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Article

Facial Regioselective Synthesis of Novel Bioactive Spiropyrrolidine/Pyrrolizine-Oxindole Derivatives via a Three Components Reaction as Potential Antimicrobial Agents

by
Huwaida M. E. Hassaneen
1,
Elshimaa M. Eid
1,
Hamid A. Eid
1,
Thoraya A. Farghaly
1,2,* and
Yahia Nasser Mabkhot
3,*
1
Chemistry Department, Faculty of Science, Cairo University, Giza 12613, Egypt
2
Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University, Makkah Almukkarramah 21514, Saudi Arabia
3
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh-11451, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Molecules 2017, 22(3), 357; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22030357
Submission received: 9 January 2017 / Revised: 19 February 2017 / Accepted: 24 February 2017 / Published: 26 February 2017
(This article belongs to the Collection Heterocyclic Compounds)
Browse Figures
Versions Notes

Abstract

:
This article presents the synthesis of new derivatives of spirooxindole-spiropiperidinone- pyrrolidines 6a–j and spirooxindole-spiropiperidinone-pyrrolizines 8a–j, through a 1,3-dipolar cycloaddition reaction of azomethineylides generated from isatin, sarcosine, and l-proline, through a decarboxylative route with dipolarophile 4a–j. All of the newly synthesized compounds were evaluated for their antimicrobial activities and their minimum inhibitory concentration (MIC) against most of the test organisms. The tested compounds displayed excellent activity against all of the tested microorganisms.

1. Introduction

In recent years, multicomponent reactions (MCR’s) [1,2,3] leading to interesting heterocyclic scaffolds have emerged as powerful tools for the diverse needs in combinatorial approaches for the synthesis of bioactive compounds, creating distinct chemical libraries of drug-like molecules for biological screening [4,5]. Spiro compounds represent an important class of naturally occurring substances with highly pronounced biological properties [6,7]. Spirooxindole ring systems are found in a number of alkaloids, such as horsifiline, spirotryprostain, and (+) elacomine [8], and are used in biological applications as antimicrobial and antitumor agents and as inhibitors of the human NKI receptor [9]. Additionally, spirooxindole rings containing the pyrrolidine and pyrrolizine ring system are found in various natural products as fundamental nuclei and are well recognized for exhibiting a wide range of pharmacological and biochemical behaviors [10,11,12]. The 1,3-cycloaddition methodology is one of the simplest tools for the construction of five-membered heterocycles. The ease of generating a 1,3-dipolar, coupled with the observed highly regio- and stereoselective nature of cycloaddition, has led to a number of syntheses that utilize such a reaction [13]. Combining several transformations into a one-pot reaction has proved to be an excellent strategy for increasing the efficiency of organic synthesis [14]. In particular, azomethineylides have been used to synthesize pyrrolidines and pyrrolizines with various substitutions, allowing the introduction of several functional groups in a single operation [15]. As part of our own interest in the synthesis of biologically active heterocyclic compounds [16,17,18,19,20,21,22,23], we report an efficient methodology for the synthesis of novel spirooxindole-spiropiperidinone-pyrrolidine and spirooxindole-spiropiperidinone-pyrrolizine derivatives, and investigate their antimicrobial activities.

2. Results and Discussion

2.1. Chemistry

In our initial endeavor, the Knoevenagel adduct 4 was prepared via condensation of 1-ethoxycarbonyl-4-piperidinone with aromatic aldehydes, in the presence of a base catalyst [24]. Then, the three-component reaction of isatin 1, sarcosine 2, and ethyl 3,5-bis[phenylmethylidene]-4-oxopiperidine-N-carboxylate 4a, as a simple model substrate, was investigated, to establish the feasibility of the strategy and optimize the reaction conditions. The reaction was carried out at 60 °C in methanol and took around 120 min. It was then cooled to room temperature and the solid that formed was filtered and recrystallized from methanol to furnish the functional dispiropyrrolidinyloxindole in a highly regioselective manner, to afford 6a (Scheme 1).
The dispiroheterocyclic ring structure of products 6a–j was confirmed by spectroscopic data (IR, 1H- and 13C-NMR) and elemental analysis. The IR spectrum of 6a revealed the presence of a carbonyl stretching vibration band at 1685 cm−1, showing an increase of 18 cm−1 from the normal value observed for 3,5-bis[phenylmethylidene]-4-oxipiperidine-N-carboxylate 4a, indicating the loss of conjugation from one side. It also exhibited two bands at 1718 and 1710 cm−1, due to the carbonyl group of oxindole and ester moiety, respectively. On the other hand, the 1H-NMR spectrum of 6a showed a sharp singlet signal at δ 1.98 for thepyrroline-N-CH3 proton. The benzylic proton Ha exhibited a doublet of the doublet in the region δ 4.82, with (J = 10.7, 9.0 Hz). The Hc and Hb protons appeared as a doublet of the doublet in the region δ 3.91 with (J = 10.7, 7.0 Hz) and 3.43 with (J = 9.0, 7.0 Hz), respectively. The protons of the piperidinone ring appeared as a multiple signal in the region δ 3.32–3.39. Also, the olefinic proton was observed at δ 7.59 as a singlet, whereas the aromatic protons appeared as multiplets in the region δ 6.75–7.47, and as a broad singlet at δ 8.56 for the NH proton of the oxindole ring.
The regiochemistry of the product 6a was confirmed by the 1H-NMR spectra. The benzylic proton Ha was observed at δ 4.82, as a doublet of the doublet. If the other isomer was formed, one would expect a singlet, instead of a doublet of the doublet, for this benzylic proton. The 13C-NMR spectra of 6a exhibited peaks at δ 64.2 ppm and 76.2 ppm for two spiro carbons. The peaks at δ 154.32, δ 173.2, and at δ 202.12 ppm, were due to the ester carbonyl, oxindole carbonyl, and keto carbonyl carbons of the N-COOEt piperidinone ring system, respectively. The mass spectra of 6a showed a molecular ion peak at m/z 521 (M+), which further confirmed the formation of a mono-adduct.
After the formation of the mono-adduct, the reaction failed to give the bis-adduct, even with an excess of 1,3-dipole and a prolonged reaction time. This may be due to the steric hindrance and fixing of the geometry of the spiropyrrolidine ring, which prevents afurther attack of 1,3-dipole on the other exocyclic double bond. However, bis-adducts are formed if a small 1,3-dipole is generated [25].
This reaction proceeds through the decarboxylative condensation of isatin 1 with sarcosine 2, to generate an azomethineylide 3. The generated 1,3-dipole cycloaddition with the dipolarophile 4a–j produces novel dispiro-oxindolopyrrolidines 6a–j (Scheme 1).
To enhance the yield, endeavors were made to streamline other response parameters, including the solvents and reaction temperature. Thus, the reaction was studied in different solvents that included THF, toluene, CH3CN, CH3OH, EtOH, and H2O (Table 1, entries 1–6). To our satisfaction, the reaction in methanol led to the desired product with an almost quantitative yield (92%) (Table 1, entry 4), while ethanol as a solvent produced the product with only an 85% yield (Table 1, entry 5). In general, the reactions carried out in protic solvents yielded better results than those in aprotic solvents. However, when water was employed as the solvent, no product was detected (Table 1, entry 6). This might be caused by the poor solubility of isatin 1 and ethyl 3,5-bis[phenylmethylidene]-4-oxopiperidine-N-carboxylate 4a–j in water.
The temperature influenced the rate of the reaction. Reducing the reaction temperature resulted in low reactivity (Table 1, entries 7 and 8), while elevating the temperature to 60 °C provided the best results. Based on the comprehensive consideration of the reaction temperature and yield, the optimal reaction conditions were established, as shown in Table 1 entry 4.
Considering the optimized conditions, the 1,3-dipolar cycloaddition reaction of Knoevenagel adducts with different structures, was investigated. As shown in Table 2, a variety of Knoevenagel adducts proved to be excellent dipolarophiles for this reaction and provided the corresponding spiropyrrolidine bisoxindoles with good yields (up to 95%) (Table 2, entries 1–10).
Substituents on the aryl groups lightly influenced the yields. Generally, Knoevenagel adducts with electron-donating groups produced lower yields than those with electron-withdrawing groups (Table 2).
On the basis of the above mentioned results, we extended our protocol to the synthesis of dispiro-oxindolopyrrolizines 8a–j from isatin 1, aminoacid 7 such as l-proline, and ethyl 3,5-bis[arylmethylidene]-4-oxipiperidine-N-carboxylate 4a–j in methanol, to yield a single product, as evidenced by TLC and spectral analysis (Scheme 2).
For example, in the 1H-NMR spectra of 8a, the Ha appears as a doublet at δ 3.26 ppm (J = 10.2 Hz) and multiplet of Hb at δ 2.51–2.55 ppm. The singlet at δ 8.53 ppm is due to the NH of the oxindole ring. The 13C-NMR spectra of 8a exhibited peaks at δ 62.1 ppm and 71.4 ppm for two spiro carbons. The peaks at δ 154.5, δ 173.2, and δ 199.2 ppm were due to the ester carbonyl, oxindole carbonyl, and keto carbonyl carbons of the N-COOEt piperidinone ring system, respectively. The mass spectra of 8a showed a molecular ion peak at m/z 547 (M+), which further confirmed the formation of a mono-adduct.
Several advantages, such as a high yield, and simple experimental and isolation procedures, render the methanol an efficient route for the synthesis of spiro-frameworks from isatin, sarcosine, and l-proline, that are important compounds in organic and medicinal chemistry.

