxybenzoyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydro-2H-spiro[acenaphthylene-1,3′-pyrrolizin]-2-one (
6c
). Yellow solid, yield: 71%. m.p: 134°C.  IR (KBr): 3038, 1727, 1642  cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.73–1.91 (m, 4H), 1.94–2.01 (m, 2H), 2.27 (m, 2H), 2.54 (m, 2H), 3.79 (m, 2H), 4.08 (m, 2H), 4.82 (d, J = 11.4 Hz, 2H), 5.19 (m, 2H), 6.59–7.83 (m, 24H). 12.17 (s, 2H). 13C NMR (75 MHz); ppm 26.09, 29.38, 48.10, 53.37, 63.98, 71.69, 76.22, 119.92, 120.34, 121.79, 121.98, 122.37, 123.78, 125.47, 126.76, 128.98, 129.15, 131.18, 132.75, 136.20, 140.81, 141.82, 197.18, 205.43  MS (EI); m/z = 841.2 (M+). Anal. Calcd for: C56H44N2O6: C, 79.98; H, 5.27; N, 3.33; O, 11.42; found; C, 80.09; H, 5.31; N, 3.24.(2′R,3R)-2′-Benzoyl-1′-(4-((2′S,3S)-2′-benzoyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (
8a
). Yellow solid, yield: 61%. m.p: 152°C. IR (KBr): 1712, 1644 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.49–1.57 (m, 4H), 1.70–1.77 (m, 2H), 1.89 (m, 2H), 2.01 (m, 2H), 2.19 (m, 2H), 2.54 (m, 2H), 3.55 (d, J = 11.10 Hz, 2H), 4.18 (m, 2H), 6.41–7.66 (m, 22H), 8.10 (s, 2H). 13C NMR (75 MHz); ppm 25.12, 26.39, 31.41, 42.03, 52.75, 62.19, 71.17, 120.93, 122.76, 125.53, 126.92, 127.52, 128.81, 129.08, 129.93, 131.61, 136.90, 140.08, 141.17, 181.33, 199.81. MS (EI); m/z = 739.2 (M+). Anal. Calcd for: C48H42N2O4: C, 78.03; H, 5.73; N, 7.58; found; C, 78.12; H, 5.81; N, 7.42.(2′R,3R)-2′-(4-Methylbenzoyl)-1′-(4-((2′S,3S)-2′-(4-methylbenzoyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (
8b
). Yellow solid, yield: 64%. m.p: 192°C. IR (KBr): 1712, 1656 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.65–1.72 (m, 4H), 1.78 (m, 2H), 1.96 (m, 2H), 2.01 (S, 6H), 2.48 (m, 2H), 3.81 (m, 2H), 4.04 (m, 2H),  4.85 (d, J = 11.10 Hz, 2H), 5.18 (m, 2H), 6.52–7.41 (m, 20H), 8.32 (s, 2H). 13C NMR (75 MHz); ppm 24.12, 27.45, 30.02, 36.91, 45.52, 54.57, 62.38, 71.17, 118.37, 125.51, 126.37, 128.23, 128.91, 130.15, 130.79, 132.24, 135.47, 137.76, 179.82, 201.31. MS (EI); m/z = 767.6 (M+). Anal. Calcd for: C50H46N4O4: C, 78.30; H, 6.05; N, 7.31; found; C, 78.37; H, 6.11; N, 7.27.(2′R,3R)-2′-(2-Hydroxybenzoyl)-1′-(4-((2′S,3S)-2′-(2-hydroxybenzoyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-yl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (
8c
). Yellow solid, yield: 68%. m.p: 146°C. IR (KBr): 3052, 1739, 1619 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.67–1.78 (m, 4H), 1.78 (m, 2H), 1.86–1.92 (m, 2H), 2.22 (m, 2H), 2.53 (m, 2H), 2.95 (m, 2H), 3.82 (m, 2H),  4.49 (d, J = 11.10 Hz, 2H), 5.67 (m, 2H), 6.59–7.62 (m, 20H), 8.07 (s, 2H). 12.08 (s, 2H), 13C NMR (75 MHz); ppm 26.48, 29.44, 37.48, 49.97, 54.35, 67.72, 70.97, 118.38, 120.07, 121.37, 121.84, 122.58, 123.39, 125.17, 127.15, 128.81, 130.07, 131.18, 133.83, 136.62, 136.91, 178.82, 202.11. MS (EI); m/z = 771.4 (M+). Anal. Calcd for: C48H42N4O6: C, 74.79; H, 5.49; N, 7.27; found; C, 74.85; H, 5.41; N, 7.22.


