Chapter 2: Synthesis of Amaryllidaceae Alkaloids (Ismine, Trisphaeridine and Bicolorine)

CHAPTER 2: SYNTHESIS OF AMARYLLIDACEAE ALKALOIDS (ISMINE, TRISPHAERIDINE AND BICOLORINE)

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2.1 Introduction Plants and natural products have played a long a crucial role in the treatment of various illnesses. They provide valuable sources of compounds with a wide variety of chemical structures and biological activities and have provided important prototypes for the development of novel drugs.1 It is impossible to overrate the importance of natural extracts as potential sources of new drugs. It is estimated that the plant kingdom comprises about 250,000 species, of which approximately 7% have been studied for biological activity and about 17% phytochemically.2 The Amaryllidaceae family consists of about 75 genera, whose 1100 species are widely spread in several countries around the world. Plants from the Amaryllidaceae family are used for the production of volatile oil. They are also cultivated as ornamental plants for their beautiful ﬂowers. Amaryllidaceae plants are extensively used in traditional medicine throughout the tropics. They are used for their pharmacological effects and are frequently associated with several typical synthesized alkaloids.3 With the isolation of lycorine from N. pseudonarcissus,4 the study of Amaryllidaceae alkaloids began in 1877 and the interest around this group of naturally occurring compounds has increased with time because of their effective antitumoral and antiviral activities. Lycorine (3) (Figure 1) is a pyrrolo[de] phenathridine ring type alkaloid extracted from different Amaryllidaceae species, whose structure was elucidated by Nagakawa et al. in 1956.5 Lycorine got increasing attention due to its ability to inhibit ascorbic acid synthesis in vivo,6 the chemical and biological properties of this interesting alkaloid are being further investigated.7 The alkaloids of the Amaryllidaceae family are mainly differentiated in to nine structural types which are compounds of crinine, galanthamine, lycorine, lycorenine, montanine, narciclasine and tazettine as shown in Figure 1.8 Hundreds of new alkaloids isolated from different parts and in different vegetative phases of ca. 150 species belonging to 36 genera can be grouped into 12 distinct ring types (Table 1).9,10 The structures of a representative alkaloid of each ring type are shown in Figures 1, 2, 3, and 7. Plants of the Amaryllidaceae family are a well known source of tetrahydroisoquinoline alkaloids with a wide range of biological activities, including antitumoral, antiviral, psychopharmacological, antiparasitic, and acetylcholinesterase inhibitory, among others.11 Extracts from the plants are applied in ethnopharmacology for different diseases. One of the most important compounds is galanthamine (32) (Figure 3), an inhibitor of acetyl cholinesterase, which is registered as a drug for Alzheimer’s disease.12

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Figure-1: Ring types and representative Amaryllidaceae alkaloids.

Narwedine (4) potentiates the pharmacological effects of caffeine, carbazole, arecoline, and to a lesser extent nicotine in laboratory animals. Nnarwedine and vittatine (12) potentiate the analgesic effects of suboptimal doses of morphine.13 Number of Amaryllidaceae alkaloids cause a transient fall in blood pressure in laboratory animals in high doses. Narwedine (4), galanthamine (32) and epigalanthamine (33) can produce signiﬁcant hypotensive effects in mice. Galanthamine show the acetylcholinesterase inhibitory activity and ability to amplify the nerve-muscle transfer.14 Extracts of several Amaryllidaceae plants were also found to possess pronounced antibacterial and antifungal activities.15 A number of species of the genus Amaryllis have been used in folk medicine, including Amaryllis belladonna L. (also named Hippeastrum equestre), which is cultivated in Egypt as an ornamental plant.16 Six alkaloids have been isolated from the bulbs of Amaryllis species, namely, lycorine (3, Figure 1), hippeastrine, pancracine, vittatine, 11-hydroxyvittatine, and amarbellisine (10-14, Figure 2).17 Amarbellisine, pancracine, vittatine, and 11-hydroxyvittatine have antibacterial activity against Gram-ve Escherichia coli, while pancracine also showed activity against Pseudomonas aeruginosae. Furthemore, all alkaloids from Amaryllis species, especially

128 lycorine, amarbellisine, and hippeastrine, showed antifungal activity against Candida albicans.17

Table -1: Ring types and representative Amaryllidaceae alkaloids. Ring Type Alkaloid Structure I N-(3,4-dioxybenzyl)-4-oxyphenethylamine norbelladine 1 II N-(3.4-dioxybenzyl)-3,4dioxyphenethylamine rystilline 2 III pyrrolo[de]phenanthridine lycorine 3 IV lycorenine hippeastrine 10 V galanthamine narwedine 4 VI 5,10b-ethanophenanthridine haemanthamine 5 VII 1,2-epoxy-5,10bethanophenanthridine 1,2-epoxy- 6 ambelline VIII pretazettine pretazettine 7 IX tetrahydroisoquinoline cherylline 8 X phenanthridone/lignoid crinasiadine 43 XI clivimine clivimine 18 XII ismine ismine 9

Amaryllidaceae alkaloids are antitumor potential and, also exhibited in vivo activity against various human viruses. Some members from the Amaryllidaceae family are toxic and cause symptoms such as dizziness, nausea, headaches, heartbeat irregularities, excessive salivation, visual disturbance, and dermatitis. Alkaloids from Ammocharis family are widely distributed genus, generally inhabit seasonal wet places. Ammocharis coronica contains biochemicals and triterpenoids in its bulbs, which is known for toxicity. Therefore, instead of oral administration, fresh, wet scales are cooked and used as enemas for blood cleansing or applied topically to open wounds or boils.18 Plants of the Bophane genus produce large bulbs and characteristic alkaloids. Bophane disticha (L. f.) herb is toxic plant containing compounds with alleged hallucinogenic potential. Bulb scales or infusions not only used on septic wounds and external sores, but also for rheumatism and relief of pain. Decoctions are also used for the treatment of headaches, cramps, and internal pains.18

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Figure-2: Alkaloids from A. belladonna L.; hippeastrine (10), pancracine (11), vittatine (12), 11-hydroxyvittatine (13), and amarbellisine (14).