2.2. Antimicrobial Activity

The in vitro antimicrobial screening of compounds prepared in this study was carried out using the cultures of four bacteria species, namely, the Gram positive bacteria, Streptococcus pneumonia (RCMB 010010) (SP) and Bacillis subtilis (RCMB 010067) (BS), and the Gram negative bacteria, Pseudomonas aeruginosa (RCMB 010043) (PS), and Escherichia coli (RCMB 010052) (EC); as well as four fungal strains, including Aspergillus fumigates (RCMB 02568) (AF), Syncephalastrumracemosum (RCMB 05922) (SR), Geotricumcandidum (RCMB 05097) (GC), and Candida albicans (RCMB05036) (CA). Amphotericin B as an antifungal agent, Ampicillin as an antibacterial agent for gram (+) bacteria, and Gentamicin as an antibacterial agent for gram (−) bacteria, were used as references to evaluate the potency of the tested compounds under the same conditions. Most of the newly synthesized compounds showed excellent results with respect to the control drugs. The results of antimicrobial activities are shown in Table 3. The data in Table 3 revealed that most of the compounds have a superior and significant antibacterial potency to antifungal activity. It is clear from the data in Table 3 that compounds 8b, 8e, 8g and 6c exhibited the highest potency against tested organisms, with respect to the reference drugs. The other derivatives showed moderate activity against the microorganisms used. The compounds 6c and 8g exhibited antimicrobial activity against all of the tested microorganisms. It has also been observed that the newly synthesized compounds exhibited more promising antibacterial activity against Gram positive bacteria than Gram negative bacteria, displaying the highest activity against Bacillis subtilis (RCMB 010067).
The minimum inhibitory concentration (MIC) of the most active synthesized compounds against highly inhibited organisms, is reported in Table 4. Compounds 8b, 8e, 8g and 6c revealed the lowest MICs (0.06, 0.015, 0.015, and 0.015 µg/mL) against Bacillis subtilis (RCMB 010067), respectively.
The inhibitory concentration of the most active synthesized compounds against 50% of microorganism growth (IC50), is reported in Table 5. Compounds 8b, 8e, 8g and 6c revealed the lowest IC50 values (2.92, 1.34, 1.36, and 1.24 µg/mL) against Bacillis subtilis (RCMB 010067), respectively.
Then, a study of the structure-activity relationships (SAR) showed that 8 compounds with a phenyl-hexahydro-1H-pyrrolizine ring, were more capable of improving the antimicrobial activity than compounds 6 with (-CH3) a pyrrolidine ring.
According to the antimicrobial data, we evaluated the effect of introducing different substituent group(s) at phenyl rings in synthesized compounds. The introduction of electron-withdrawing groups (halogen groups or -NO2) showed better antimicrobial effects than the electron-donating group (4-OCH3, 3,4,5-(OCH3)3).

3. Experimental Section

3.1. General Procedures

Melting points were determined using a Gallenkamp electro-thermal apparatus and wereuncorrected. IR spectra were recorded as KBr disc, using a Shimadzu FTIR-prestige 21 spectrophotometer. 1H- and 13C-NMR spectra were recorded in DMSO-d6 as solvents (at 300 MHz for 1H and 75 MHz for 13C) on a Varian Mercury NMR spectrometer, using TMS as the internal standard. Chemical shifts δ are reported in parts per million units (ppm), and J values are given in hertz. The mass spectra were recorded on a GCeMS-QP1000 EX mass spectrometer at 70 eV. Elemental analyzes were measured using a German made elementary vario LIII CHNS analyzer. Antibacterial activity was studied at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt. Compounds 4a–c, 4e and 4h were prepared as previously reported in the respective literature [24].

3.2. General Procedure for the Preparation of 3,5-bis[Arylmethylidene]-4-oxopiperidine-N-carboxylate (4d,f,g,i,j)

A mixture of 1-ethoxycarbonyl-4-piperidinone (1.71 mL, 10 mmol) and aromatic aldehydes (20 mmol) in methanol (20 mL), in the presence of potassium hydroxide, was used as the base catalyst (1 g). The mixture was stirred at room temperature for 30 min. The solid formed was collected, washed with methanol, and crystallized from proper solvent, to give the compounds, as listed below:
Ethyl 3,5-bis[4-fluorophenyl-methylidene]-4-oxopiperidine-N-carboxylate (4d). Yellow solid, m.p. 210–212 °C (EtOH); IR (KBr): 1717, 1687 (2CO) cm−1. 1H-NMR (DMSO-d6); 1.12 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 3.92 (s, 4H, 2CH2 of piperidinone), 4.75 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 7.40–7.55 (m, 8H, Ar-H), 7.67 (s, 2H, 2CH=). 13C-NMR (DMSO-d6); 13.2, 59.5, 45.5, 117.0, 128.2, 133.2, 142.3, 145.9, 164.2, 155.3, 185.1. MS m/z (%): 385 (M+ + 2, 40), 384 (M+ + 1, 23), 383 (M+, 45), 206 (14), 97 (30), 57 (15). Anal. for C22H19F2NO3 (383.39): calcd. C, 68.92; H, 5.0; N, 3.65. Found C, 68.65; H, 5.10; N, 3.54.
Ethyl 3,5-bis[3,4-dichlorophenyl-methylidene]-4-oxopiperidine-N-carboxylate (4f). Yellow solid, m.p. 208–210 °C (EtOH/Dioxane); IR (KBr): 1720, 1688 (2CO) cm−1. 1H-NMR (DMSO-d6): δ 1.13 (t, 3H, J = 7 Hz, CH3 of CH2CH3), 3.93 (s, 4H, 2CH2 of piperidinone), 4.73 (q, 2H, J = 7 Hz, CH2 of CH2CH3), 7.45–7.56 (m, 6H, Ar-H), 7.66 (s, 2H, 2CH=). MS m/z (%): 487 (M+ + 2, 50), 486 (M+ + 1, 30), 485 (M+, 32), 167 (15), 147 (10). Anal. For C22H17Cl4NO3 (485.18): calcd. C, 54.46; H, 3.54; N, 2.89. Found: C, 54.45; H, 3.82; N, 2.63.
Ethyl 3,5-bis[2,4-difluorophenyl-methylidene]-4-oxopiperidine-N-carboxylate (4g). Yellow solid, m.p. 167–168 °C (EtOH/Dioxane); IR (KBr): 1715, 1686 (2CO) cm−1. 1H-NMR (DMSO-d6): δ 1.2 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 3.91 (s, 4H, 2CH2 of piperidinone), 4.56 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 7.53–7.76 (m, 6H, Ar-H), 7.84 (s, 2H, 2CH=). 13C-NMR (DMSO-d6): δ 14.22, 59.3, 46.7, 105, 112.2, 119.2, 130.4, 140.2, 145.9, 154.7, 160.3, 165.1, 185.8. MS m/z (%): 421 (M+ + 2, 5), 420 (M+ + 1, 23), 419 (M+, 42), 206 (12), 97 (10), 57 (9). Anal. for C22H17F4NO3 (419.38): calcd. C, 63.00; H, 4.27; N, 3.62. Found: C, 63.11; H, 4.26; N, 3.42.
Ethyl 3,5-bis[4-trifluoromethylphenyl-methylidene]-4-oxo-piperidine-N-carboxylate (4i). Yellow solid, m.p. 147–149 °C (EtOH/Dioxane); IR (KBr): 1709, 1691 (2CO) cm−1. 1H-NMR (DMSO-d6): δ 1.19 (t, 3H, J = 7.1 Hz, CH3 of CH2CH3), 3.86 (s, 4H, 2CH2 of piperidinone), 4.24 (q, 2H, J = 7.1 Hz, CH2 of CH2CH3), 7.21–7.53 (m, 8H, Ar-H), 7.83 (s, 2H, 2CH=). MS m/z (%): 485 (M+ + 2, 25), 484 (M+ + 1, 38), 483 (M+, 62), 158 (23), 145 (20). Anal. for C24H19F6NO3 (483.41): calcd. C, 59.63; H, 3.96; N, 2.90. Found: C, 59.91; H, 4.00; N, 2.71.
Ethyl 3,5-bis[3,4,5-trimethoxyphenyl-methylidene]-4-oxopiperidine-N-carboxylate (4j). Yellow solid, m.p. 126–127 °C (EtOH); IR (KBr): 1718, 1686 (2CO) cm−1. 1H-NMR (DMSO-d6): δ 1.2 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 2.49 (s, 4H, 2CH2 of piperidinone), 3.17 (s, 6H, CH3 of OCH3), 3.65 (s, 6H, CH3 of OCH3), 3.72 (s, 6H, CH3 of OCH3), 4.8 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 6.84 (s, 4H, Ar-H), 7.24 (s, 2H, 2CH=). 13C-NMR (DMSO-d6): δ 13.6, 58.9, 46.4, 56.1, 56.5, 105, 129.2, 140.1, 151.2, 140.5, 145.7, 154.9, 186.7. MS m/z (%): 529 (M+ + 2, 6), 528 (M+ + 1, 20), 527 (M+, 63), 97 (15), 57 (9). Anal. for C28H33NO9 (527.57): calcd. C, 63.75; H, 6.31; N, 2.66. Found: C, 63.50; H, 6.41; N, 2.69.