## 2.3.  General Procedure for Synthesis of Cycloadducts,10a–c and 11a–c

To a solution of acenaphthenequinone5/isatin 7 (2 eq), pipecolinic acid 9 (3 eq) in dry toluene (15 mL) bischalcones 3a–c (1eq) was added. The solution was refluxed until the completion of the reaction (6–8 h) as evidenced by TLC analysis. The solvent was removed in vacuo, and the crude product was subjected to column chromatography on silica gel (100–200 mesh) using petroleum ether/ethyl acetate (8 : 2) as eluent.(1′R,2′R,8a′S)-2′-Benzoyl-1′-(4-((1S,1′S,2′S,8a′R)-2′-benzoyl-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizin]-2-one (
10a
). Yellow solid, yield: 72%. m.p: 138°C. IR (KBr): 1721, 1658 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.19–1.31 (m, 5H), 1.35–1.42 (m, 2H), 1.78 (m, 3H), 2.22 (m, 2H), 2.36 (m, 2H), 3.68 (m, 2H), 4.03 (m, 2H),  4.45 (d, J = 9.6 Hz, 2H), 4.77 (m, 2H), 6.96–8.27 (m, 26H). 13C NMR (75 MHz); ppm 23.74, 25.74, 30.66, 45.86, 52.00, 61.49, 66.07, 76.11, 120.50, 121.67, 122.08, 123.43, 124.56, 127.11, 127.39, 127.67, 128.50, 128.61, 128.77, 129.37, 131.68, 131.82, 132.02, 132.64, 132.69, 133.54, 136.85, 137.64, 138.33, 142.40, 143.81, 144.79, 198.00, 210.04. MS (EI); m/z = 837.9 (M+). Anal. Calcd for: C58H48N2O4: C, 83.23; H, 5.78; N, 3.35; found; C, 83.34; H, 5.67; N, 3.28.(1′R,2′R,8a′S)-2′-(4-Methylbenzoyl)-1′-(4-((1S,1′S,2′S,8a′R)-2′-(4-methylbenzoyl)-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizin]-2-one (
10b
). Yellow solid, yield: 65%. m.p: 120°C.  IR (KBr): 1739, 1657 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.25–1.51 (m, 5H), 1.73 (m, 3H), 1.85 (m, 2H), 1.97 (s, 6H), 2.25 (m, 2H), 2.42 (m, 2H),   3.67 (m, 2H), 4.12 (m, 2H),  4.43 (d, J = 9.3 Hz, 2H), 4.52 (m, 2H), 6.53–8.28 (m, 24H). 13C NMR (75 MHz); ppm 21.68, 23.75, 25.74, 29.67, 45.88, 52.11, 61.30, 66.07, 77.50, 120.47, 122.08, 123.48, 124.50, 126.53, 127.47, 128.12, 128.66, 129.32, 132.67, 134.49, 137.66, 142.85, 143.56, 197.62, 205.61. MS (EI); m/z = 865.6 (M+). Anal. Calcd for: C60H52N2O4: C, 83.30; H, 6.06; N, 3.24; found; C, 83.37; H, 6.11; N, 3.16.(1′R,2′R,8a′S)-2′-(2-Hydroxybenzoyl)-1′-(4-((1S,1′S,2′S,8a′R)-2′-(2-hydroxybenzoyl)-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H,2H-spiro[acenaphthylene-1,3′-indolizin]-2-one (
10c
). Yellow solid, yield: 70%. m.p: 134°C.  IR (KBr): 3021, 1729, 1662 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.25–1.47 (m, 5H), 1.70 (m, 2H), 1.79 (m, 3H), 2.20 (m, 2H), 2.34 (m, 2H),   3.65 (m, 2H), 3.99 (m, 2H),  4.43 (d, J = 9.0 Hz, 2H), 4.58 (m, 2H), 5.92–8.29 (m, 24H), 11.85 (s, 2H). 13C NMR (75 MHz); ppm 23.70, 29.36, 29.70, 46.00, 51.39, 60.63, 66.42, 77.25, 117.55, 118.85, 122.08, 124.62, 127.74, 128.58, 129.76, 131.01, 131.98, 132.66, 133.44, 135.35, 145.85, 193.32, 206.12. MS (EI); m/z = 869.4 (M+). Anal. Calcd for: C58H48N2O6: C, 80.16; H, 5.57; N, 3.22; found; C, 80.27; H, 5.49; N, 3.19.(1′R,2′R,3R,8a′S)-2′-Benzoyl-1′-(4-((1′S,2′S,3S,8a′R)-2′-benzoyl-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizin]-2-one (