The BrunsVigia genus produces large bulbs containing a number of alkaloids having signiﬁcant antineoplastic, antimalarial, and cytotoxic activity. The bulbs are applied as antiseptic dressings on fresh wounds, while bulb decoctions are administered for the treatment of abdominal, colds, coughs, renal, and liver complaints.18 CliVia species, (CliVia miniata or CliVia nobilis) are cultivated in Egypt as ornamental plants for their beautiful ﬂowers. A root infusion of C. miniata regel is used to treat snake bites and wounds. A South African woman takes roots and leaves during pregnancy and child birth. Aqueous leaf extracts have proven to augment or induce labor. Bulb decoctions are used against infertility and urinary complaints.18 A number of CliVia species are reported to contain Amaryllidaceae alkaloids represented by the 3a,4-dihydrolactone[2]-benzopyrano[3,4g]indole ring system. This contains clivonine (15, Figure 3), isolated from C. miniata and clivatine, clivimine, nobilisine, and nobilisitine A and B (16, 18, 19, 20 and 17, Figure 3) isolated from C. nobilis.(+)-8- Demethylmaritidine (21, Figure 3), a crinine type alkaloid, was also isolated from this species.10 The antimicrobial activity of the alkaloid extract from C. nobilis was tested against Gram +ve (S. aureus) and Gram –ve (E. coli and P. aeruginosae) bacterial strains as well as fungi (C. albicans). All alkaloids with the exception of clivimine showed antibacterial activity against Gram -ve S. aureus. The same compounds, in particular, nobilisitine B, exhibited antifungal activity against C. albicans.19

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Figure-3: Alkaloids from Clivia spp., clivonine (15), clivatine (16), clivimine (18), nobilisine (19), nobilisitine A (20) and B (17), and (+)-8-demethylmarilidine (21).

Two new pyrrolophenanthridone alkaloids, pratorimine and pratosine (24 and 25, Figure 4), were isolated from the bulbs of Crinum latofolium and characterized by spectral analysis, chemical transformations, and synthesis.20 The bulbs of C. jagus and crinum glaucum are used in traditional medicine for memory loss in southern Nigeria and other mental symptoms associated with aging. Alkaloids from each species show inhibition of acetylcholinesterase. The most active alkaloids are hyamine (23, Figure 4) and lycorine (3, Figure 1), while other alkaloids show comparatively less activity, such as haemanthamine and crinamine (5, Figure 1 and 22, Figure 4). Cholinesterase activity seems to be due to the presence of two free hydroxyl groups in this structural type of Amaryllidaceae alkaloids.21 The use of Crinum extends to animals, such as the treatment of weight loss, low milk production, milk loss, or for healthy calves and placenta retention in cattle.22 The structures of the Crinum alkaloids are derived in to three fundamental nuclei i.e., N- (3,4-dioxybenzyl)-4-oxyphenethylamine (norbelladine, 1, Figure 1), pyrrolo[de]

131 phenanthridine (lycorine, 3, Figure 1), and 5,10b-ethanophenanthridine (vittatine or crinine, 12, Figure 2, 29 and 6).

Figure-4: Selected alkaloids from Crinum spp.: crinamine (22), hyamine (23), pratorimine (24), and pratosine (25).

The genus Cyrtanthus species are used in South African as a traditional medicine. C. mackenii show activity against storms and evil. Other related species are used to treat diseases like chronic coughs, scrofula, cystitis, headache, and leprosy and also used during pregnancy and child birth. The Cyrtanthus plants are divided in to 51 species in southern tropical Africa.23

Figure-5: Alkaloids from N. Tazetta L.: homolycorine (26), its 9-O-demethyl derivative (27), and tazettine (28).

The Gethyllis genus comprises 32 species found mainly in southern Africa (Namibia and South Africa) are known for its chemistry and biological activities. Gethyllis species are used traditionally in South African as folk medicine. G. ciliaris is used in the Cape as a remedy for colic, flatulence, and indigestion. The fruit pods of many Gethyllis species are boiled or administered as an alcohol infusion of brandy for stomach disorders, while flowers extraction are used for toothache.23 Organic extracts of Cyrtanthus species show antibacterial and anti-inflammatory activities, with the most potent obtained from leaves and roots of C. falcatus and G. ciliaris.24

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Figure-6: Alkaloids from Leucojum aestivum, N. Tazetta ssp. Tazetta, and pancratium maritimum exhibiting antimalarial activity: crinine (29), 3-epi-hydroxybulbospermine (30), 6-hydroxyhacmanthamine (31), galanthamine (32), and 3-epi-galanthamine (33).

Alkaloids from the Haemanthus genus are used against coughs, dropsy asthma, and as topical antiseptics. Some species have shown RNA antiviral (Poliovirus) and antineoplastic activities. Alkaloids from this genus are also reported for their toxicity.18 N. tazetta L. grows widely in Egypt. It is also cultivated as an ornamental plant and for the production of volatile oil. A number of alkaloids extracted12 from this species are important because of their effective antitumor and antiviral activities, mainly against the choriomeningitis virus.24 Wild plants were found to contain lycorine (3, Figure 1), homolycorine, 9-O-demethylhomolycorine, and tazettine (26, 27, and 28, Figure 5).25 The alkaloids isolated from N. tazzetta ssp. tazetta as well as Pancratium maritimum and Leucojum aestiVum appear to belong to four groups, namely, lycorine, crinine, tazettine, and galanthamine types. Isolated lycorine, haemanthamine (1 and 5, Figure 1), tazettine (28, Figure 5), crinine, 3-epihydroxy- bulbispermine, and 6- hydroxyhaemanthamine (29-31, Figure 6) were tested for antimalarial activity against Plasmodium falciparum. All four groups of alkaloids exhibited antimalarial activity, albeit at different potencies. 6-Hydroxyhaemanthamine, haemanthamine, and lycorine were found to be the most potent inhibitors of P. falciparum, while galanthamine and tazettine were least potent.26 The Pancratium genus alkaloids possess attention due to their complex structure 25 and some of them show signiﬁcant therapeutic activity.27

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Figure-7: Phenanthridiunium betain alkaloids from Pancratium and Zephyranthes spp.: ungeremine (34), zeflabetain (35), zefbetaine (36), iso-zefbetaine (37), pseudolycorine (38), α-dihydrolycorine (39), maritidine (40), and (+)-epi-maritidine (41), anhydrolycorinium chloride (42) and anhydrolyeorine lactam (43).

The Egyptian P. maritimum L., alkaloids earlier study resulted in the isolation of lycorine, galanthamine, sickenbergine, tazettine, lycorenine, pancracine, homolycorine, haemanthidine, demethylhomolycorine, hippadine, and trispheridirine28 and also pseudolycorine, haemanthamine, and 11-hydroxyvittatine.29,30 Two 2- oxyphenathridinium betaine type alkaloids, ungeremine and zefbetaine (34 and 36, Figure 7) are also isolated31 having biological activities like cytotoxic, antibiotic, and plant growth-regulator activities. 32-34 The structure of zefbetaine was determined from spectroscopic and chemical analysis study. It was also supported by the partial synthesis of zefbetaine from pseudolycorine (38, Figure 7), and R-dydrolycorine (39, Figure 7) 29 and by comparison with its unnatural isomer iso-zefbetaine (37, Figure 7).31,32 Ungeremine is also found in Z. ﬂaVa.,32 Ungernia minor,36 Crinum americanum,37 and C. Asiaticum.38 Ungeremine can be obtained by microbial transformation of lycorine performed by a Pseudomonas sp. isolated from the rizosphere of S. lutea. Two other phenanthridinuim derivatives, anhydrolycorinium chloride (42, Figure 7) and the dihydro derivative of ungeremine were isolated and characterized. Many microbially aromatized compounds, including lactam 43 (criasiadine, Figure 7), showed good antibiotic activity when tested against Corynebacterium fascians.34 The genus Zephyrantus, Z. parulla Killip used for treating tumors in Peru, while Z. Rosea Lindll used in China for breast cancer treatment.1 Z. ﬂaVa Roem and Schult is a deciduous herb from America, which is naturalized in India. The extract of its bulbs is used as medicine for a variety of therapeutic purposes, i.e. in the treatment of diabetes,

