Synthesis of diversely substituted bis-pyrrolizidino/ thiopyrrolizidino oxindolo/acenaphthyleno curcuminoids via sequential azomethine ylide cycloaddition

Curcumin has been transformed to several diversely substituted bis-pyrrolizidino/thiopyrrolizidino oxindolo/acenaphthyleno curcuminoids via a sequential azomethine ylide cycloaddition reaction using isatins/acenaphthoquinone and proline/thioproline as the reagents. The products were separated via extensive chromatography and characterized by 1D/2D NMR and HRMS analysis.


Introduction
Incorporating diversity in the synthesis of combinatorial libraries of small molecules for biological screening is an emerging eld. 1 Rather than being directed toward a single biological target, diversied libraries can be used to identify new ligands for a variety of targets. It is hoped that the range of molecular architectures and potential bonding interactions present in a diversied library can provide interesting and specic biological activity across a range of targets. Although various chemical libraries are now available commercially, these remain focused primarily on so called 'drug-like' compounds. 2 Because these libraries are concentrated in a relatively narrow region of chemical structure space, it seems unlikely that they will provide useful probes for all biological targets of interest. 3 The crucial factor for achieving success in drug discovery is not the size of the library but its structural diversity. 4 Several different strategies for library design have therefore been developed to target the biologically relevant regions of chemical structure space. DOS has provided powerful probes to investigate biological mechanisms and also served as a new driving force for advancing synthetic organic chemistry.
To provide cyclic and heterocyclic compounds with a high degree of structural complexity as well as skeletal and stereochemical diversity, dipolar cycloaddition reaction has emerged as a potential tool. 5 Its ability to generate new stereocenters has allowed it to contribute very much to the development of stereo structure-activity relationships during screening campaigns. In particular, the sequential multicomponent reaction 6 and sequential azomethine ylide (1,3 dipole) cycloaddition approach has emerged as one of the efficient strategies which can provide diverse spirooxindoles in an operationally simple procedure from readily available chemical reagents. 7 Keeping the above facts in mind we started our journey of preparing diversely functionalized heterocycles via azomethine ylide cycloaddition using simple commercially available 8 or synthetic dipolarophiles. 9 We then extended it to a new dimension by employing dipolarophiles available from nature like andrographolide, 10 withaferin A, 11 curcumin 12 etc. Several compounds have been prepared and biological activity evaluation revealed some very promising increment in activity. 13,14 In continuation of our molecular diversity programme, very recently we have synthesized various spirooxindolo super curcumin analogues 12 to overcome the drawbacks related to the bioavailability of curcumin (less water solubility, easy metabolism and excretion) with comparable or better efficacy. An equally compelling motivation for their synthesis lies in their unique and formidable structure, the central feature being the biologically important curcumin and isatin (oxindole) units 12 likewise done by various other group by synthsizing various diversied heterocyclic analogs of curcumin and their bioevaluation. [15][16][17][18] We have so far succeeded in synthesizing a library of pyrrolizidino spirooxindolo curcumins, some with better and equal cytotoxic/antioxidant and antibacterial activity 19 but with much more specicity and solubility compared to curcumin. These results of biological evaluation encouraged us to construct a better diversied library applying the sequential azomethine ylide cycloaddition strategy, coupling isatin, substituted isatins or acenapthoquinone with proline or thioproline as the amino acid component.
The products (4Aa-Ad) were characterized from detailed spectral studies. All gave the same pseudo molecular ion peaks at m/z 895 [M + H] + and 917 [M + Na] + in ESI-Q-TOF MS, indicating them to be isomeric. In the 13 C NMR spectra, they displayed 45 carbon signals due to the absence of symmetry (present in the previously reported 12 diastereomers) due to the difference in substitution pattern in the two oxindole rings (originating from isatin substitution).
However, as in the previous publication, 12 the signals for the aromatic ring of curcumin remained virtually unaltered in the spectra of the products. The chemical shis for the nuclei belonging to the a,b-unsaturated-diketone part of curcumin were of course distinctly perturbed, with C3/25 and C4/26 suffering profound alteration from downeld to upeld resonance positions.
It is obvious from Table 1 that in the entries 17-20 and 21-24 only the sequence of addition of 5-uoroisatin (2E) and 5,7dimethylisatin (2F) has changed. This leads to the formation of (AE)-4Ea-Ed and (AE)-4Fa-Fd. From a simple analysis of the reaction prole and structure of the product it could be easily concluded that 4Ea, 4Eb are identical with 4Fa, 4Fb respectively whereas 4Ec, 4Ed are not identical with 4Fc and 4Fd due to different substitution present in oxindole ring. In fact this was also proved from the detailed NMR spectral comparison of all the products. Detailed possibilities in change of sequential addition with different isatins have been schematically Scheme 1 Synthesis of mixed bis-pyrrolizidino dispiro-oxindolo curcuminoids.  represented using 5-uoroisatin (2E) and 5,7-dimethylisatin (2F) as model example in Fig. 1. Because of the possibility of formation of two other diastereomers (AE)-4(A-F)c 0 and (AE)-4(A-F)d 0 (Fig. 1), positional isomers of (AE)-4(A-F)c and (AE)-4(A-F)d due to different substitution in isatin ring, there is the extra advantage of more diversication. But it is necessary to separate (AE)-3(A-F)a and 3(A-F)b/(AE)-3(A-F) a 0 and 3(A-F)b 0 synthesized in the rst step to avoid formation of complex mixture of compounds in one pot sequence reaction, difficult to separate.
Following the success in the synthesis of mixed bispyrrolizidino dispiro-oxindolo curcuminoids we wanted to synthesize mixed pyrrolizidino-thiopyrrolizidino dispirooxindolo curcuminoids (Scheme 2). But to conrm that the reaction indeed proceeds with thioproline (thiazolidine-4carboxylic acid), we treated curcumin and isatins with thiazolidine-4-carboxylic acid in 1 : 2 : 2 mole ratio. In this case we obtained only two diastereomers (AE)-5Aa and (AE)-5Ab in around 70% yield in 15-16 h; both are symmetric in nature (conrmed from the 21 carbon peaks present in 13 C NMR).
In case of the reactions where thioproline was used, the reaction rate and also the yield of the product were lower compared to those with proline perhaps due to lower solubility of thiazolidine-4-carboxylic acid (Table 2) in methanol.
As usual the products were characterized from detailed spectral studies. Both the products (AE)-6Aa and (AE)-6Ab gave the same peaks at m/z 817 [M + H] + /839 [M + Na] + in the ESI-Q-TOF MS spectrum for the pseudomolecular ions, indicating them to be isomeric. Both showed 45 carbon signals in 13 C NMR spectra indicating a break in symmetry due to the change in amino acid as well as for the difference in substitution in the isatin ring.
Scheme 3 Synthesis of mixed pyrrolizidino-thiopyrrolizidino dispirooxindolo curcuminoids. Table 2 Yields of mixed bis-thiopyrrolizidino dispiro-oxindole 6(A-E)(a-b) derived from curcumin, isatins, proline and thioproline It is worth mentioning here that the adjacent hydrogens on both side of the diketone functionality of the curcumin unit are anti to each other, whereas they were in syn orientation with the spirocyclic oxindole bond of the respective sides.
The products were again characterized from detailed spectral studies. The product (AE)-7B showed peak at m/z 930 ascribed to [M + H]