3.3. General Synthesis of Dispiropyrrolidine Oxindole Derivatives (6a–j)

A reaction mixture of isatin 1 (1.47 g, 10 mmol), sarcosine 2 (0.89 g, 10 mmol), and Ethyl 3,5-bis(arylmethylidene)-N-carboxyl-4-piperidinone 4a–j (10 mmol), was produced by refluxing it in methanol for 120 min at 60 °C, and was then poured on water. The solid formed was collected and crystallized from a suitable solvent to produce white to brown crystals of compounds (6a–j).
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-benzylidene-1′′-N-carboxylate-4′′-piperidinone-4-phenyl-pyrrolidine (6a). White crystals, m.p. 160–162 °C (EtOH/Dioxane); IR (KBr): 3253 (NH), 1718, 1710, 1685 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.15 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 1.98 (s, 3H, NCH3), 3.32, 3.39 (2s, 4H, 2CH2 of piperidinone ring), 3.43 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.91 (dd, J = 10.7, 7.0 Hz, 1Hc), 4.39 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 4.82 (dd, J = 10.7, 9.0 Hz, 1Ha), 6.75–7.47 (m, 14H, Ar-H), 7.59 (s, 1H, CH=), 8.56 (s, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.7, 29.9, 38.1, 43.2, 46.7, 55.3, 59.3, 64.2, 76.2, 121.6, 124.1, 126.3, 126.4, 126.5, 127.4, 127.9, 128.2, 128.5, 136.2, 136.4, 138.2, 139.5, 141.2, 153.9, 154.3, 173.2, 202.1. MS m/z (%): 523 (M+ + 2, 9), 522 (M+ + 1, 25), 521 (M+, 35), 130 (14), 77 (30). Anal. for C32H31N3O4 (521.62): calcd. C, 73.68; H, 5.99; N, 8.06.Found C, 73.53; H, 5.94; N, 8.02.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(4-nitro)benzylisdene-1′′-N-carboxylate-4′′-piperidinone-4-(4-nitro)phenyl-pyrrolidine (6b). Yellow crystals, m.p. 175–176 °C (Dioxane); IR (KBr): 3262 (NH), 1714, 1701, 1692(3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.18 (t, 3H, J = 7 Hz, CH3 of CH2CH3), 2.03 (s, 3H, NCH3), 3.22 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.30, 3.45 (2s, 4H, 2CH2 of piperidinone ring), 3.84 (dd, J = 10.7, 7.0 Hz, 1Hc), 4.18 (q, 2H, J = 7 Hz, CH2 of CH2CH3), 4.79 (dd, J = 10.7, 9.0 Hz, 1Ha), 6.76–7.82 (m, 12H, Ar-H), 8.40 (s, 1H, CH=), 11.01 (s, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.5, 29.9, 37.8, 43.5, 46.9, 55.8, 58.9, 64.3, 76.5, 121.6, 124.1, 126.1, 126.4, 126.6, 127.1, 127.4, 128.6, 128.8, 128.9, 136.2 , 136.3, 138.2, 139.5, 141.5, 153.9, 158.3, 175.2, 201.5. MS m/z (%): 613 (M+ + 2, 8), 612 (M+ + 1, 35), 611 (M+, 98), 242 (12), 205 (15), 97 (15). Anal. For C32H29N5O8 (611.61): calcd. C, 62.84; H, 4.78; N, 11.45. Found: C, 62.78; H, 4.69; N, 11.36.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(4-chloro)benzylidene-1′′-N-carboxylate-4′′-piperidinone-4-(4-chloro)phenyl-pyrrolidine (6c). Yellow crystals, m.p. 185–187 °C (EtOH/Dioxane); IR (KBr): 3251 (NH), 1716, 1708, 1686 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.16 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 1.97 (s, 3H, NCH3), 3.11 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.29, 3.38 (2s, 4H, 2CH2 of piperidinone ring), 3.89 (dd, J = 10.7, 7.0 Hz, 1Hc), 4.20 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 4.72 (dd, J = 10.7, 9.0 Hz, 1Ha), 6.72–7.49 (m, 12H, Ar-H), 7.56 (s, 1H, CH=), 10.48 (s, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.8, 29.8, 37.8, 43.2, 46.6, 55.5, 59.1, 63.9, 76.1, 121.2, 124.1, 126.4, 127.2,128.4, 128.6, 128.8, 130.1, 131.1, 133.4, 133.8, 136.6 , 137.6, 139.3, 139.7, 152.6, 154.0, 172.3, 203.9. MS m/z (%): 592 (M+ + 2, 6), 591 (M+ + 1, 34), 590 (M+, 88), 242 (12), 97 (15). Anal. For C32H29Cl2N3O4 (589.50): calcd. C, 65.09; H, 4.95; N, 7.12.Found C, 65.01; H, 4.86; N, 7.06.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(4-fluoro) benzylidene-1′′-N-carboxylate-4′′-piperidinone-4-(4-fluoro)phenyl-pyrrolidine (6d). Brown crystals, m.p. 215–217 °C (Dioxane); IR (KBr): 3256 (NH), 1720, 1711, 1684 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.19 (t, 3H, J = 7.4 Hz, CH3 of CH2CH3), 2.18 (s, 3H, NCH3), 3.17 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.36, 3.48 (2s, 4H, 2CH2of piperidinone ring), 3.86 (dd, J = 10.5, 7.0 Hz, 1Hc), 4.19 (q, 2H, J = 7.4 Hz, CH2 of CH2CH3), 4.71 (dd, J = 10.5, 9.0 Hz, 1Ha), 6.75–7.33 (m, 12H, Ar-H), 7.40 (s, 1H, CH=), 10.5 (s, 1H, D2O-exchangeable, NH).13C-NMR (DMSO-d6): δ 13.5, 29.7, 37.9, 42.9, 46.6, 54.7, 59.1, 63.7, 76.1, 114.7 ,115.3, 121.4, 124.3, 126.2, 128.1, 128.5, 130.2, 130.9, 136.4, 136.7, 137.9, 139.5, 159.8, 162.2, 152.6, 154.4, 173.4, 201.6. MS m/z (%): 559 (M+ + 2, 3), 558 (M+ + 1, 35), 557 (M+, 85), 206 (10), 97 (8). Anal for C32H29F2N3O4 (557.60): calcd. C, 68.93; H, 5.24; N, 7.54.Found C, 68.87; H, 5.21; N, 7.49.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(4-methoxy) benzylidene-1′′-N-carboxylate-4′′-piperidinone-4-(4-methoxy)phenyl-pyrrolidine (6e). Yellow crystals, m.p. 118–120 °C (Methanol); IR (cm−1): 3251 (NH), 1716, 1702, 1674 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.13 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 2.09 (s, 3H, NCH3), 3.25 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.33, 3.49 (2s, 4H, 2CH2 of piperidinone ring), 3.85, 3.87 (2s, 6H, 2OCH3), 3.89 (dd, J = 10.8, 7.0 Hz, 1Hc), 4.22 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 4.79 (dd, J = 10.8, 9.0 Hz, 1Ha), 6.72–7.59 (m, 12H, Ar-H), 7.65 (s, 1H, CH=), 10.41 (s, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.9, 29.6, 38.0, 43.4, 46.9, 54.9, 55.9, 56.2, 59.3, 64.2, 76.2, 113.4, 114.1, 121.3, 124.2, 126.3, 127.3, 128.4, 129.4, 129.6, 133.4, 136.1, 138.3, 139.6, 152.8, 154.2, 157.6, 159.8, 172.0, 202.5. MS m/z (%): 583 (M+ + 2, 4), 582 (M+ + 1, 35), 581 (M+, 90), 244 (10), 206 (8), 95 (19). Anal. For C34H35N3O6 (581.67): calcd. C, 70.21; H, 6.07; N, 7.22. Found C, 70.19; H, 6.10; N, 7.20.