134 for ear and chest ailments, and against viral infections.40 Investigations carried out showed presence of eight tertiary alkaloids, crinamine, haemanthamine, lycorine, maritidine, methylpseudolycorine, pretazettine, haemanthidine, and pseudolycorine; two lactam alkaloids, narciclasine and pratorimine; three glucosyloxy alkaloids, kalbreclasine, lycorine-1-O-β-D-glucoside, and pseudolycorine-1-O-β-D-glucoside; and four phenanthridinuim betaine alkaloids, criasbetaine, ungeremine, zefbetaine, and zeflabetaine (35, Figure 7).33 Similarly Z. rosae found in upper Gangetic plain and in the Sikkim region of the Eastern Himalayas up to 1000 m. These are also grown in gardens as an ornamental flowering plant and for medicinal purposes. Extracts of flowers and bulbs are used for immunomodulators i.e. a variety of therapeutic purposes. Four alkaloids were extracted as: crinamine (22, Figure 4), haemanthamine (5, Figure 1), maritidine, and (+)-epimaritidine (40 and 41, Figure 7).41 Ismine (9, Figure 1) is a minor alkaloid isolated from several Amaryllidaceae 42 plants. It has been proved that ismine is a catabolic product. It is derived from the C15 crinane skeleton after the loss of the ethane bridge which is not retained as the N-methyl group by labelled experiments.43 Ismine, in turn shows a significant hypotensive effect on the arterial pressure of normotensive rats and is cytotoxic against Molt-4 lymphoid and LMTK fibroblastic cell lines.44 Fully aromatized benzo[c]phenanthridine alkaloids have a broad range of potent pharmacological activities such as antitumour and antiviral activities, and inhibition of DNA topoisomerase I. Trisphaeridine (44, Figure 8) display of biological activity like antifungal, antitumor, antibacterial, nematodical, and cytotoxic activities.45 Trisphaeridine displays excellent antiproliferative effects on both human and mouse cell lines.46 The structure of this polycyclic Trisphaeridine is quite remarkable because the three aromatic rings have different electron densities, which makes them more or less susceptible to electrophilic attack and facilitates regioselective substitution. Such compounds possess extended π conjugation and have been extensively used in optical and electronic devices, such as light-emitting diodes, photovoltaic cells, and thin-film transistors, due to their optoelectronic properties.47 Trisphaeridine and pretazettine increased the intracellular Rh-123 concentration 30- and 50-fold, respectively as compared to the non-treated cells, and 2-O-acetyllycorine and trisphaeridine were demonstrated by means of the checkerboard method to enhance the antiproliferative activity of doxorubicin on L5178 MDR mouse lymphoma cells. The MTT assay revealed that pretazettine, trisphaeridine and 2-O-acetyllycorine displayed excellent antiproliferative effects on both the human and the mouse cell lines. The apoptosis-

135 inducing activities of selected agents (2-O-acetyllycorine and trisphaeridine) were measured via acridine orange and ethidium bromide dual staining and flow cytometry of the subG1 population.48 The N-methylated compounds of ungeremine and their analogs for ex. Bicolorine (45) shows activity against leukemiya.49

Figure-8: Structure of Trisphaeridine (44) and Bicolorine (45)

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2.1.1 Review of Literature Literature search revealed that there are many reports are available for the synthesis of Trisphaeridine, but very few reports are available on the synthesis of Ismine and Bicolorine, which some are described below. Richard K. Hill and Carlson (1964)50 Richard et al. used one step synthesis of unsymmetrical biphenyls as a key step for the synthesis of Ismine. The addition of trans, trans-l, 4-diacetoxybutadiene (47) to dienophile, (Z)-5-(2-nitrovinyl)benzo[d][1,3]dioxole (46) furnishes cyclo adducts which on heating at 100-110 oC, leads directly to aromatized products (48). Reaction leads directly to benzene derivatives without isolation of the intermediate Diels-Alder adducts. 3,4-Methylenedioxy- 2'-nitrobiphenyl (48) on treatment with formaldehyde and hydrogen chloride in acetic acid, followed by hydrolysis with bicarbonate gave the hydroxy methyl derivative (47). Catalytic hydrogenation yielded the amine (50). Amine 50 was treated with ethyl chloro carbonate in pyridine to form compound 51, which on reduction with LiAlH4, give ismine 9.

Scheme-1: Reagents and conditions: (a) 100 oC, 45%; (b) HCHO, HCl, AcOH,

NaHCO3; (c) H2, Pd/C; (d) ClCOOEt, Pyridine; (e) LiAlH4.

Hill and Carlson (1965)51 Carlson et al. again synthesized Ismine with modified scheme and yield. Biphenyl compound 48 was brominated to gice bromo compound 52 with pyridiniym tribromide in acetic acid, followed by nitrile substitution with cuprous cynide in dry pyridine to give compound 53. Cyanide 53 was kept for decomposition in benzene- ammonia solution to give amide 54 which on hydrolysis gives acid 55. Acid was reduced to corresponding alcohol 49 using sodium bicarbonate, and also reduction of nitro group to amine functionality using platinum dioxide to give compound 56. Amine

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56 was treated with ethyl chlorocarbonate to give amide 57 which was reduced with lithium aluminium hydride to give Ismine.

o 2- Scheme-2: Reagent and conditions: (a)Heat, sealed tube, 110-120 C; (b) C5H6Br3N , AcOH; (c) CuCN, dry pyridine; (d) C6H6/NH3; (e) Conc HCl, ETOH-dixoane; (f) dioxane-aq. NaHCO3; (g) PtO2; (h) CH3CH2OCOCl; (i) LiAlH4.

P. Sudaresan (1985)52 Sudaresan et al. synthesized Ismine using Suzuki coupling reaction between 6- bromobenzo[d][1,3]dioxole-5-carbonyl chloride (58) and N-phenylhydroxylamine (59) to give the coupling product, N-hydroxy amine 60 which was then converted to borane complex 61 photochemicaly. Compound 61 gives cyclised product, N-hydroxy lactam

62 on breakdown of borane complex. Lactam 62 was oxidized with NaIO4/dioxane to give acid 63 which on reduction gives Ismine.