Conclusion
In conclusion, curcumin has been successfully used in a two step sequential 1,3-dipolar azomethine ylide cycloaddition reaction to produce novel mixed bis-pyrrolizidino/thiopyrrolizidino dispiro-oxindolo/acenaphthyleno curcuminoids. The structures of the products were determined by 1D/2D NMR analysis and MS. The unaltered pharmacophores of curcumin along with the newly generated diversely mixed spiro oxindolo pyrrolizidine moiety might add to the biological effectiveness of the scaffold with increased polarity and solubility.

General information
All the compounds evaluated in this work were synthesized in one-pot sequences. Melting points were determined in capillaries and are uncorrected. IR spectra were recorded as KBr Table 3 Yields of mixed bis-pyrrolizidino/-thiopyrrolizidino dispirooxindolo-acenaphthylino curcuminoids 7A-D a Unless otherwise noted, the reaction was performed with 1.76 mmol of 3A-D(a) (AE), isatins and proline in 50.0 mL of MeOH under reux for 8 h. b Determined aer isolation. Scheme 4 Synthesis of mixed bis-pyrrolizidino/-thiopyrrolizidino dispiro-oxindolo-acenapthylino curcuminoids. pellets using a JASCO 410 FTIR spectrometer. The NMR spectra were recorded using a Bruker 600 DPX spectrometer operating at 600 MHz for 1 H and 150 MHz for 13 C in pyridine-d 5 and the chemical shis are reported in d units. Mass spectra (positive mode) were obtained on a ESI-Q-TOF micro mass spectrometer in the electrospray ionization mode. Curcumin was isolated from Curcuma longa in the usual way. Isatins, acenaphthoquinone and a-amino acids were purchased from Alfa-Aesar Company. All other solvents and chromatographic absorbents were procured from E. Merck (Germany) and SRL (India) Ltd. unless otherwise indicated. Thin layer chromatography was performed on pre-coated silica gel 60 F 254 aluminum sheets (E. Merck, Germany) using the solvent system 5% MeOH in CHCl 3 and spots were observed using UV irradiation and iodine. Compounds were separated using AKROS -"Automatic TLC Smart Flash" of Yamazen Corporation.
Typical experimental procedure for synthesis of mono cycloaddition products (AE)-3Aa A mixture of 1 (13.6 mmol, 5 g), isatin (13.6 mmol, 2.00 g) and proline (13.6 mmol, 1.56 g) was taken in a round bottom ask, dissolved in 200 mL methanol, and heated to reux for specied time period. Aer completion of the reaction as evident from TLC, the solvent was removed and the crude product was subjected to ash chromatography using increasing concentration of methanol in chloroform as eluant. The product was crystallized from chloroform-methanol mixture (85%, 6.56 g).