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(3,4-dichloro)benzylidene-1′′-N-carboxylate-4′′- piperidinone-4-(3,4 dichloro)phenyl-pyrrolidine (6f). Yellow crystals, m.p. 213–214 °C (Dioxane);; IR (cm−1): 3400 (NH), 1715, 1713, 1692 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.12 (t, 3H, J = 7.1 Hz, CH3 of CH2CH3), 1.98 (s, 3H, NCH3), 3.18 (dd, J = 9.1, 7.0 Hz, 1Hb), 3.30, 3.45 (2s, 4H, 2CH2 of piperidinone ring), 3.88 (dd, J = 10.6, 7.0 Hz, 1Hc), 4.28 (q, 2H, J = 7.1 Hz, CH2 of CH2CH3), 4.73 (dd, J = 10.6, 9.1 Hz, 1Ha), 6.80–7.65 (m, 10H, Ar-H), 7.66 (s, 1H, CH=), 10.4 (s, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.6, 29.2, 38.2, 42.9, 46.7, 54.7, 59.3, 63.2, 76.1, 121.6, 124.1, 126.4, 127.9, 129.5, 129.8, 128.9, 130.2, 130.3, 136.1, 132.5, 132.6, 133.4, 134.8, 137.8, 139.5, 140.6, 153.9, 154.5, 173.1, 202.8. MS m/z (%): 661 (M+ + 2, 48), 660 (M+ + 1, 34.5), 659 (M+, 89), 657 (70), 357 (10), 339 (20), 338 (5), 145 (5). Anal. For C32H27Cl4N3O4 (659.38): calcd. C, 58.29; H, 4.13; N, 6.37.Found C, 58.28; H, 4.11; N, 6.36.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(2,4-difluoro)-benzylidene-1′′-N-carboxylate-4′′-piperidinone-4-(2,4-difluoro)phenyl-pyrrolidine (6g). Yellow crystals, m.p. 130–132 °C (EtOH/Dioxane); IR: 3255 (NH), 1716, 1692, 1660 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.15 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 1.98 (s, 3H, NCH3), 3.29 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.34, 3.46 (2s, 4H, 2CH2 of piperidinone ring), 3.82 (dd, J = 10.7, 7.0 Hz, 1Hc), 4.29 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 4.73 (dd, J = 10.7, 9.0 Hz, 1Ha), 6.73–7.21 (m, 10H, Ar-H), 7.52 (s, 1H, CH=), 10.52 (br., 1H, D2O-exchangeable, NH). MS m/z (%): 595 (M+ + 2, 6), 594 (M+ + 1, 35), 593 (M+, 78), 339 (20), 325 (8), 306 (7), 113 (5). Anal. For C32H27F4N3O4 (593.58): calcd. C, 64.75; H, 4.59; N, 7.08. Found: C, 64.69; H, 4.50; N, 7.02.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(3,4-dimethoxy)benzylidene-1′′-N-carboxylate-4′′-piperidinone-4-(3,4-dimethoxy)phenyl-pyrrolidine (6h). Brown crystals, m.p. 95–98 °C (Methanol); IR: 3260 (NH), 1712, 1695, 1659 (3CO) cm−1. 1H-NMR (DMSO-d6); δ 1.18 (t, 3H, J = 6.9 Hz, CH3 of CH2CH3), 2.04 (s, 3H, NCH3), 3.23 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.33, 3.47 (2s, 4H, 2CH2of piperidinone ring), 3.67, 3.69 (2s, 6H, 2OCH3), 3.79, 3.82 (2s, 6H, 2OCH3), 3.87 (dd, J = 10.5, 7.0 Hz, 1Hc), 4.22 (q, 2H, J = 6.9 Hz, CH2 of CH2CH3), 4.69 (dd, J = 10.5, 9.0 Hz, 1Ha), 6.55–7.59 (m, 10H, Ar-H), 7.62 (s, 1H, CH=), 10.3 (s, 1H, D2O-exchangeable, NH). 13C-NMR (75 MHz, δ ppm, DMSO-d6); 13.9, 29.7, 37.8, 43.1, 46.9, 54.7, 56.3, 56.4, 56.8, 56.9, 59.0, 63.8, 76.7, 111.5, 113.7, 114.7, 115.4, 119.8,120.2, 121.5, 124.5, 126.3, 128.4, 128.6, 134.4, 136.0, 137.8, 139.7, 146.6, 148.9, 149.0, 149.8, 152.9, 154.5, 172.2, 204.1. MS m/z (%): 643 (M+ + 2, 8), 642 (M+ + 1, 39), 641 (M+, 70), 329 (10), 318 (9), 137 (4). Anal. For C36H39N3O8 (641.72): calcd. C, 67.38; H, 6.13; N, 6.55. Found: C, 67.31; H, 6.08; N, 6.48.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(4-trifluro-methyl)benzylidene-1′′-N-carbo-xylate-4′′-piperidinone-4-(4-trifluro-methyl)phenyl-pyrrolidine (6i). Green crystals, m.p. 136–137 °C (EtOH/Dioxane); IR: 3367 (NH), 1709, 1701, 1654 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.15 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 2.09 (s, 3H, NCH3), 3.24 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.32, 3.46 (2s, 4H, 2CH2of piperidinone ring), 3.82 (dd, J = 10.7, 7.0 Hz, 1Hc), 4.23 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 4.76 (dd, J = 10.7, 9.0 Hz, 1Ha), 6.51–7.71 (m, 12H, Ar-H), 7.75 (s, 1H, CH=), 10.39 (s, 1H, D2O-exchangeable, NH). MS m/z (%): 659 (M+ + 2, 7), 658 (M+ + 1, 39), 657 (M+, 90), 344 (12), 339 (20), 145 (8). Anal. For C34H29F6N3O4 (657.61): calcd. C, 62.1; H, 4.44; N, 6.39.Found: C, 62.13; H, 4.36; N, 6. 37.
Ethyl 1-N-methyl-spiro[2.3′]oxindole-spiro[3.3′′]5′′-(3,4,5-trimethoxy)benzylidene-1′′-N-carboxylate-4′′-piperidinone-4-(3,4,5-trimethoxy)phenyl-pyrrolidine (6j). Yellow crystals, m.p. 115–116 °C (EtOH/Dioxane); IR: 3309 (NH), 1715, 1696, 1655 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.20 (t, 3H, J = 7 Hz, CH3 of CH2CH3), 2.12 (s, 3H, NCH3), 3.26 (dd, J = 9.0, 7.0 Hz, 1Hb), 3.27, 3.44 (2s, 4H, 2CH2 of piperidinone ring), 3.69, 3.71 (2s, 6H, 2OCH3), 3.74, 3.75 (2s, 6H, 2OCH3), 3.80, 3.82 (2s, 6H, 2OCH3), 3.87 (dd, J = 10.7, 7.0 Hz, 1Hc), 4.23 (q, 2H, J = 7 Hz, CH2 of CH2CH3), 4.75 (dd, J = 10.7, 9.0 Hz, 1Ha), 6.43–6.81 (m, 8H, Ar-H), 7.25 (s, 1H, CH=), 9.88 (s, 1H, D2O-exchangeable, NH). MS m/z (%): 703 (M+ + 2, 9), 702 (M+ + 1, 43), 701 (M+, 92), 364 (12), 368 (19), 339 (20) 167 (7). Anal. For C38H43N3O10 (701.77): calcd. C, 65.04; H, 6.18; N, 5.99. Found C, 64.95; H, 6.11; N, 5.92.