Scheme-3: Reagent and conditions: (a) No details; (b) BF3-OEt2, Et2O; (c) C6H6, hν; (d) EtOH, H2O; (e) NaIO4, H2O, dioxane; (f) NaBH4, TiCl4, CH2(OMe)2; (g) NaHCO3, MeI, C6H6, MeOH

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M.A. Siddiqui (1988)53 Siddiquie et al. also used Suzuki reaction for the synthesis of Ismine. 6-bromo- N-methylbenzo[d][1,3]dioxole-5-carboxamide (65) and (2-((tert-butoxy carbonyl) amino)- phenyl) boronic acid (66) on Suzuki coupling gives biphenyl compound (67). Amine compound 65 was subjected to N-methylation, followed by chemoselective reanduction and Boc deprotection to give Ismine.

Scheme-4: Reagent and conditions: (a) Toluene, reflux; 75% (b) i) NaH, MeI, THF, o 95%; ii) LiEt3BH, THF, 0 C-rt, 72%; (c) i. TBDMSOTf, CH2Cl2, 2,6-lutidine, rt, 30 min; ii) TBAF, H2O:THf, reflux, 85%.

A. M. Lobo (1997)54 Lobo et al. synthesized Norismine 72, using radical cyclization as the key step. Alkylation of o-nitrophenol 69 with 2-bromo-3,4-methylenedioxybenzylchloride 68 gives the benzyl phenylether 70, which on catalytic hydrogenation of gives aminobenzylether 71. On exposure to the combined action of AIBN and TBTN, the compound 71 yielded a mixture of products including Norismine 72 (Figure 9).

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Figure-9

Robert K. (1978)49 Robert et al. used photochemical cyclization as a key step for the synthesis of Bicolorine 45. Benzo[d][1,3]dioxole-5-carbaldehyde ( Piperonal, 73) was transformed in to 6-bromobenzo [d][1,3]- dioxole-5-carbonyl chloride 58 by reported methods. Acid chloride 58 was condensed with N-methyl aniline to produce compound 74. Bromo amide 74 was irradiated in a 500 mL quartz photochemical immersion well with a 450- W medium-pressure Hg lamp to get cyclised product 75. Lactam 75 first treated with

B2H6 and then air was passed through the ethanolic HCl solution to get the final product Bicolorine (figure 10).

Figure-10

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Takashi Harayama (2001)55 Takashi et al. used Pd catalysed cyclization reaction as a key step for the synthesis of Trisphaeridine 44. 6-bromopiperonylic acid (77) on successive treatment with oxalyl chloride and anilines in the presence of triethylamine give 78 which were protected as methoxymethylated secondary amine 79. Palladium catalyst is used to cyclise the amide 79 to give cyclic product, lactam 80 which on reduction, treated with HCl to produce Trisphaeridine 44 as final product (Figure 11).

Figure-11

Martin G. Banwell (2004)56 Martin et al. used Palladium[0]-mediated Ullmann cross coupling of 1-bromo-2- nitrobenzene and its derivatives with a range of β-halo-enals, -enones, or -esters readily affords the corresponding β-aryl derivatives, which are converted into the corresponding quinolines, 2-quinolones, phenanthridines, or 6(5H)-phenanthridinones on reaction with dihydrogen in the presence of Pd/C or with TiCl3 in aqueous acetone. 6- bromobenzo[d][1,3]dioxole-5-carbaldehyde 81 was cross coupled with 1-bromo-2- nitrobenzene 82 to give biphenyl 83 which on reductive cyclization gives Trisphaeridine (Figure 12).

Figure-12

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J. C. Walton (2008)57 Walton used dioxime oxalates as a useful precursors for the clean generation of iminyl radicals by sensitised UV photolysis which can be adapted for serviceable preparations of 3,4-dihydro-2H-pyrroles and phenanthridines. The biphenyl derivatives 85 was obtained in good yields by Suzuki coupling of the aromatic carbonyl compound 82 with phenyl boronic acid 84. Aldehyde 85 was treated with hydroxylamine to give oxime 86 which was treated with oxalyl dichloride to form dioxime oxalates 87. When oxalate 87 was irradiated with UV light, cyclization takes place through iminyl radicals to give Trisphaeridine (Figure 13). Walton also used pyrex glass for the photolysis reaction to cyclise the acetoxy oximes (88) (Figure 14).

Figure-13.

Figure-14

R. A. Rossi58 and C. M. Williams59 Rossi et al. synthesized N-((6-iodobenzo[d][1,3]dioxol-5-yl)methyl)aniline 90, which was aromatised by using ammonia, photon and t-butoxide which was then oxidised to Trisphaeridine (Scheme 4). Williams also synthesized benzyl phenyl amine 90, and cyclised with quarts, photon and acetonitrile to give Trisphaeridine (Scheme 5).

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Scheme-4: Reagent and conditions: (a) NaHCO3, H2O, 85%; (b) NH3, hν, t-BuOK, DMSO. o Scheme-5: Reagent and conditions: (a) KI, CH3CN, microwave, 170 C, 15 min; (b) CH3CN, hν, 254nm, quartz, 2 h, 82%.

Chao Chen (2015)60 Chen et al. developed a novel copper-catalyzed tandem C−S/C−C bond-forming reaction of 2-biaryl isothiocyanates with diaryliodonium salts. Amine 93 was prepared using Suzuki coupling reaction between benzo[d][1,3]dioxol-5-ylboronic acid 91 and 2- bromoaniline 92, followed by isothiocyanate formation obtained the precursor 94. Copper-catalyzed tandem reaction of 2-biaryl isothiocyanates 94 with diaphenyliodonium salts provides an effficient approach to construct cyclised compound 95, which on desulfurization by Raney Ni afford the natural alkaloid trisphaeridine (Figure 15).

Figure-15

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Wei-Lin Chen (2015)61 Wei-Lin et al. used hydride-induced anionic cyclization as a key step for the synthesis of Bicolorine. 6-Bromopiperonal 81 was converted to nitrile 96 and successive conversation into bromo complex 97. Compound 97 was treated with 1- bromo-2-iodobenzene in presence of palladium chloride complex to give biphenyl compound 98 which was cyclised using hydride-induced anionic method to give Trisphaeridine 44. Compound 44 was treated with methyl iodide to form the Bicolorine 45.

Scheme-6: Reagent and conditions: (a) NaN3/TfOH, MeCN, rt, 88%; (b) o bis(Pinacolato)diboron, PdCl2(dppf)/KOAc, 1,4 dioxane, 80 C, 36 h, 85%; (c) 1- o bromo-2-iodobenzene, PdCl2(PPh3)2, K3PO4.nH2O, toluene, 100 C, 24h, 67%; (d) o Li(Et)3BH/THF, 100 C, N2, 48 h; (e) i) MeI, toluene, rt, 10h; ii) AgCl/H2O, rt, 2h.

