Efficient synthesis of some new antiproliferative N-fused indoles and isoquinolines via 1,3-dipolar cycloaddition reaction in an ionic liquid

Syntheses of some new pyrrolo-fused pyrrolo[1,2-a] indole derivatives have been achieved by combining N-allyl-indole-2-carbaldehyde with a variety of N-alkyl-glycine esters as well as tetrahydroisoquinolines in an ionic liquid, triethylammonium acetate (TEAA), a recyclable reaction medium, via intramolecular [3+2] cycloaddition reaction. This new method is highly efficient, and the ionic liquid employed is recyclable. The stereochemistry of all the compounds was confirmed by 2D NMR NOESY and in some cases single crystal X-ray diffraction data. The in vitro screening of all new candidates against various bacterial strains and representative human solid tumor cell lines, A549 (lung), HeLa (cervix), SW1573 (lung), T-47D (breast) and WiDr (colon), revealed that many of them have good antibacterial, antifungal and antitubercular and antiproliferative activities.


Introduction
The heterocycles with a N-fused indole/isoquinoline-heterocyclic framework as one of their integral structural units constitute an important class of bioprofiles, and have attracted the interest of many in the research fields of medicines and pharmaceuticals. 1 Containing the pyrrolo-indole nucleus, terpenoidal alkaloids 2 and mitomycins exhibited antitumor activity (Fig. 1A). 3 With pyrrolo [1,2-a]indole as a specific fusion, cyclopropamitosenes revealed cytotoxicity through bacterial cell division and DNA alkylation. 4 Likewise, antimalarial Flinderole B, 5 bioactive Isatisine A (Fig. 1B) 6 and hallucinogenic Yuremamine (Fig. 1C), which are potential candidates of this class have attracted the interest of many chemists and biochemists. 7 Pyrrolo-isoquinoline alkaloids, on the other hand, showed promising results for depression in animals, with the pyrroloisoquinoline nucleus. 8 A tricyclic hydropyrrolo [2, 1-a] isoquinoline is a key fused-ring system with (2)-trolline structure, which can be derived from Trollius Chinese flowers 9 and its Portulaca oleracea L weed-derived antipode (+)-oleracein E. 10,11 Studies showed that (2)-trolline has remarkable activity against influenza viruses A and B, and so revealed antiviral properties. (+)-Oleracein E displayed DPPH-radical scavenging activity. 10 In addition, the members of the trolline family are active against respiratory Staphylococcus aureus and pneumonia bacteria. 9 Like trolline, (+)-crispine A (Fig. 1D) also incorporates an analogous heterocyclic framework and has potential anti-proliferative activity against SKOV3, KB, and HeLa human cancer lines. 12 Finally, pyrrolizidine present in many heterocycles represents another useful framework of biological interest. In addition to anti-inflammatory and analgesic properties, it confers heterocycles with effective aromatase and tumour growth inhibitory action. 13 Besides, 5-HT 2c receptor agonists, known to be effective in hyperglycaemia and other diseases, come from the same family and make it an interesting source of bioactive scaffolds. 14 In view of the above, it follows that incorporation of N-fused indole, N-fused isoquinoline and pyrrolizidine into a molecular assembly helps generate a new and useful class of heterocycles, finding a great importance in medicinal chemistry. Development and enlargement of a molecular library of these heterocycles is thus desirable and worth studying too.
Intermolecular alkylation, 15 radical cyclization, 16 trans annulation reaction, 17 carbene-based rearrangement, 18 etc. are general synthetic approaches for N-fused indoles. Cycloaddition reaction involving in situ azomethine ylide generation, nevertheless, seems to be seldom studied for such heterocycles. 19 This protocol is highly atom economic, and has been used so far for many complex heterocyclic systems. Many aldehydes reacted with amino acid derivatives 20 to afford 6-5 fused-ring systems. Indole-based substrates however are very few in number for designing 5-5 fusedring systems. 21 The ring system exists in biotin, an important naturally occurring heterocycle. 21d As part of our research program, we have reported the synthesis of many pyran-heterocycles. 20h including aminobenzopyrans 20h,i via 1,3 dipolar cycloaddition. Here, we describe pyrroloindole and pyrrolo-isoquinoline derivatives as new bioprofiles, constructed from N-allyl-3-chloro-indole-2-carbaldehyde 1 with a variety of a-amino acid esters as well as tetrahydroisoquinolines, in ionic liquid TEAA as an effective reaction medium.
Use of ionic liquids (ILs) helped translate many synthetic routes into green methodologies, with adopting requisite practice not only as economic one, but as environmentally friendly too. 22 To the best of our knowledge, very few reports appeared on use of ionic liquid in 1,3-dipolar cycloaddition reaction. 23 And those reported in ILs still suffer from preparation cost, use and recyclability of ILs, and, of course, promotion of the reaction. 23c Conventional methods in general suffers from many disadvantages like use of conventional solvent, longer reaction time and tedious work-up procedure. 24