3.4. General Synthesis of Dispiropyrrolizine Oxindole Derivatives (8a–j)

A reaction mixture of isatin 1 (1.47 g, 1.0 mmol), l-proline 7 (1.15 g, 10 mmol), and ethyl 3,5-bis(aryl-methylidene)-N-carboxyl-4-piperidinone 4a–j (1.0 mmol), was producedby refluxing it in methanol for 120 min at 60 °C, and was then poured on water. The solid that formed was collected and crystallized from suitable solvent to get white to green crystals of compounds 8a–j.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-phenylmethylidene-tetra-hydro-4'(1H)-piperidinone-1-phenyl-hexahydro-1H-pyrrolizine (8a). Yellow crystals, m.p. 127–128 °C (EtOH/Dioxane); IR: 3251 (NH), 1720, 1690, 1600 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.15 (t, 3H, J = 6.9 Hz, CH3 of CH2CH3), 1.78–1.90 (m, 4H, 2CH2 of pyrrolizline), 2.40–2.48 (m, 2H, CH2 of pyrrolizine), 2.51–2.55 (m, 1Hb), 3.26 (d, J = 10.1 Hz, 1Ha), 3.53, 3.77 (2s, 4H, 2CH2 of piperidinone ring), 4.11 (q, 2H, J = 6.9 Hz, CH2 of CH2CH3), 6.68-7.53 (m, 14H, Ar-H), 7.64 (s, 1H, CH=), 8.53 (br, 1H, D2O-exchangeable, NH).13C-NMR (DMSO-d6): δ 13.9, 24.2, 28.5, 51.6, 32.7, 42.9, 46.6, 59.2, 61.1, 62.1, 71.4, 121.5, 124.5, 126.2, 126.3, 126.6, 128.2, 128.3, 128.4, 128.5, 128.7, 135.7, 136.1, 137.5, 139.4, 139.5, 152.9, 154.5, 173.2, 199.2. MS m/z (%): 549 (M+ + 2, 5), 548 (M+ + 1, 37), 547 (M+, 65), 470 (3), 457 (9), 412 (5), 302 (34), 257 (30), 206 (10), 158 (9), 97 (8), 77 (3). Anal. For C34H33N3O4 (547.65): calcd. C, 74.57; H, 6.07; N, 7.67. Found C, 74.33; H, 6.1; N, 7.6.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(4-nitro)phenylmethylidene-tetrahydro-4'(1H)-piperidinone-1-(4-nitro)phenyl-hexahydro-1H-pyrrolizine (8b). Yellow crystals, m.p. 160–161 °C (Dioxane); IR: 3255 (NH), 1701, 1692, 1620 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.15 (t, 3H, J = 7 Hz, CH3 of CH2CH3), 1.78–1.98 (m, 4H, 2CH2 of pyrrolizline), 2.32–2.38 (m, 2H, CH2 of pyrrolizine), 2.71–2.75 (m, 1Hb), 3.28 (d, J = 10.3 Hz, 1Ha), 3.52, 3.71 (2s, 4H, 2CH2 of piperidinone ring), 4.21 (q, 2H, J = 7 Hz CH2 of CH2CH3), 6.76–7.52 (m, 12H, Ar-H), 7.66 (s, 1H, CH=), 8.62 (br, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.9, 23.7, 29.5, 31.6, 42.5, 46.7, 50.6, 59.1, 60.9, 61.5, 71.2, 120.9, 121.1, 121.4, 124.3, 126.5, 127.4, 128.3, 129.2, 136.0, 137.8, 139.7, 141.3,145.3, 145.7, 147.7, 152.7, 154.6, 172.6, 203.2. MS m/z (%): 639 (M+ + 2, 10), 638 (M+ + 1, 35), 637 (M+, 89), 503 (9), 494 (4), 242 (12), 205 (9), 122 (5), 97 (10). Anal. For C34H31N5O8 (637.65): calcd. C, 64.04; H, 4.90; N, 10.98. Found: C, 63.96; H, 4.84; N, 10.86.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(4-chloro)phenylmethylidene-tetrahydro-4'(1H)-piperidinone-1-(4-chloro)phenyl-hexahydro-1H-pyrrolizine (8c). Yellow crystals, m.p. 128–129 °C (EtOH/Dioxane); IR: 3251 (NH), 1701, 1678, 1616 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.15 (t, 3H, J = 7.4 Hz, CH3 of CH2CH3), 1.78–1.89 (m, 4H, 2CH2of pyrrolizline), 2.33–2.38 (m, 2H, CH2 of pyrrolizine), 2.51–2.56 (m, 1Hb), 3.35 (d, J = 10.4 Hz, 1Ha), 3.78, 3.84 (2s, 4H, 2CH2 of piperidinone ring), 4.39 (q, 2H, J = 7.4 Hz, CH2 of CH2CH3), 6.72–7.49 (m, 12H, Ar-H), 7.56 (s, 1H, CH=), 8.48 (s, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.7, 23.8, 29.2, 32.0, 42.5, 46.0, 51.5, 59.0, 60.8, 61.8, 71.1, 121.4, 124.3, 126.7, 127.8, 128.0, 128.6, 128.9, 129.6, 131.6, 133.4, 133.7, 136.3, 137.4, 137.6, 139.8, 152.7, 154.4, 172.5, 200.2. MS m/z (%): 617 (M+ + 2, 67), 616 (M+ + 1, 59), 615 (M+, 80), 494 (7), 482 (14), 337 (20), 292 (15), 219 (8), 206 (20), 97 (10), 57 (6). Anal. For C34H31Cl2N3O4 (616.54): calcd. C, 66.24; H, 5.07; N, 6.82. Found: C, 66.13; H, 5.02; N, 6.80.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(4-fluoro)phenylmethylidene-tetrahydro-4'(1H)-piperidinone-1-(4-fluoro)phenyl-hexahydro-1H-pyrrolizine (8d). White crystals, m.p. 185–187 °C (Dioxane); IR: 3394 (NH), 1715, 1692, 1620 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.18 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3),1.75–1.96 (m, 4H, 2CH2 of pyrrolizline), 2.49–2.55 (m, 2H, CH2 of pyrrolizine), 2.51–2.57 (m, 1Hb), 3.26 (d, J = 10.2 Hz, 1Ha), 3.46, 3.77 (2s, 4H, 2CH2 of piperidinone ring), 4.24 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 6.79–7.35 (m, 12H, Ar-H), 7.36 (s, 1H, CH=), 8.35 (s, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.7, 23.9, 28.9, 32.7, 42.4, 46.8, 50.8, 59.0, 60.8, 61.6, 71.2, 115.2, 115.3, 121.3, 124.2, 126.0, 128.2, 128.2, 129.9, 130.8,134.9, 136.5, 137.3, 139.7, 152.8, 154.4, 160.3, 162.2,173.1, 199.9. MS m/z (%): 585 (M+ + 2, 7), 584 (M+ + 1, 37), 583 (M+, 86), 206 (10), 97 (10). Anal. For C34H31F2N3O4 (583.64): calcd. C, 69.97; H, 5.35; N, 7.20. Found: C, 69.90; H, 5.28; N, 7.15.