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2.1.2 Present work 2.1.2.1 Objective Amaryllidaceae alkaloids show activity as like antifungal, antitumor, antibacterial, nematodical, and cytotoxic activities. Still today number of methods has been developed for the synthesis of these alkaloids but there is lack of general method for the synthesis of different type of alkaloids from different structural groups. We are interested to synthesize Amaryllidaceae alkaloids Ismine 9, Trissphaeridine 44 and Bicolorine 45 via a common intermediate 6-(6-(hydroxymethyl)benzo[d][1,3]dioxol-5- yl)cyclohex-3-en-1-yl 4-methylbenzenesulfonate 104 to develop Wittig-Claisen rearrangement protocol as shown in figure-16.

Figure-16

2.1.3 Result and discussion We have developed Wittig-Claisen rearrangement protocol for the synthesis of different alkaloids from Amaryllidaceae family. The synthetic scheme is described below. Synthesis of Ismine 9 is shown in scheme 7, while scheme 8 shows synthesis of Trisphaeridine 44 and Bicolorine 45. We started our synthesis with piperonal 73, which was subjected to Wittig-Claisen rearrangement to give 4-pentinal 100. Pentinal subjected to Barbier reaction to give diene 101 followed by Grubb’s cyclization to give cyclic product 102. After tosylation of compound 102, we get compound 103, which was subjected to formylation under Vilsmeier Haack reaction conditions to give aldehyde which was immediately reduced to corresponding alcohol 104. Alcohol 104 was refluxed with methyl amine for substitution reaction go give sec. amine 105 which was further aromatized to Ismine 9 using DDQ.

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- + Scheme-7: Reagents and conditions: (a) Cl Ph3P CH2-O-CH2CH=CH2, t-BuONa, o o toluene, 0 C, 1h, 90%; (b) toluene, 120 C, 10h, 98 %; (c) C3H5Br, zn-dust, THF, reflux, 4h, 70%; (e) Grubb’s II gen. catalyst, DCM, rt, 3h, 90%; (e) TsCl, Py., ME, o DCM, rt, 5h, 80%; (f) i) POCl3, DMF, 60%; ii) NaBH4, MeOH, 0 C, 1h, 85%; (g) o o NH2CH3, DCM, 40 C, 75%; (h) DDQ, dioxane, 7 h, 90 C, 81%.

For the synthesis of Trisphaeridine and Bicolorine, alcohol 104 was treated with phthalimide under Mitsunobu reaction conditions to get phthalimide 106 which was deprotected to corresponding amine 107. Amine 107 was subjected to cyclization using different bases. Triethylamine, pyridine gives modrate yield (20 and 30%) with recovery of starting material. Reaction in presence of NaH gives good yield (77%) to produce 108, which was further aromatised to Trisphaeridine 44 using DDQ. Trisphaeridine further treated with methyl iodide to form methylated compound Bicolorine 45.

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Scheme-8: Reagents and conditions: (a) TPP, DIAD, NHPhth, THF, 0 oC, 87%; (b) o o NH2NH2.H2O, EtOH; (c) NaH, THF/DMF, 100 C, 77%; (d) DDQ, dioxane, 90 C, 78%; (e) MeI, toluene, rt, 12h, 73%.

5-(2-(allyloxy)vinyl)benzo[d][1,3]dioxole (99)

1H NMR spectrum of the compound 99

Piperanol 73 was used for Wittig-Claisen rearrangement protocol62 to synthesize E and Z allyl vinyl ether 99. In the IR spectrum peak at 2935 cm‒1 shows olefinic double bond and peaks at 1693, 927 cm‒1 shows presence of benzene ring. Peak at 1090 cm‒1 indicates the ether linkage.

147

13C NMR spectrum of the compound 99

In the 1H NMR spectrum, multiplet at δ 7.5 attributed to 0.28 proton and multiplet at δ 7.25 attributed to 0.65 protons. These two multiplets combines together (δ 7.58‒7.10) to give integration for one proton which is ortho to both, side chain and dioxole i.e. 2ʹ proton. A multiplet integrated between δ 6.70‒6.50 attributed to total three protons, that is one methine proton from the internal double bond attached to carbon atom linked to oxygen atom, and two aromatic protons at 5ʹ and 6ʹ position. A multiplet resonating between δ 6.10‒5.70 attributed to two methylene protons from dioxole, and one internal methine proton from the terminal double bond. Two methylene protons from dioxole ring shows singlet while methine proton from terminal olefin shows multiplet at the same δ value. Two methine protons from the terminal double bond and one benzylic methine proton from the internal double bond showed multiplet between δ 5.35‒5.00. A multiplet resonating between δ 4.40‒4.12 attributed to two allylic methylene protons attached to oxygen atom. In the 13C NMR spectrum, peaks resonating at δ 142.72 and 141.92 were due to the 3′ carbon and 4′ carbon from the benzene ring. A peak resonated at δ 141.17 corresponding to methine carbon attached to oxygen atom from the internal double bond. Peaks resonating at δ 133.79 and δ 117.16 were due to methine and methylene carbons from terminal double bond. Peaks resonated at δ129.21, 126.98, 112.87, 106.11

148 were attributed to carbons from benzene ring at 1′, 6′, 2′ and 5′ positions. Methylene carbon from dioxole ring resonated at δ 101.66 and benzylic methine carbon resonated at δ 101.37. Peat resonating at δ 72.62 was attributed to methylene carbon allylic to terminal double bond.

2-(benzo[d][1,3]dioxol-5-yl)pent-4-enal (100) The isomeric mixture of allyl vinyl ether 99 as such was refluxed in toluene to affect the Claisen rearrangement62 and produced 4-pentenal 100 in racemic form. IR spectrum showed strong absorption bands at 2938 cm-1 and 1725 cm-1 indicating the presence of aldehyde functional group. The peaks appeared at 1641 and 910 cm-1 were due to terminal olefin. In the 1H NMR spectrum, peak resonated at δ 9.52 attributed to one proton from aldehyde group. A multiplet resonating between δ 6.82‒6.42 corresponded to three protons from aromatic ring. Two methylene protons from dioxole ring show singlet resonated between δ 6.02‒5.75. A multiplet resonating between δ

1H NMR spectrum of the compound 100

5.75‒5.50 attributed to one methine proton from terminal double bond. Multiplet resonated between δ 5.10‒4.80 were corresponded to two methylene protons from terminal double bond. A multiplet resonated between δ 3.50‒3.38 was due to one

149 benzylic methine proton. Two protons from allylic methylene show two multiplets resonated between δ 2.80‒2.50 and δ 2.45‒2.25.

13C NMR spectrum of the compound 100

In the 13C NMR spectrum, peak at δ 199.27 corresponded to carbon atom from the aldehyde functionality. Carbon atoms at 3′ and 4′ positions resonated at δ 149 and δ 147.04. Methine carbon and methylene carbon from the terminal olefin resonated at δ 136.23 and 115.32 respectively. Aromatic carbons at 1′, 6′, 2′, 5′ position resonated respectively at δ 133.88, 122.31, 110.77, and 109.19. Methylene carbon from dioxole was observed to be present at δ 101.5. Methine carbon bearing aldehyde functionality was resonated at δ 54.71 while allylic methylene carbon resonated at δ 35.72.