11a
). Yellow solid, yield: 65%. m.p: 198°C.  IR (KBr):  1718, 1639 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.18–1.36 (m, 4H), 1.42 (m, 3H), 1.81 (m, 4H), 2.26 (m, 2H), 2.34 (m, 2H),   3.37 (m, 2H), 3.75 (m, 2H),  4.33 (d, J = 9.9 Hz, 2H), 4.57 (m, 2H), 6.75–7.86 (m, 22H), 8.42 (s, 2H). 13C NMR (75 MHz); ppm 24.48, 26.38, 30.07, 45.48, 51.97, 59.37, 65.78, 71.37, 120.39, 121.44, 124.39, 125.78, 127.01, 127.67, 128.18, 128.97, 129.58, 132.78, 133.86, 137.15, 139.39, 141.15, 178.93, 197.39. MS (EI); m/z = 767.4 (M+). Anal. Calcd for: C50H46N4O4: C, 78.30; H, 6.05; N, 7.31; found; C, 78.43; H, 6.01; N, 7.26.(1′R,2′R,3R,8a′S)-2′-(4-Methylbenzoyl)-1′-(4-((1′S,2′S,3S,8a′R)-2′-(4-methylbenzoyl)-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizin]-2-one (
11b
). Yellow solid, yield: 67%. m.p: 194°C.  IR (KBr): 1721, 1646 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.18–1.38 (m, 3H), 1.51 (m, 3H), 1.88 (m, 2H), 1.92 (s, 6H), 2.09 (m, 2H), 2.32 (m, 2H),   3.22 (m, 2H), 3.61 (m, 2H), 4.09 (m, 2H),  4.40 (d, J = 9.9 Hz, 2H), 4.59 (m, 2H), 6.39–7.85 (m, 20H), 8.30 (s, 2H). 13C NMR (75 MHz); ppm 22.63, 25.90, 29.47, 30.91, 44.61, 50.44, 59.69, 64.33, 71.49, 107.77, 121.74, 123.36, 125.76, 126.70, 127.11, 127.11, 127.65, 128.34, 129.91, 133.96, 137.65, 139.65, 142.11, 179.86, 196.12. MS (EI); m/z = 795.5 (M+). Anal. Calcd for: C52H50N4O4: C, 78.56; H, 6.34; N, 7.05; found; C, 78.62; H, 6.28; N, 7.13.(1′R,2′R,3R,8a′S)-2′-(2-Hydroxybenzoyl)-1′-(4-((1′S,2′S,3S,8a′R)-2′-(2-hydroxybenzoyl)-2-oxo-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizine]-1′-yl)phenyl)-2′,5′,6′,7′,8′,8a′-hexahydro-1′H-spiro[indoline-3,3′-indolizin]-2-one (
11c
). Yellow solid, yield: 66%. m.p: 186°C.  IR (KBr): 3037, 1718, 1651 cm−1; 1H NMR (CDCl3, 300 MHz): δ 1.27–1.42 (m, 4H), 1.56 (m, 3H), 1.98 (m, 3H), 2.39 (m, 2H), 2.48 (m, 2H),  3.57 (m, 2H), 3.67 (m, 2H), 4.45 (d, J = 9.6 Hz, 2H), 4.76 (m, 2H), 6.57–7.89 (m, 20H), 8.39 (s, 2H), 11.91 (s, 2H). 13C NMR (75 MHz); ppm 23.60, 25.50, 30.52, 45.70, 51.36, 60.26, 65.19, 73.04, 118.62, 119.20, 119.79, 120.19, 123.13, 126.17, 127.30, 128.49, 129.24, 133.48, 136.42, 143.55, 144.82, 180.43, 202.91. MS (EI); m/z = 799.6 (M+). Anal. Calcd for: C50H46N4O6: C, 75.17; H, 5.80; N, 7.01; found; C, 75.24; H, 5.78; N, 7.08.