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(4-methoxy)phenylmethylidene-tetrahydro-4'(1H)-piperidinone-1-(4-methoxy)phenyl-hexahydro-1H-pyrrolizine (8e). Yellow crystals, m.p. 158–160 °C (EtOH/Dioxane); IR: 3421 (NH), 1715, 1662, 1600 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.14 (t, 3H, J = 6.9 Hz, CH3 of CH2CH3), 1.78–1.96 (m, 4H, 2CH2 of pyrrolizline), 2.44–2.50 (m, 2H, CH2 of pyrrolizine), 2.59–2.65 (m, 1Hb), 3.25 (d, J = 10.2 Hz, 1Ha), 3.68, 3.82 (2s, 4H, 2CH2 of piperidinone ring), 3.69,3.75 (2s, 6H, 2OCH3), 4.25 (q, 2H, J = 6.9 Hz, CH2 of CH2CH3), 7.05–7.52 (m, 12H, Ar-H), 7.65 (s, 1H, CH=), 8.8 (s, 1H, D2O- exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.8, 24.1, 29.1, 32.5 , 42.5, 46.7, 50.6, 56.1, 58.8, 58.9, 60.8, 61.6, 71.2, 114.2, 114.3, 121.5, 124.1, 126.9, 127.5, 127.7, 128.2, 129.2, 131.7, 136.3, 137.2, 139.6, 152.7, 154.2, 157.9, 159.9, 173.0, 202.2. MS m/z (%): 609 (M+ + 2, 8), 608 (M+ + 1, 97), 607 (M+, 45), 333 (20), 288 (30), 215 (15), 120 (8). Anal. For C36H37N3O6 (607.71): calcd. C, 71.15; H, 6.14; N, 6.91. Found: C, 71.13; H, 6.12; N, 6.87.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(3,4-dichloro)phenylmethylidene-tetrahydro-4'(1H)-piperidinone-1-(3,4-dichloro)phenyl-hexahydro-1H-pyrrolizine (8f). White crystals, m.p. 158–159 °C (EtOH/Dioxane); IR: 3255 (NH), 1701, 1681, 1620 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.16 (t, 3H, J = 7.2 Hz, CH3 of CH2CH3), 1.77–1.98 (m, 4H, 2CH2 of pyrrolizline), 2.46–2.52 (m, 2H, CH2 of pyrrolizine), 2.55–2.75 (m, 1Hb), 3.26 (d, J = 10.2 Hz, 1Ha), 3.34, 3.62 (2s, 4H, 2CH2 of piperidinone ring), 4.32 (q, 2H, J = 7.2 Hz, CH2 of CH2CH3), 6.79–7.35 (m, 10H, Ar-H), 7.38 (s, 1H, CH=), 8.45 (s, 1H, D2O-exchangeable, NH). MS m/z (%): 685 (M+, 49), 684 (100), 683 (78), 371 (21), 353 (14), 327 (14), 143 (8). Anal. For C34H29Cl4N3O4 (685.42) calcd. C, 59.58; H, 4.26; N, 6.13. Found: C, 59.56; H, 4.19; N, 6.07.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(2,4-difluoro)phenylmethylidene-tetrahydro-4'(1H)-piperidinone-1-(2,4-difluoro)phenyl-hexahydro-1H-pyrroliz-ine (8g). Pale green crystals, m.p. 132–133 °C (Methanol); IR: 3433 (NH), 1705, 1681, 1616 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.13 (t, 3H, J = 7.4 Hz, CH3 of CH2CH3), 1.72–1.89 (m, 4H, 2CH2 of pyrrolizline), 2.42–2.49 (m, 2H, CH2 of pyrrolizine), 2.69–2.75 (m, 1Hb), 3.30 (d, J = 10.0 Hz, 1Ha), 3.45, 3.88 (2s, 4H, 2CH2 of piperidinone ring), 4.30 (q, 2H, J = 7.4 Hz, CH2 of CH2CH3), 6.71–7.61 (m, 10H, Ar-H), 7.66 (s, 1H, CH=), 8.48 (s, 1H, D2O-exchangeable, NH). MS m/z (%): 621 (M+ + 2, 7), 620 (M+ + 1, 38), 619 (M+, 55), 294 (23), 393 (9), 221 (4), 93 (4). Anal. for C34H29F4N3O4 (619.62): calcd. C, 65.91; H, 4.72; N, 6.78. Found: C, 65.88; H, 4.68; N, 6.73.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(3,4-dimethoxy)phenylmethylidene-tetrahydro-4'(1H)-piperidinone-1-(3,4-dimethoxy)phenyl-hexahydro-1H-pyrro-lizine (8h). Yellow crystals, m.p. 231–233 °C (EtOH/Dioxane); IR: 3421 (NH), 1700, 1697, 1616 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.14 (t, 3H, J = 7.1 Hz, CH3 of CH2CH3), 1.78–2.02 (m, 4H, 2CH2 of pyrrolizline), 2.38–2.48 (m, 2H, CH2 of pyrrolizine), 2.59–2.73 (m, 1Hb), 3.33 (d, J = 10 Hz, 1Ha), 3.52, 3.65 (2s, 4H, 2CH2of piperidinone ring), 3.69, 3.71 (2s, 6H, 2OCH3), 3.79, 3.82 (2s, 6H, 2OCH3), 4.21 (q, 2H, J = 7.1 Hz, CH2 of CH2CH3), 6.79–7.65 (m, 10H, Ar-H), 7.72 (s, 1H, CH=), 8.31 (s, 1H, D2O-exchangeable, NH). 13C-NMR (DMSO-d6): δ 13.7, 23.6, 29.5, 31.9, 42.5, 46.6, 50.9, 56.2, 56.4, 56.7, 56.8, 59.5, 61.3, 61.4, 71.3, 111.7, 113.2, 115.2, 119.8, 121.6, 121.2, 124.7, 126.5, 128.4, 128.5, 132.6, 136.4, 137.3, 139.2, 147.2, 149.1, 149.7, 149.9, 152.9, 154.5, 173.0, 200.9. MS m/z (%): 669 (M+ + 2, 10), 668 (M+ + 1, 43), 667 (M+, 92), 362 (31), 318 (9), 243 (10), 90 (20), 93 (10). Anal. For C38H41N3O8 (667.76): calcd. C, 68.35; H, 6.19; N, 6.29. Found C, 68.28; H, 6.14; N, 6.24.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(4-trifluromethyl)phenyl-methylidene-tetrahydro-4'(1H)-piperidinone-1-(4-trifluromethyl)phenyl-hexahydro-1H-pyrrolizine (8i). Green crystals, m.p. 137–139 °C (EtOH/Dioxane); IR: 3371 (NH), 1701, 1690, 1616 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.18 (t, 3H, J = 7 Hz, CH3 of CH2CH3), 1.76–1.98 (m, 4H, 2CH2 of pyrrolizline), 2.46–2.50 (m, 2H, CH2 of pyrrolizine), 2.59–2.74 (m, 1Hb), 3.96 (d, J = 10.3 Hz, 1Ha), 3.35, 3.77 (2s, 4H, 2CH2 of piperidinone ring), 4.32 (q, 2H, J = 7 Hz, CH2 of CH2CH3), 6.79–7.35 (m, 12H, Ar-H), 7.42 (s, 1H, CH=), 8.42 (s, 1H, D2O-exchangeable, NH).MS m/z (%): 685 (M+ + 2, 7), 684 (M+ + 1, 39), 683 (M+, 92), 370 (13), 325 (7), 251 (13), 145 (6). Anal. For C36H31F6N3O4 (683.65): calcd. C, 63.25; H, 4.57; N, 6.15. Found: C, 63.18; H, 4.51; N, 6.08.
Ethyl spiro[3.3′′]-oxindole-spiro[2.3′]1'-carboxylate-5'-(3,4,5-trimethoxy)phenyl-methylidene-tetrahydro-4'(1H)-piperidinone-1-(3,4,5-trimethoxy)phenyl-hexahydro-1H-pyrrolizine (8j). Yellow crystals, m.p. 123–124 °C (EtOH/Dioxane); IR: 3464 (NH), 1701, 1692, 1620 (3CO) cm−1. 1H-NMR (DMSO-d6): δ 1.15 (t, 3H, J = 6.9 Hz, CH3 of CH2CH3), 1.82–2.09 (m, 4H, 2CH2 of pyrrolizline), 2.44–2.50 (m, 2H, CH2 of pyrrolizine), 2.72–2.80 (m, 1Hb), 3.25 (d, J = 10.1 Hz, 1Ha), 3.46, 3.65 (2s, 4H, 2CH2 of piperidinone ring), 3.69, 3.71 (2s, 6H, 2OCH3), 3.74,3.76 (2s, 6H, 2OCH3), 3.82, 3.84 (2s, 6H, 2OCH3), 4.19 (q, 2H, J = 6.9 Hz, CH2 of CH2CH3), 6.39–7.55 (m, 8H, Ar-H), 7.62 (s, 1H, CH=), 8.37 (s, 1H, D2O-exchangeable, NH). MS m/z (%): 729 (M+ + 2, 2), 728 (M+ + 1, 10), 727 (M+, 42), 726 (94), 347 (19), 392 (25), 275 (17), 167 (9). Anal. For C40H45N3O10 (727.81): calcd. C, 66.01; H, 6.23; N, 5.77. Found: C, 65.94; H, 6.14; N, 5.65.