5-(benzo[d][1,3]dioxol-5-yl)octa-1,7-dien-4-ol (101) Pentinal 100 was subjected to Barbier reaction63 to get diene 101. In the IR spectrum peak at 3470 cm‒1 indicates the presence alcohol. Peaks appearing at 2937, 1639 and 910 cm-1 corresponded to terminal double bond. In the 1H NMR spectrum, multiplet resonating between δ 6.88‒6.65 was attributed to three aromatic protons from the benzene ring. Two methylene protons from the dioxole ring show singlet at δ 5.97. Multiplet resonating between δ 5.88-5.70, integrating for two protons was assigned to the methine protons from the both terminal olefins. Multiplet resonating between δ

150

5.28‒5.08 was assigned to four methylene protons from the both terminal double bonds. Methine proton from the carbon carrying alcohol functionality was resonated as multiplet between δ 3.90‒3.78 while benzylic methine proton was resonated between δ 3.50‒3.35. Two allylic methylene protons resonated as two multiplets between δ 2.80‒2.70 and δ 2.70‒2.57 attributed to one proton each. Methylene protons adjacent to carbon bearing hydroxy group was resonated between δ 2.41‒2.27 and δ 2.27‒2.17 as multiplet integrating for one proton each.

1H NMR spectrum of the compound 101

13C NMR spectrum of the compound 101

151

In the 13C NMR spectrum, peak resonating at δ 143.01, and 142.17 attributed to 3′ and 4′ carbons from the benzene ring. Peaks resonated at 133.32 and 129.92 were assigned as two methine carbons from two terminal olefins. Methylene carbons from two terminal olefins resonated at δ 117.54. Peak resonated at δ 129.69, 127.35, 113.49, 104.97attributed to 1′, 6′, 2′ and 5′ carbons from benzene ring. Methylene carbon from dioxole resonated at δ 103.29. Peak at 68.50 was assigned as methine carbon carrying hydroxy functionality while peak at δ 39.05 attributed to benzylic methine carbon. Methylene carbon adjacent to carbon carrying hydroxy was resonated at δ 38.21. Peak resonated at δ 36.37 attributed to methylene carbon adjacent to benzylic carbon.

6-(benzo[d][1,3]dioxol-5-yl)cyclohex-3-enol (102)

1H NMR spectrum of the compound 102

Diene 101 was subjected to Grubb’s cyclization64 to give cyclohexenol 102. In the IR spectrum, peak at 3412 cm‒1 indicates the presence of hydroxy functionality. Peaks at 2930, 1442, 912 indicate the presence of non aromatic double bond. In the 1H NMR spectrum, multiplet resonating between δ 6.85‒6.58 was attributed to three aromatic protons. Singlet resonated at δ 6.00 was attributed to two methylene protons from dioxole ring. Multiplet resonating between δ 5.90‒5.75 and 5.75‒5.57 was attributed to two olefinic protons from cyclohexene ring. Methine proton from carbon carrying hydroxy functionality was resonated between δ 4.17‒4.02 as multiplet. Multiplet resonating between δ 3.00‒2.89 was attributed to benzylic methine

152 proton. Multiplet resonated between δ 2.60-2.38 was attributed to one 6HA methylene proton Multiplet resonating between δ 2.38-2.07 was attributed to one 6HB methylene proton, and one 3HA methylene proton. Multiplet resonated between δ1.72-1.48 attributed for one 3HB methylene proton.

13C NMR spectrum of the compound 102

In the 13C NMR spectrum, peaks resonated at δ 147.61, and 146.20 attributed to 3′- carbon and 4′-carbon. 1′-carbon resonated at δ 137.00. Peak resonated at δ 135.00, and 121.45 attributed to 4 and 5-methine carbons. 6′, 2′, 5′-carbons resonated at δ 117.52, 115.92, 108.10 respectively. Methylene carbon from dioxole ring resonated at δ 100.08. Peaks resonating at δ 74.03 attributed to 2-methine carbon bearing hydroxy functionality. Peaks resonated at δ 51.35, 39.72, 36.04 attributed to 1, 3, and 6-carbons.

6-(benzo[d][1,3]dioxol-5-yl)cyclohex-3-en-1-yl 4-methylbenzenesulfonate (103) Cyclohexenol 102 was protected as its tosyl ether.65 In the 1H NMR spectrum, multiplet resonated between δ 7.63‒7.49 was attributed to two ortho substituted protons from tosyl group. Multiplet resonated between δ 7.22‒7.10 was attributed to two meta substituted protons from tosyl group. Three aromatic protons show multiplet between δ 6.60‒6.50. Multiplet between 5.97‒5.75 attributed to two methylene protons from dioxole ring and two protons from methine of cyclohexene olefin. One proton from the tosyl substituted carbon (C-2) show multiplet at δ 5.70‒5.51. Methine proton at C-1 position resonated between δ 3.05‒2.91 as a multiplet. One 6HA and one 6HB proton

153 show multiplet resonated between δ 2.64‒2.50. One 3HA and three methyl protons from tosyl combine to resonate between δ 2.50‒2.33. Multiplet resonated between δ

2.33‒2.20 attributed to one 3HB proton.

1H NMR spectrum of the compound 103

13C NMR spectrum of the compound 103

154

In the 13C NMR spectrum, peaks resonated at δ 147.37, 146.30 attributed to 3′ and 4′ carbons. Signal resonated at 144.07 corresponded to 1′′ carbon attached to sulphur from tosyl benzene ring. Para carbon in the tosyl group resonated at 134.58. Peak resonated at 129.38 was due to C1′. Para carbons from tosyl ring resonated at 127.59. Peak resonated at δ 126.65 attributed to ortho carbons from tosyl ring and C5. Signal at 122.39 and 121.13 attributed to C4 and C6′. Peaks resonated at 108.41 and 107.94 corresponded to C2′ and C5′ carbons. Methylene carbon from dioxole ring resonated at 100.81. Peak at 81.07 corresponded to C2 carbon bearing tosyl group. Peaks resonated at 42.34, 31.38, 29.70 corresponded to C1, C3 and C6 carbons. Methyl carbon from the tosyl ring resonated at δ 21.53.