## 3. Materials and Methods for Antimicrobial Studies

We have carried out bioactivity studies for the synthesized compounds against six microorganisms,Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella typhi, Proteus vulgaris, and Proteus mirabilis which were obtained from the Microbial Metabolite Lab Culture collection, University of Madras, Chennai.The agar diffusion method was used for the determination of antibacterial activity of dispiropyrrolizidines against microorganism listed above. About 9 mL of nutrient agar media were poured into petri plates (9 cm in diameter) and inoculated with respective test organism. Wells are made with cork borer on the solid agar and loaded with 20–60 µg/mL of the test compound with tetracycline as control. Petri dishes were incubated at 37°C for 24 h, and the average diameter of the inhibition zone surrounding the wells was measured after specified incubation period.


## 4. Results and Discussion

### 4.1. Chemistry

The bischalcones used as dipolarophiles for the present study were prepared according to the Claisen-Schmidt reaction, by reacting various substituted acetophenones1a–c with terephthalaldehyde 2 and NaOH (60%) as a base to give pale yellow products 3a–c (80–86%) (Scheme 1).Scheme 1The products3a–c were assigned the (E)-configuration based on the chemical shift value of olefinic protons in accordance with the literature data [18–22].We have explored the reactivity of bischalcone derivatives as efficient dipolarophiles for the synthesis of a rare class of dispiroheterocycles. The azomethine ylide generatedin situ through decarboxylative condensation reaction from L-proline 4 and acenaphthenequinone 5/isatin 7reacted with bischalcone derivatives 3a–c as dipolarophiles in refluxing toluene under the Dean-Stark reaction condition to afford a series of novel dispiro pyrrolizidines 6a–c and 8a–c (Scheme 2).Scheme 2The structures of these products were also confirmed by1H NMR, 13C NMR, DEPT, COSY, NOESY, and mass spectral analysis. The 1H NMR spectrum of 6b showed a multiplet at δ 5.22 for benzylic H2 proton. The benzoyl proton H1 resonated as a doublet at δ 4.91 (J = 11.10 Hz), and H3 proton appeared as a multiplet at δ 4.19–4.26. The stereochemistry of the cycloadduct 6b was deduced on the basis of 2D NOESY experiments. The strong NOESY between H2 and H3 shows cisstereochemistry, and when there is no NOESY between H1 and H2 of 6b shows transstereochemistry. The 13C NMR spectrum of 6b showed a signal at 76.44 ppm for the spiro carbon. The peaks at 52.19 ppm and 63.18 ppm correspond to the benzylic –CH– carbon and N–CH– carbon of 10b and were confirmed by DEPT 135 spectrum. Acenaphthenequinone carbonyl carbon appeared at 205.33 ppm, and the benzoyl carbon resonated at 196.86 ppm. Moreover, the cycloadduct 6b exhibited a peak at m/z = 837.3 (M+) in the mass spectrum.With a view to explore the potential of the cycloaddition reaction of 1,3-dipole for the synthesis of spiroheterocycles, we have carried out the reaction of the azomethine ylide generated from pipecolinic acid9and acenaphthenequinone 5/isatin 7with bischalcones  as dipolarophiles to give dispiro pyrrolizidines 10a–c and 11a–c (Scheme 3).Scheme 3The IR spectrum of the cycloadduct11c showed peaks at 1651 and 1718 cm−1 which correspond to the amide and benzoyl carbonyl groups, respectively. The 1H NMR spectrum of 11c showed a multiplet at δ 4.76 for benzylic H2 proton. The benzoyl proton H1 resonated as a doublet at δ 4.45 (J = 9.6 Hz), and H3 proton appeared as a multiplet at δ 3.95–4.01. The stereochemistry of the cycloadduct 11c was deduced on the basis of 2D NOESY experiments. The strong NOESY between H2 and H3 shows cisstereochemistry, and when there is no NOESY between H1 and H2 of 11c shows transstereochemistry. The –NH proton of the oxindole moiety appeared as a singlet at δ 8.39, and the –OH peak appeared as a singlet at δ 11.91. The 13C NMR spectrum of 11c showed a signal at 73.04 ppm for the spiro carbon. The N–CH carbon of 11c resonated at 60.26 ppm and was confirmed by DEPT 135 spectrum. The peaks at 202.91 and 180.43 ppm correspond to the benzoyl and amide carbonyl groups. Moreover, the cycloadduct 11cexhibited a peak at m/z = 799.6 (M+) in the mass spectrum.