Results and discussion
Chemistry All requisite secondary amines 2-6, except tetrahydro-isoquinolines 6; N-methyl/ethyl/benzyl/(4-morpholinyl) ethyl glycine esters, are liquids and prepared by methods reported elsewhere. 25 Teterahydroisoquinolines 6 were used as received from commercial sources. The substrate, N-allyl-3-chloro-indole-2-carbaldehyde 1, was obtained by reacting 3-chloro-indole-2-carbaldehyde with ally bromide in the presence of anhydrous K 2 CO 3 , suspended in DMF (dimethylformamide) solution (Scheme 1), in 95% yield. 3-Chloroindole-2-carbaldehyde was obtained as the Vilsmeier-Haack product of (phenylglycine)-O-carboxylic acid, with excellent purity. 26 The reaction between amine 3a and aldehyde 1 was taken as a model to optimize the reaction conditions (Table 1). Initially, we heated the aldehyde and amine in refluxing methanol (entry 1), acetonitrile (entry 2), toluene (entries 3 and 4) and xylene (entries 5 and 6), in the presence and absence of Na 2 SO 4 . Although results in toluene and xylene using Na 2 CO 3 were good, prolonged heating was discouraging to accept this method (6 h). Even when tried reported methods, they failed to run present conversion effectively. 22c,d Thus, we opted for heating in a solvent-free environment at 100 1C. Here, the reaction time could be improved but not the yields (entry 7). The conventional way was thus abandoned in favour of the TEAA promoted one (entry 8). It showed improved results in yield and reaction time at 80 1C that were improved further at 100 1C (entry 9). Above 100 1C, however no further improvement was seen. This improved method was then generalised to receive other products, too (Scheme 2 and Table 2). The advantage of the present protocol is that it takes relatively less reaction time (3 h) than others. 24c It should be noted further that the present combination allows pyrrolizidine to act as a highly significant bioactive unit to incorporate effectively into N-fused indole/isoquinoline heterocycles.
A plausible mechanism of the reaction has been depicted in Scheme 3. Addition of electrons from the tethered-alkene terminal carbon on imine follows the addition of the enolized ester on the other end of the alkene via a semi bicyclic transition state that may force the system to adopt the most favoured cis-fusion product. The exo or endo attack of dipolarophile alkene on azomethine ylide dipole determines the stereoselectivity of the reaction. The spectral data support the cis-fusion between central pyrrolidine rings in all heterocycles, hence favouring the endo transition state. In 11 0 a-b, however, the cis-fusion may involve a further isomerization of the ylide. 27 Scheme 1 Synthesis of N-allyl-3-chloro-indole-2-carbaldehyde 1, the reagents and conditions (1) K 2 CO 3 , allyl bromide, DMF, 10-12 h, room temp. The proposed structures of all newly synthesized heterocycles fully agreed with the observed elemental analyses, mass, IR and NMR spectral data. In the 1 H NMR spectra, all compounds except 7 showed a multiplet in the region d 2.93-4.09 ppm, due to diastereotopic methylene protons of the pyrrolidine ring. In 7, instead the N-methyl proton appeared as a singlet at d B 2.75 ppm. The carbonyl group in 7-10 showed a characteristic IR band at B1730 cm À1 , and a 13 C NMR signal Bd 173 ppm. In all compounds, a doublet in the d 4.00-4.80 ppm range, with the J value in the 7.6-8.4 Hz range, can be attributed to a bridge-head proton 10b or 12b, suggestive of cis-fusion between central pyrrolidine rings. Another bridgehead proton 3a or 5a appeared as a multiplet at d B 3.80 ppm is therefore orientated cis to this 10b or 12b proton. Analysing cross-peaks in 2D NMR NOESY (nuclear Overhauser effect spectroscopy) and DQF-COSY (double quantum filtered correlation spectroscopy) of representative 8a ( Fig. 2 and 3), a similar relationship between these protons could be confirmed. The bridge-head proton 2 or 4b is however trans with respect to proton 10b or 12b, appearing at d 4.00-4.40 ppm, except in 11 0 a-b. In 11 0 a-b, it is cis oriented.
Finally, with the single-crystal X-ray diffraction data of 9b and 11 0 b, we could be able to establish important stereochemical considerations (Fig. 4).