6-(6-(hydroxymethyl)benzo[d][1,3]dioxol-5-yl)cyclohex-3-en-1-yl-4-methylbenzenesulfonate (104)

1H NMR spectrum of the compound 104

Compound 103 was formylated66 and reduced to give alcohol 104. IR spectrum show peak at 3430, 1084 cm-1 indicates the hydroxy group. Peaks at 2930, 1605, 1460 and 951 cm-1 indicates non aromatic double bond. 1H NMR spectrum show multiplets resonated at δ 7.56 attributed to two ortho substituted protons while multiplet resonated at 7.18 attributed to two meta substituted protons from tosyl ring. Peaks resonated at δ

155

6.86 and 6.81 attributed to H3′ and H6′ protons. Singlet resonated at δ 5.92 attributed to two methylene protons from dioxole ring. Multiplet resonated between δ 5.91‒5.78 and 5.64‒5.05 attributed to methine protons from cyclohexene ring. Singlet resonated at δ 4.86 attributed to two benzylic methylene protons. Multiplet resonated between

3.07‒2.92 attributed to one H1 proton. Multiplet resonated between 2.66‒2.50 attributed to one H6 and one H3 proton. Multiplet resonated between δ 2.50‒2.30 attributed to three methyl protons from tosyl ring along with one H6 and one H3 proton.

13C NMR spectrum of the compound 104

In the 13C NMR spectrum, signals resonating at δ 147.40, 146.32, 134.62, 126.64, 108.38, and 107.93 attributed to aromatic carbons from dioxole benzene ring. Peaks resonated at δ 147.40, 146.32, 144.02, 129.40 attributed to aromatic carbons from tosyl ring. Peaks resonated at 127.56, 122.35 attributed to olefin carbons. Peak resonated at 100.71 attributed to methylene carbon from dioxole ring. Peaks resonated at 81.05 and 42.32 attributed to C2 and C1 carbons. Peak at 58.53 attributed to methylene carbon bearing hydroxy functionality. Signals resonated at 31.40 and 29.65 attributed to C3 and C6 carbons. Methyl carbon show signal at δ 21.50.

156

(6-(6-(methylamino)cyclohex-3-en-1-yl)benzo[d][1,3]dioxol-5-yl)methanol (105)

1H NMR spectrum of the compound 105

13C NMR spectrum of the compound 105

Tosyl ether 104 was treated with methyl amine to give sec. amine 105. In the IR spectrum peak 3435, 1110 cm-1 indicates the presence of hydroxy group. 1H NMR spectrum show two singlet resonated at a δ 6.96 and 6.94 attributed to H3′ and H6′ protons. Methylene protons from dioxole ring show singlet resonated at 5.93. Methine

157 protons from isolated double bond show singlet resonated between 5.85‒5.50. Peak resonated at 4.83 attributed to two benzylic methylene protons. Methyl protons resonated at 3.31. Methine protons show multiplet between 3.10‒3.00. Methylene protons from cyclohexene ring show multiplet resonated between δ 2.60-2.27. In the 13C NMR spectrum, signals resonated at δ 147.37, 146.30, 134.58 127.59, 108.41, 107.94 attributed to C5′, C4′, C2′, C1′, C6′, and C3′ carbons respectively. Peaks resonated at 129.38 and 126.65 attributed methine carbons from isolated double bond. Methylene carbon from dioxole ring resonated at 100.81. Methine carbons bearing amine group resonated at 68.60 and methine C1 carbon resonated at 42.34. Peak observed at 62.51attributed to methylene carbon carrying hydroxy group. Methyl amine show signal at 31.38. Two methylene carbons from cyclohexene ring resonated at δ 29.70 and 21.53.

Ismine [(6-(2-(methylamino)phenyl)benzo[d][1,3]dioxol-5-yl)methanol] (9)

1H NMR spectrum of the compound 9

Compound 105 was subjected to aromatization67 using DDQ to produce Ismine. In the IR spectrum peak at 3430, 1084 cm-1 indicates the presence of hydroxy group. Peaks at 2930, 1605, and 1460 show the aromatic ring. In the 1H NMR spectrum, peak resonating between δ 7.22‒7.10 attributed to one H4 proton. Multiplet resonated between 7.00-6.96 attributed to one H6 proton and one H3′ proton. Multiplet resonated

158 between 6.86‒6.77 attributed to one H5 proton and one H3 proton. Singlet resonated at 6.59 was attributed to one H6′ proton. Singlet resonated at 5.97 was attributed to two methylene protons from the dioxole ring. Singlet resonated at 4.17 was attributed to two protons from methylene bearing hydroxy group. Methyl protons attached to nitrogen atom was resonated at δ 2.78 as singlet.

13C NMR spectrum of the compound 9

In the 13C NMR spectrum, peak resonated at δ 147.53, 147.38, 147.23 attributed to C5′, C4′, C2 carbons. Peak resonated at 134.89 attributed to C2′ carbon. Peaks resonated at 130.51, 129.66, 128.92, 127.90 attributed to C1′, C6, C4, and C1 carbons. Peak resonated at 117.85 attributed to C5 carbon. Signals resonated at 111.09, 110.39, 109.89 were attributed to C3, C6′, C3′ carbons. Methylene carbon from dioxole ring resonated at 109.19. Peak resonated at 63.17 was attributed to methylene carbon bearing hydroxy functionality. Methyl carbon resonated at δ 32.14.

6-(6-((1,3-dioxoisoindolin-2-yl)methyl)benzo[d][1,3]dioxol-5-yl)cyclohex-3-en-1-yl- 4-methylbenzenesulfonate (106) Alcohol 104 was subjected to Mitsunobu reaction68, 69 to synthesize phthaloyl derivative 106. In the 1H NMR spectrum, multiplet resonated between δ 7.92‒7.67 attributed to four protons from phthalimide ring. Ortho and meta substituted protons from the tosyl ring was resonated between 7.62‒7.50 and 7.27‒7.18 attributed to two protons each. Two singlet observed between the range 6.92‒6.78 attributed to two

159 protons from benzodioxole ring. Two methylene protons from the dioxole ring were observed to be resonated at 5.93 as singlet. Multiplet resonating between 5.90‒5.80 corresponded to two methine protons from isolated double bond. Methine proton from carbon bearing tosyl ether was resonated between 5.66‒5.55 while methine proton at C2 position was resonated between 3.12‒3.00. A singlet resonated at 4.80 was corresponded to two methylene protons from the carbon carrying phthalimide group.

1H NMR spectrum of the compound 106

13C NMR spectrum of the compound 106

160

Multiplet resonated between 2.64‒2.39 was attributed to one C3 methylene proton

(3HA) and one C6 methylene proton (6HA). Three methyl protons from tosyl ring and two methylene protons from 3 and 6 positions resonated between δ 2.39‒2.25 as multiplet. In the 13C NMR spectrum, peak resonated at δ 168.02 was attributed to carbonyl carbon from phthalimide ring indicates the presence of phthalimide ring in the molecule. Signals resonated at 147.45, 146.07, 129.02, 122.85, 114.13, and 111.07 attributed to carbons from dioxolebenzene ring. Peaks resonated at δ139.92 121.89, 121.05 attributed to aromatic carbons form phthalimide ring. Peaks resonated at δ 139.39, 127.93, 127.12, 123.00 attributed to carbons form tosyl benzene ring. Signal resonated at 101.02 was attributed to methylene carbon form dioxole ring. Methine carbon bearing tosyl group resonated at 72.40 and methine carbon at C1 position was resonated at δ 40.89. Methylene carbon carrying phthalimide group resonated at 42.96. Peaks observed at 31.89 and 29.98 were attributed to methylene carbons C3 and C6. Methyl carbon form tosyl ring resonated at 21.87.