## 4.1. Chemistry

The bischalcones used as dipolarophiles for the present study were prepared according to the Claisen-Schmidt reaction, by reacting various substituted acetophenones1a–c with terephthalaldehyde 2 and NaOH (60%) as a base to give pale yellow products 3a–c (80–86%) (Scheme 1).Scheme 1The products3a–c were assigned the (E)-configuration based on the chemical shift value of olefinic protons in accordance with the literature data [18–22].We have explored the reactivity of bischalcone derivatives as efficient dipolarophiles for the synthesis of a rare class of dispiroheterocycles. The azomethine ylide generatedin situ through decarboxylative condensation reaction from L-proline 4 and acenaphthenequinone 5/isatin 7reacted with bischalcone derivatives 3a–c as dipolarophiles in refluxing toluene under the Dean-Stark reaction condition to afford a series of novel dispiro pyrrolizidines 6a–c and 8a–c (Scheme 2).Scheme 2The structures of these products were also confirmed by1H NMR, 13C NMR, DEPT, COSY, NOESY, and mass spectral analysis. The 1H NMR spectrum of 6b showed a multiplet at δ 5.22 for benzylic H2 proton. The benzoyl proton H1 resonated as a doublet at δ 4.91 (J = 11.10 Hz), and H3 proton appeared as a multiplet at δ 4.19–4.26. The stereochemistry of the cycloadduct 6b was deduced on the basis of 2D NOESY experiments. The strong NOESY between H2 and H3 shows cisstereochemistry, and when there is no NOESY between H1 and H2 of 6b shows transstereochemistry. The 13C NMR spectrum of 6b showed a signal at 76.44 ppm for the spiro carbon. The peaks at 52.19 ppm and 63.18 ppm correspond to the benzylic –CH– carbon and N–CH– carbon of 10b and were confirmed by DEPT 135 spectrum. Acenaphthenequinone carbonyl carbon appeared at 205.33 ppm, and the benzoyl carbon resonated at 196.86 ppm. Moreover, the cycloadduct 6b exhibited a peak at m/z = 837.3 (M+) in the mass spectrum.With a view to explore the potential of the cycloaddition reaction of 1,3-dipole for the synthesis of spiroheterocycles, we have carried out the reaction of the azomethine ylide generated from pipecolinic acid9and acenaphthenequinone 5/isatin 7with bischalcones  as dipolarophiles to give dispiro pyrrolizidines 10a–c and 11a–c (Scheme 3).Scheme 3The IR spectrum of the cycloadduct11c showed peaks at 1651 and 1718 cm−1 which correspond to the amide and benzoyl carbonyl groups, respectively. The 1H NMR spectrum of 11c showed a multiplet at δ 4.76 for benzylic H2 proton. The benzoyl proton H1 resonated as a doublet at δ 4.45 (J = 9.6 Hz), and H3 proton appeared as a multiplet at δ 3.95–4.01. The stereochemistry of the cycloadduct 11c was deduced on the basis of 2D NOESY experiments. The strong NOESY between H2 and H3 shows cisstereochemistry, and when there is no NOESY between H1 and H2 of 11c shows transstereochemistry. The –NH proton of the oxindole moiety appeared as a singlet at δ 8.39, and the –OH peak appeared as a singlet at δ 11.91. The 13C NMR spectrum of 11c showed a signal at 73.04 ppm for the spiro carbon. The N–CH carbon of 11c resonated at 60.26 ppm and was confirmed by DEPT 135 spectrum. The peaks at 202.91 and 180.43 ppm correspond to the benzoyl and amide carbonyl groups. Moreover, the cycloadduct 11cexhibited a peak at m/z = 799.6 (M+) in the mass spectrum.