3.5. Microbiological Assay

An agar diffusion well method was used to determine the antimicrobial activity. The microorganism inoculums were uniformly spread using a sterile cotton swab on a sterile Petri dish containing Malt extract agar (for fungi) and nutrient agar (for bacteria). Each sample (100 μL) was added to each well (6 mm diameter holes cut in the agar gel, 20 mm apart from one another). The systems were incubated for 24–48 h at 37 °C (for bacteria) and at 28 °C (for fungi). After incubation, microorganism growth was observed. The inhibition of bacterial and fungal growth was measured in mm. Tests were performed in triplicate [26].

4. Conclusions

In conclusion, we synthesized new derivatives of spirooxindole-spiropiperidinone- pyrrolidinesnd-spirooxindole-spiropiperidinone-pyrrolizinesthrougha 1,3-dipolar cycloaddition reaction of azomethineylides, generated from isatin, sarcosine, and l-proline through a decarboxylative route with dipolarophile 4a–j. All of the newly synthesized compounds were evaluated for their antimicrobial activities and the minimum inhibitory concentration (MIC) of the most active compounds against the test organisms. Four compounds from the series have emerged as potent antibacterial and antifungal agents.

Acknowledgments

The authors extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this Prolific Research group (PRG-1437-29).

Author Contributions

Huwaida M. E. Hassaneen, Hamid A. Eid and Thoraya A. Farghaly conceived and designed the experiments; Huwaida M. E. Hassaneen and Elshimaa M. Eid carried out the experiments; Huwaida M. E. Hassaneen, Elshimaa M. Eid and Yahia Nasser Mabkhot analyzed and interpreted the data; Huwaida M. E. Hassaneen, Thoraya A. Farghaly and Yahia Nasser Mabkhot prepared the manuscript. All authors have read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Nishiyama, Y.; Katahira, C.; Sonoda, N. A new method for the synthesis of 1,3-oxathiolan-2-ones by the reaction of epoxides with sulfur and carbon monoxide. Tetrahedron Lett. 2004, 45, 8539–8540. [Google Scholar] [CrossRef]
  2. Hassaneen, H.M.E. A novel one-pot three-components reaction: Synthesis of indeno[2′,1′:5,6]pyrido[2,3:4″,5″]pyrimido[2″,1″-c]triazole-5,7-dione. A new ring system. ARKIVOC 2007, 2007, 154–163. [Google Scholar]
  3. Hassaneen, H.M.E.; Abdelhamid, I.A. One-Pot Synthesis of 5-Unsubstituted-6-Aroyl[1,2,4]triazolo[1,5-a]pyrimidine Utilizing Biginelli-Like Multicomponent Reaction ofEnaminones with 3-Amino-1,2,4-triazole as the Urea Component. Curr. Org. Synth. 2013, 10, 974–976. [Google Scholar] [CrossRef]
  4. Weber, L. Multi-component reactions and evolutionary chemistry. Drug Discov. Today 2002, 7, 143–147. [Google Scholar] [CrossRef]
  5. Domling, A. Recent advances in isocyanide-based multicomponent chemistry. Curr. Opin. Chem. Biol. 2002, 6, 306–313. [Google Scholar] [CrossRef]
  6. Kobayashi, J.; Tsuda, M.; Agemi, K.; Shigemori, H.; Ishibashi, M.; Sasaki, T.; Mikami, Y. Purealidins B and C, new bromotyrosine alkaloids from the Okinawan marine sponge Psammaplysilla purea. Tetrahedron 1991, 47, 6617–6622. [Google Scholar] [CrossRef]
  7. James, D.M.; Kunze, H.B.; Faulkner, D.J. Two New Brominated Tyrosine Derivatives from the Sponge Druinella (=Psammaplysilla) purpurea. J. Nat. Prod. 1991, 54, 1137–1140. [Google Scholar] [CrossRef] [PubMed]
  8. Hilton, S.T.; Ho, T.C.; Pljevalijcic, G.; Jones, K. A New Route to Spirooxindoles. Org. Lett. 2000, 17, 2639–2641. [Google Scholar] [CrossRef]
  9. Pavlovska, T.L.; Redkin, R.G.; Lipson, V.V.; Atamanuk, D.V. Molecular diversity of spirooxindoles. Synthesis and biological activity. Mol. Divers. 2016, 20, 299–344. [Google Scholar] [CrossRef] [PubMed]
  10. Cravotto, G.; Giovenzana, G.B.; Pilati, T.; Siste, M.; Palmisano, G. Azomethine Ylide Cycloaddition/Reductive Heterocyclization Approach to Oxindole Alkaloids: Asymmetric Synthesis of (−)-Horsfiline. J. Org. Chem. 2001, 66, 8447–8453. [Google Scholar] [CrossRef] [PubMed]
  11. Daly, J.W.; Spande, T.W.; Whittaker, N.; Highet, R.J.; Feigl, D.; Noshimori, N.; Tokuyama, T.; Meyers, C.W. Alkaloids from dendrobatid frogs: Structures of two omega-hydroxy congeners of 3-butyl-5-propylindolizidine and occurrence of 2,5-disubstituted pyrrolidines and a 2,6-disubstituted piperidine. J. Nat. Prod. 1986, 49, 265–280. [Google Scholar] [CrossRef] [PubMed]
  12. Waldmann, H. Amino Acid Esters: Versatile Chiral Auxiliary Groups for the Asymmetric Synthesis of Nitrogen Heterocycles. Synlett 1995, 133–141. [Google Scholar] [CrossRef]
  13. Grigg, R.; Sridharan, V. Advances in Cycloaddition; Curran, D.P., Ed.; JAI Press: Greenwich, London, UK, 1993; pp. 161–204. [Google Scholar]
  14. Tsuge, O.; Kanemasa, S.; Katrizky, A.R. Advances in Heterocyclic Chemistry; Academic Press: New York, NY, USA, 1989; pp. 232–349. [Google Scholar]
  15. Grigg, R.; Surendrakumar, S.; Thiapatanagul, S.; Vipond, D. The decarboxylative route to azomethineylides. Stereochemistry of 1,3-dipole formation. J. Chem. Soc. Chem. Commun. 1987, 47–49. [Google Scholar] [CrossRef]
  16. Farghaly, T.A.; Abdallah, M.A.; Masaret, G.S.; Muhammad, Z.A. New and efficient approach for synthesis of novel bioactive [1,3,4]thiadiazoles incorporated with 1,3-thiazole moiety. Eur. J. Med. Chem. 2015, 97, 320–333. [Google Scholar] [CrossRef] [PubMed]
  17. Shawali, A.S.; Farghaly, T.A.; Hussein, S.M.; Abdalla, M.M. Site-selective reactions of hydrazonoyl chlorideswith cyanoacetichydrazide and its N-arylidene derivatives and anti-aggressive activity of prepared Products. Arch. Pharm. Res. 2013, 36, 694–701. [Google Scholar] [CrossRef] [PubMed]
  18. Farghaly, T.A.; Mahmoud, H.K. Site- and Regioselectivity of the Reaction of Hydrazonoyl Chlorides with Perimidine-ketene Aminal. Antimicrobial Evaluation of the Products. J. Heterocycl. Chem. 2015, 52, 86–91. [Google Scholar] [CrossRef]
  19. Elwan, N.M.; Hassaneen, H.M.E.; Hassaneen, H.M. Novel Synthesis of 4,5-dihydrospiropyrazole-5,2′-indane-1′,3′-diones. Synth. Comm. 2002, 32, 3047. [Google Scholar]
  20. Farghaly, T.A.; Abdallah, M.A.; Muhammad, Z.A. 2,7-Diarylidene-cycloheptanone, Hydrazonoyl Chlorides and Heterocyclic Amines as Precursors for Synthesis of Bioactive new Fused Cycloheptapyrimidine Derivatives. Curr. Org. Synth. 2016, 13, 291–299. [Google Scholar] [CrossRef]