Biological results
Table 3 summarizes in vitro antimicrobial screening test results of all the compounds. The majority of compounds displayed good resistance against bacteria, at least, in line with one of the standard reference drugs ampicillin. The activity of some of them was found to be equal to that of a more potent drug. Analyzing results in terms of maximum how many bacterium species a compound can kill effectively revealed that the compound can cover a maximum of five species at least with the potency equivalent to ampicillin. Examples include 7c, 9c, Scheme 2 Synthesis of all newly pyrrolo-fused indoles and isoquinolines (1) TEAA, 100 1C. 10e and 11b. Among them, 7c revealed excellent activity against Gram-positive Clostridium tetani (reaching to potency of even more potent norfloxacin) and Gram-negative Escherichia coli bacteria (reaching the potency of even more potent chloramphenicol). Similarly, compound 9c resembled more potent standard norfloxacin drug in activity, against Grampositive Bacillus subtilis bacteria. Compounds 7a, 7d, 8a, 8c, 8d, 9e, 10a, 10b, 10c and 11a, on the other hand, had recorded better resistance against at least four types of bacteria. Among them, 7a, 7d, 8c, and 10a registered excellent activity against Gram-positive Clostridium tetani bacteria, with MIC values in line with ciprofloxacin which is more potent than ampicillin. Compounds 7d and 10a with chloramphenicolequivalent potency showed good results against Bacillus subtilis and Salmonella typhi bacteria respectively. It is noted that both ciprofloxacin and compound 7d recorded similar MIC values against Bacillus subtilis bacteria. Next, those with at least activity against three types of bacterium species include 7b, 7e, 8b, 8e, 9b, 9d and 10d. Among them 8b, 8c and 9d are very close to standard ciprofloxacin in potency, against Clostridium tetani bacteria. Further, a few of the compounds showed good antifungal activity, particularly against Candida albicans fungus. Examples include 7a, 7e, 8c, 8e, 9c and 10d all having griseofulvin-equivalent power.
As anti-fungal agents, compounds 7d, 10a and 10b are relatively more active.
Scheme 3 A plausible mechanism of the reaction between aldehyde 1 and secondary amine via 1,3-dipolar cycloaddition reaction.