## 5. Biology

### 5.1. Antimicrobial Activity of Dispiropyrrolizidines (Agar Diffusion Assay)

The agar diffusion method [23] was used for the determination of antibacterial activity of dispiropyrrolizidines against microorganism listed above. The results of the antimicrobial screening of the twelve compounds have been listed in Tables 1, 2, 3, 4, 5, and 6.Table 1
Effect of dispiro pyrrolizidines on the growth of human pathogenProteus mirabilis.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Proteus mirabilis
6a
—
14
19
6b
—
13
18
6c
—
17
21
8a
—
10
18
8b
—
15
23
8c
11
19
22
10a
—
10
15
10b
—
—
9
10c
—
18
22
11a
—
11
18
11b
—
14
19
11c
12
18
23
Tetracycline
16
20
24Table 2
Effect of dispiro pyrrolizidines on the growth of human pathogenProteus vulgaris.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Proteus vulgaris
6a
—
12
18
6b
—
13
19
6c
—
15
21
8a
—
14
20
8b
—
13
21
8c
10
18
23
10a
—
9
13
10b
—
—
7
10c
—
19
23
11a
—
10
17
11b
9
14
19
11c
12
19
23
Tetracycline
18
22
25Table 3
Effect of dispiro pyrrolizidines on the growth of human pathogenSalmonella typhi.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Salmonella typhi
6a
—
11
17
6b
—
9
11
6c
—
15
21
8a
—
13
18
8b
—
12
19
8c
11
19
25
10a
—
9
12
10b
—
8
12
10c
—
16
21
11a
—
11
18
11b
—
14
19
11c
12
20
26
Tetracycline
17
21
26Table 4
Effect of dispiro pyrrolizidines on the growth of human pathogenStaphylococcus aureus.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Staphylococcus aureus
6a
—
15
19
6b
—
9
11
6c
—
17
21
8a
—
10
18
8b
—
15
22
8c
9
17
24
10a
—
10
15
10b
—
—
9
10c
—
18
23
11a
—
11
18
11b
—
14
19
11c
10
19
23
Tetracycline
18
21
24Table 5
Effect of dispiro pyrrolizidines on the growth of human pathogenBacillus subtilis.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Bacillus subtilis
6a
—
12
18
6b
—
9
14
6c
—
15
21
8a
—
10
18
8b
—
15
22
8c
9
17
21
10a
—
8
14
10b
—
—
13
10c
—
18
23
11a
—
11
18
11b
—
14
19
11c
9
19
22
Tetracycline
14
19
21Table 6
Effect of dispiro pyrrolizidines on the growth of human pathogenEscherichia coli.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Escherichia coli
6a
—
11
18
6b
—
7
15
6c
—
15
21
8a
—
15
22
8b
—
10
18
8c
9
18
22
10a
—
10
15
10b
—
—
9
10c
—
19
23
11a
—
14
19
11b
—
11
17
11c
11
19
23
Tetracycline
16
20
23It was observed that the synthetic compounds6c, 8b, 8c, 10c, and 11cexhibited good antimicrobial activity and inhibition zones against the pathogen Proteus mirabilis while 6a, 6b, 8a,10a,11a, and 11b showed moderate activity. All other derivatives did not show appreciable activity at low concentration. For P. vulgaris the compounds 11c,10c, and 8c showed very good antibacterial activity which is comparable to that of reference compound tetracycline, and6a, 6b,6c,8a,8b,11a, and 11bshowed moderate inhibitory activity against this pathogen. The compounds 11c and 8cshowed very good antimicrobial activity against the pathogen Salmonella typhi while 6c,10c,8b,8a,6a,11b, and 11a showed moderate activity. The compounds 8c, 11c,10c, and 8b exhibited good antibacterial activity, while 6c, 6a, 8a,11a, and 11bshowed moderate activity against the pathogenStaphylococcus aureus. All other compounds showed low activity. The inhibitory activity of the compounds 10c,11c,8b,8c, and 6c against the bacteria Bacillus subtilis was observed to be good while 6a,8a, 11a, and 11b showed moderate activity against this pathogen. The compounds 11c,10c,8c,6c, and 8ashowed good antimicrobial activity against the pathogenEscherichia coliwhile 6a,8b,11a, and 11b showed moderate activity, and all other compounds showed low activity.Some of the oxindole derived compounds and acenaphthenequinone derived compounds showed good activity against all the pathogens in low concentration, which is comparable to the reference control. It was observed that all the antibacterial activities of the presently studied compounds are dose dependent. The oxindole derived compounds11cand 8cshowed good activity against all the test pathogens. The activity was very much comparable to the reference drug. The acenaphthenequinone derived compounds 6c and 10c showed good activity against all the test pathogens. Hydroxy substituted dispiropyrrolizidines 11c,10c,8c, and 6c showed very good antibacterial activity against all the pathogens studied. Further clinical studies are required to validate the compounds of the present study as antimicrobial agents. The results are summarized in Tables 1, 2, 3, 4, 5, and 6.