  21. Hassaneen, H.M.; Hassaneen, H.M.E.; Mohammed, Y.S.; Pagni, R.M.Z. Synthesis, reactions and antibacterial activity of 3-acetyl[1,2,4]triazolo[3,4-a]isoquinoline derivatives using chitosan as heterogeneous catalyst under microwave irradiation. Naturforsch 2011, 66, 299–310. [Google Scholar] [CrossRef]
  22. Gomha, S.M.; Hassaneen, H.M.E. Synthesis and Antimicrobial Activity of Some New Pyrazoles, Fused Pyrazolo[3,4-d]pyrimidine and 1,2-Dihydroimidazo-[2,1-c][1,2,4]triazin-6-one Derivatives. Molecules 2011, 16, 6549–6560. [Google Scholar] [CrossRef] [PubMed]
  23. Farghaly, T.A.; Hassaneen, H.M.E. Synthesis of pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones 3 as potential antimicrobial agents. Arch. Pharm. Res. 2013, 36, 564–572. [Google Scholar] [CrossRef] [PubMed]
  24. Ameen, M.A.; Ahmed, E.K.H. Iodine catalyzed synthesis of α,α′-bis(substituted benzylidene)-1-carbethoxy-4-piperidone. J. Heterocycl. Chem. 2008, 45, 579–581. [Google Scholar] [CrossRef]
  25. Raj, A.A.; Raghunathan, R.A. High Regioselective Synthesis of Novel Dispiro[oxindole-cyclohexanone]pyrrolidines. Synth. Commun. 2003, 33, 421–426. [Google Scholar] [CrossRef]
  26. Esma, G.K.; Hatice, Ö.; Songül, A. Determination of the effect of gentamicin against staphylococcus aureus by using microbroth kinetic system. ANKEM Derg. 2009, 23, 110–114. [Google Scholar]
  • Sample Availability: Samples of the compounds 4, 6 and 8 are available from the authors.
Molecules 22 00357 sch001 550
Scheme 1. Synthesis of spiropyrrolidine bisoxindoles.
Scheme 1. Synthesis of spiropyrrolidine bisoxindoles.
Molecules 22 00357 sch001
Molecules 22 00357 sch002 550
Scheme 2. Synthesis of spiropyrrolizinebisoxindoles.
Scheme 2. Synthesis of spiropyrrolizinebisoxindoles.
Molecules 22 00357 sch002
Table
Table 1. Optimization of reaction condition.
Table 1. Optimization of reaction condition.
EntrySolventTemp (°C)Yield a (%)
1THF6054
2Toluene60trace
3CH3CN6072
4CH3OH6092
5EtOH6085
6Water60NR
7CH3OH3058
8CH3OH4566
a isolated yield based on isatin.
Table
Table 2. Yields of spiropyrrolidine and spiropyrrolizinebisoxindoles.
Table 2. Yields of spiropyrrolidine and spiropyrrolizinebisoxindoles.
EntryAr6 & 8Yield a (%)
1C6H56a92
24-NO2C6H46b95
34-ClC6H46c91
44-FC6H46d92
54-OCH3C6H46e84
63,4-Cl2C6H36f87
72,4-F2C6H36g89
83,4-OCH3·C6H36h79
94-F3CC6H46i86
103,4,5-OCH3 C6H26j81
11C6H58a90
124-NO2C6H48b88
134-ClC6H48c82
144-FC6H48d85
154-OCH3C6H48e80
163,4-Cl2C6H38f82
172,4-F2C6H38g83
183,4-OCH3·C6H38h81
194-F3CC6H48i79
203,4,5-OCH3 C6H28j87
a isolated yield based on isatin.
Table
Table 3. Antimicrobial activity of the synthesized compounds.
Table 3. Antimicrobial activity of the synthesized compounds.
Compd. No.Gram Positive BacteriaGram Negative BacteriaFungi
S. pneumoniaeB. subtilisP. aeruginosaE. coliA. fumigatusS. racemosumG. candidumC. albicans
6b42.34 ± 0.1574.25 ± 0.42NA49.25 ± 0.5342.36 ± 0.3349.32 ± 0.2563.25 ± 0.34NA
6c96.25 ± 0.297.24 ± 0.349.25 ± 0.174.29 ± 0.396.25 ± 0.174.25 ± 0.295.36 ± 0.291.29± 0.1
6e53.21 ± 0.4363.28 ± 0.53NA24.29 ± 0.2553.24 ± 0.5863.29 ± 0.2574.16 ± 0.38NA
6g49.21 ± 0.4453.21 ± 0.67NA21.34 ± 0.4625.36 ± 0.2549.24 ± 0.3449.24 ± 0.58NA
6i74.12 ± 0.6374.95 ± 0.32NA42.34 ± 0.4653.21 ± 0.3663.24 ± 0.4478.21 ± 0.58NA
6j63.24 ± 0.6374.21 ± 0.32NA34.25 ± 0.4642.15 ± 0.3649.21 ± 0.4453.21 ± 0.58NA
8b74.52 ± 0.4491.25 ± 0.63NA49.25 ± 0.2574.32 ± 0.3982.63 ± 0.1690.33 ± 0.58NA
8c34.25 ± 0.4442.67 ± 0.25NA22.14 ± 0.3339.25 ± 0.2539.46 ± 0.5842.67 ± 0.17NA
8e78.26 ± 0.3496.25 ± 0.25NA49.25 ± 0.5874.25 ± 0.6378.23 ± 0.2782.34 ± 0.35NA
8g95.44 ± 0.4495.34 ± 0.5822.14 ± 0.5874.21 ± 0.1953.22 ± 0.3474.25 ± 0.2592.68 ± 0.58NA
8i74.25 ± 0.4496.25 ± 0.63NA34.24 ± 0.2574.25 ± 0.3949.25 ± 0.5878.34 ± 0.58NA
8j74.65 ± 0.4378.34 ± 0.53NA42.37 ± 0.3382.65 ± 0.2578.34 ± 0.2598.25 ± 0.38NA
Ampicillin96.25 ± 0.297.24 ± 0.3NANANANANANA
GentamicinNANA49.25±0.174.29 ± 0.3NANANANA
Amphotericin BNANANANA96.25 ± 0.174.25 ± 0.295.36 ± 0.291.29 ± 0.1
NA: No activity.
Table
Table 4. Antimicrobial Activity as MICS (µg/mL) of tested samples against tested microorganisms.
Table 4. Antimicrobial Activity as MICS (µg/mL) of tested samples against tested microorganisms.
Compd. No.Gram Positive BacteriaGram Negative BacteriaFungi
S. pneumoniaeB. subtilisP. aeruginosaE. coliA. fumigatusS. racemosumG. candidumC. albicans
6c0.120.01515.631.950.0151.950.0150.06
8b1.950.06NA15.631.950.490.12NA
8e0.980.015NA15.631.950.980.49NA
8g0.0150.015NA1.957.811.950.03NA
Amphotericin BNANANANA0.0151.950.0150.06
Ampicillin0.0150.007NANANANANANA
Gentamicin NANA15.631.95NANANANA
NA: No activity.
Table
Table 5. Antimicrobial Activity as IC50 (µg/mL) of tested samples against tested microorganisms.
Table 5. Antimicrobial Activity as IC50 (µg/mL) of tested samples against tested microorganisms.
Compd. No.Gram Positive BacteriaGram Negative BacteriaFungi
S. pneumoniaeB. subtilisP. aeruginosaE. coliA. fumigatusS. racemosumG. candidumC. albicans
6c3.981.3436.2517.631.9616.371.452.16
8b16.352.92NA32.8418.3610.644.29NA
8e12.361.36NA32.1216.3211.329.25NA
8g1.731.24>12515.9821.3616.382.09NA
Amphotericin BNANANANA1.2514.271.162.48
Ampicillin1.531.06NANANANANANA
Gentamicin NANA20.3116.62NANANANA
NA: No activity.

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MDPI and ACS Style

Hassaneen, H.M.E.; Eid, E.M.; Eid, H.A.; Farghaly, T.A.; Mabkhot, Y.N. Facial Regioselective Synthesis of Novel Bioactive Spiropyrrolidine/Pyrrolizine-Oxindole Derivatives via a Three Components Reaction as Potential Antimicrobial Agents. Molecules 2017, 22, 357. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22030357

AMA Style

Hassaneen HME, Eid EM, Eid HA, Farghaly TA, Mabkhot YN. Facial Regioselective Synthesis of Novel Bioactive Spiropyrrolidine/Pyrrolizine-Oxindole Derivatives via a Three Components Reaction as Potential Antimicrobial Agents. Molecules. 2017; 22(3):357. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22030357

Chicago/Turabian Style

Hassaneen, Huwaida M. E., Elshimaa M. Eid, Hamid A. Eid, Thoraya A. Farghaly, and Yahia Nasser Mabkhot. 2017. "Facial Regioselective Synthesis of Novel Bioactive Spiropyrrolidine/Pyrrolizine-Oxindole Derivatives via a Three Components Reaction as Potential Antimicrobial Agents" Molecules 22, no. 3: 357. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22030357

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