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Anti-tubercular activity study shows that compounds 9d and 10b have highest M. tuberculosis H37Rv bacterial resistance, with growth inhibition in the 90-100% range. Compounds 8a, 9a, 9c, 10a and 11 0 b have growth inhibition in the 80-90% range.
FRAP values of majority of the heterocycles are around 225 (mmol per 100 g), indicating that they are moderate in antioxidant power. However, compound 10b revealed remarkable activity.
The antiproliferative activity of 7-11 was evaluated against a panel of representative human tumor cell lines including A549 (lung), HeLa (cervix), SW1573 (lung), T-47D (breast) and WiDr (colon), using the SRB assay. 28 The experimental GI 50 values are summarized in Table 4 and compared to those of cisplatin, etoposide and camptothecin after 48 h of treatment. Taken as a whole, pyrrolo-fused-indoles 7-8 was the most active class of compounds, with activity against all cell lines tested. In this particular context, compounds 7-8 showed more activity against HeLa cells, with GI 50 values in the range 3.1-14 mM and comparable to those of cisplatin (2.0 mM) or etoposide (3.3 mM).
No significant differences were observed between methyl and ethyl ester derivatives. In contrast, the presence of a benzyl group (9) or a morpholine substituent (10) on the pyrrol nitrogen produced a severe loss in activity. From the pyrrolofused-isoquinolines 11 obtained in our investigations, the best results of antiproliferative activity were obtained for adduct 11b, which showed active against all the cell lines with GI 50 values in the range 9.5-18 mM. This is a relevant result, since the class of adduct correlates to selectivity towards cancer cell lines.
Analysing N-fused indoles derived from amino acid esters, structurally, it reveals that methyl at pyrolidine nitrogen confers heterocycles with enhanced resistivity against Clostridium tetani and Escherichia coli bacteria, when carbpropoxy moiety is present at carbon next to this nitrogen. Pyrrolidin with morpholine or ethyl moieties at nitrogen in combination with carbmethoxy group also had a similar effect against these bacteria. Carbbutoxy moiety, on the other hand, had very less effect on activity, irrespective of substituent present at pyrrolidine nitrogen. Antiproliferative activity, nevertheless, seemed to be altered very less taking any ester component with N-methyl/ N-ethyl pyrrolidine ring, against HeLa (cervix) cell lines. In addition, N-ethyl pyrrolidine with any ester component had similar effect against Widr(colon) cell lines. The N-fused indoles derived from tetrahydreoisoquinolines showed remarkable bioactivities as well. In general, heterocycles derived from electron releasing methoxy substituted-tetrahyderoisoquinoline are excellent in antibacterial, antitubercular, antioxidant and antiproliferative activities, compared to the ones derived from simple tetrahydro isoquinoline. Fig. 4 The ORTEP views of compounds 9b and 11 0 b, with displacement ellipsoids at the 40% probability level.

Conclusions
Thus, we synthesized N-fused indole and isoquinoline derivatives as new bioactive compounds via 1,3-dipolar cycloaddition reaction, using an ionic liquid, TEAA as inexpensive, environmentally friendly and recyclable reaction medium. The resistance power of 7c, 7d, 8a and 10a at least against one of the bacterial species studied is highest resembling chloramphenicol in activity with MIC 62.5 mg L À1 . The antiproliferative activity of 11b, on the other hand, was found to be excellent against all cell lines, with GI 50 values lying in the 9.5-18 mM range, comparable to standard drugs used. Besides, 10b, with the FRAP value of 225 (mmol per 100 g), showed excellent ferric reducing antioxidant power.

Materials and methods
All solvents and reagents were used as supplied from commercial sources. The recorded melting points are uncorrected. IR spectra were recorded in KBr on a Shimadzu FT-IR 8401 spectrometer and are reported in wave numbers (cm À1

General procedure for the synthesis of N-fused indoles and isoquinolines
A mixture of an aldehyde 1 (1 equiv.) and an acyclic secondary amine 2-5 (1 equiv.) or isoquinoline 6a-b (1 equiv.) in 2 mL of ionic liquid TEAA in a round bottom flask was heated at 100 1C and completion of the reaction was confirmed through TLC. The reaction mass was cooled to room temperature and poured into ice species. The oily product thus emulsified was then extracted with three 10 mL of diethyl ether portions. It yielded crude products in quantitative amounts upon removal of ether. Finally, the product was purified by column chromatography using a 90 : 10 n-hexane-ethyl acetate mixture as an eluent. The yields were in the 75-85% range. TEAA was recovered quantitatively by heating the aqueous layer-left after the ether extraction of the product-under the reduced presser at 80 1C. The recovered ionic liquid can be used again for the same. It was noticed that TEAA can be recycled at least four-times with its unaltered efficiency.