## 5.1. Antimicrobial Activity of Dispiropyrrolizidines (Agar Diffusion Assay)

The agar diffusion method [23] was used for the determination of antibacterial activity of dispiropyrrolizidines against microorganism listed above. The results of the antimicrobial screening of the twelve compounds have been listed in Tables 1, 2, 3, 4, 5, and 6.Table 1
Effect of dispiro pyrrolizidines on the growth of human pathogenProteus mirabilis.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Proteus mirabilis
6a
—
14
19
6b
—
13
18
6c
—
17
21
8a
—
10
18
8b
—
15
23
8c
11
19
22
10a
—
10
15
10b
—
—
9
10c
—
18
22
11a
—
11
18
11b
—
14
19
11c
12
18
23
Tetracycline
16
20
24Table 2
Effect of dispiro pyrrolizidines on the growth of human pathogenProteus vulgaris.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Proteus vulgaris
6a
—
12
18
6b
—
13
19
6c
—
15
21
8a
—
14
20
8b
—
13
21
8c
10
18
23
10a
—
9
13
10b
—
—
7
10c
—
19
23
11a
—
10
17
11b
9
14
19
11c
12
19
23
Tetracycline
18
22
25Table 3
Effect of dispiro pyrrolizidines on the growth of human pathogenSalmonella typhi.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Salmonella typhi
6a
—
11
17
6b
—
9
11
6c
—
15
21
8a
—
13
18
8b
—
12
19
8c
11
19
25
10a
—
9
12
10b
—
8
12
10c
—
16
21
11a
—
11
18
11b
—
14
19
11c
12
20
26
Tetracycline
17
21
26Table 4
Effect of dispiro pyrrolizidines on the growth of human pathogenStaphylococcus aureus.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Staphylococcus aureus
6a
—
15
19
6b
—
9
11
6c
—
17
21
8a
—
10
18
8b
—
15
22
8c
9
17
24
10a
—
10
15
10b
—
—
9
10c
—
18
23
11a
—
11
18
11b
—
14
19
11c
10
19
23
Tetracycline
18
21
24Table 5
Effect of dispiro pyrrolizidines on the growth of human pathogenBacillus subtilis.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Bacillus subtilis
6a
—
12
18
6b
—
9
14
6c
—
15
21
8a
—
10
18
8b
—
15
22
8c
9
17
21
10a
—
8
14
10b
—
—
13
10c
—
18
23
11a
—
11
18
11b
—
14
19
11c
9
19
22
Tetracycline
14
19
21Table 6
Effect of dispiro pyrrolizidines on the growth of human pathogenEscherichia coli.
Organism
Compound
Concentration of compound (µg/mL)
20
40
60
Zone of inhibition (mm)
Escherichia coli
6a
—
11
18
6b
—
7
15
6c
—
15
21
8a
—
15
22
8b
—
10
18
8c
9
18
22
10a
—
10
15
10b
—
—
9
10c
—
19
23
11a
—
14
19
11b
—
11
17
11c
11
19
23
Tetracycline
16
20
23It was observed that the synthetic compounds6c, 8b, 8c, 10c, and 11cexhibited good antimicrobial activity and inhibition zones against the pathogen Proteus mirabilis while 6a, 6b, 8a,10a,11a, and 11b showed moderate activity. All other derivatives did not show appreciable activity at low concentration. For P. vulgaris the compounds 11c,10c, and 8c showed very good antibacterial activity which is comparable to that of reference compound tetracycline, and6a, 6b,6c,8a,8b,11a, and 11bshowed moderate inhibitory activity against this pathogen. The compounds 11c and 8cshowed very good antimicrobial activity against the pathogen Salmonella typhi while 6c,10c,8b,8a,6a,11b, and 11a showed moderate activity. The compounds 8c, 11c,10c, and 8b exhibited good antibacterial activity, while 6c, 6a, 8a,11a, and 11bshowed moderate activity against the pathogenStaphylococcus aureus. All other compounds showed low activity. The inhibitory activity of the compounds 10c,11c,8b,8c, and 6c against the bacteria Bacillus subtilis was observed to be good while 6a,8a, 11a, and 11b showed moderate activity against this pathogen. The compounds 11c,10c,8c,6c, and 8ashowed good antimicrobial activity against the pathogenEscherichia coliwhile 6a,8b,11a, and 11b showed moderate activity, and all other compounds showed low activity.Some of the oxindole derived compounds and acenaphthenequinone derived compounds showed good activity against all the pathogens in low concentration, which is comparable to the reference control. It was observed that all the antibacterial activities of the presently studied compounds are dose dependent. The oxindole derived compounds11cand 8cshowed good activity against all the test pathogens. The activity was very much comparable to the reference drug. The acenaphthenequinone derived compounds 6c and 10c showed good activity against all the test pathogens. Hydroxy substituted dispiropyrrolizidines 11c,10c,8c, and 6c showed very good antibacterial activity against all the pathogens studied. Further clinical studies are required to validate the compounds of the present study as antimicrobial agents. The results are summarized in Tables 1, 2, 3, 4, 5, and 6.


## 6. Conclusion

In conclusion, we have achieved the synthesis of a variety of novel dispiro pyrrolizidines through 1,3-dipolar cycloaddition reaction and evaluated their structure by using different spectroscopic techniques. Most of the synthesized compounds showed good antimicrobial activity against all the selected human pathogens. In particular, cycloadducts,11c,8c,10c, and 6c, exhibited the best antibacterial activity against all the bacterial pathogens.



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*Source: 492604-2013-09-10.xml*