6-(6-(aminomethyl)benzo[d][1,3]dioxol-5-yl)cyclohex-3-en-1-yl-4-methylbenzene sulfonate (107)

1H NMR spectrum of the compound 107

161

In the 1H NMR spectrum, multiplet resonated at δ 7.60 was attributed to ortho substituted protons while multiplet resonated at 7.26 attributed to meta substituted protons from tosyl benzene ring. Two singlet observed at 6.59 and 6.57 were attributed to 3H and 6H proton. Multiplet resonated between 5.78‒5.99 attributed to two methylene protons form the dioxole ring and two methine protons from isolated double bond. 2 and 1 substituted methine proton were resonated between 5.65‒5.52 and 3.05‒2.92. Methylene protons form the carbon atom carrying amine functionality was resonated at δ 3.82 as singlet. Multiplet resonated between 2.63‒2.50 and 2.32‒2.19 attributed to one proton each form the methylene group form C6 and C3position. Multiplet resonated between 2.50‒2.32 attributed to three methyl protons form tosyl ring.

1,4,4a,5,6,11b-hexahydro-[1,3]dioxolo[4,5-j]phenanthridine (108)

1H NMR spectrum of the compound 108

Amine 107 was subjected to cyclization to produce phenanthridine 108. In the 1H NMR spectrums, singlet resonated at δ 6.90, 6.89 attributed to H-10 and H-7 protons. Singlet observed at 6.58 was due to two methylene protons form dioxole ring. Methine protons (H2, H3) form isolated double bond show multiplet resonated between 5.72‒5.57. Multiplet resonated between 3.95‒3.79 was attributed to two H6 methylene protons. Multiplet observed between 3.25‒3.12 attributed to 4Ha and 10Hb methine

162 protons. Multiplet resonated in the range 2.42‒2.29 was attributed to one 1Hα methylene proton. Multiplet resonating between 2.29‒2.17 attributed to one 1Hᵦ and one 4Hα methylene protons. Multiplet resonated between 2.17‒2.02 was due to one 4Hβ methylene proton.

Trisphaeridine (44)

1H NMR spectrum of the compound 44

13C NMR spectrum of the compound 44

163

After aromatization of compound 108, trisphaeridine 44 was obtained which is yellow solid. IR spectrum shows signals at 2918, 1660, 1494, 1466 cm-1 indicated the aromatic rings. In the 1H NMR spectrum, singlet resonated at δ 9.06 attributed to H6 proton. H1 proton show doublet at 8.33 with coupling constant 8.4 Hz. Doublet resonated at 8.13 attributed to H4 proton with coupling constant 8.4 Hz. Singlet resonated at 7.85 was due to H-10 proton. H-3 proton show tiplet of doublet at δ 7.67 with coupling constant 8.4 and 1.2 Hz. H-2 proton also show doublet of triplet at 7.60 with coupling constant 7.8 and 0.6 Hz. Singlet resonated at 7.28 attributed to H-7 proton. Singlet at 6.13 corresponded to two methylene protons from dioxole ring. In the 13C NMR spectrum, signals resonated at δ 151.7, 151.4 attributed to C-9 and C-8 carbons. Signals appeared at δ 148.1, 144.1, 130.1attributed to C-6, C-4a and C-10a carbons. Peaks resonated at δ130.0, 127.9, 126.6, 124.2 corresponded to C-3, C- 4, C-2, and C-1 carbons. Signals appeared at 123.0 and 121.9 was due to C-10a and C-6a carbons. Peaks resonating at 105.4, and 101.9 attributed to C-6 and C-10 carbons. Methylene carbon (C-11) from dioxole ring show peak at δ 99.8.

Bicolorine (45)

1H NMR spectrum of the compound 45

164

13C NMR spectrum of the compound 45

Trisphaeridine treated with methyl iodide to give Bicolorine. In the 1H NMR spectrum, singlet resonating at δ 9.95 was attributed to C-6 proton. Doublet appeared at 9.03 with coupon constant 8.4 Hz attributed to H-1 proton. Singlet resonating at 8.63 was due to H-4 proton. Doublet resonating at 8.43 attributed to H-3 proton with coupling constant 8.4 Hz. Triplet resonated at 8.08 with coupling constant 8.4 attributed to H-10 proton while that of resonated at 8.01 was attributed to H-2 proton with coupling constant 7.8 Hz. Singlet appeared at 7.90 corresponded to H-7 proton. Two H- 11 protons show singlet at δ 6.48. Methyl protons show singlet resonated at δ 4.57. In the 13C NMR spectrum, signals appeared at δ 157.2, 152.4, 150.3 corresponded to C-8, C-9 and C-6 carbons. Signals resonating at δ 134.3, 133.7 and 131.9 were attributed to C-4a, C-3 and C-10a carbons. Peaks appeared at δ129.5, 125.5, 125.1 were attributed to C-10b, C-1 and C-2 carbons. Signals resonating at δ 120.3, 119.4, 107.9, 104.2, and 101.45 were attributed to C-6a, C-4, C-7 and C-10 carbons respectively. Methylene carbon (C-11) resonated at δ 46.42.

165

2.1.4 Experimental section

5-(2-(allyloxy)vinyl)benzo[d][1,3]dioxole (99) To a suspension of Piperonal 73 (1 g, 6.66 mmol) in dry toluene, and allyloxymethylene-triphenylphosphonium-chloride salt (2.9g, 7.99 mmol), potacium tert-butoxide was added portion wise over the period of 10 min at room temperature. The reaction was stirred for 1h at the room temperature. After the completion of reaction (TLC check), toluene was removed under reduced pressure and the crude product was extracted with ethyl acetate (3x10 ml). The combined organic layer was washed with water, dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using pet ether as a mobile phase, gave pure allyl vinyl ether 99 as colourless thick liquid (1.22 g, 90%). The product in hand was the inseparable mixture of E and Z isomers.

IR (Neat, cm‒1): 2935, 1693, 1090, 927, 823. 1 H NMR (400 MHz, CDCl3): δ 7.58‒7.10 (m, 1H), 6.70‒6.50 (m, 3H), 6.10‒5.70 (m, 13 3H), 5.35‒5.00 (m, 3H), 4.40‒4.12 (m, 2H); C NMR (100 MHz, CDCl3): δ 142.72, 141.92, 141.17, 133.79, 129.21, 126.98, 117.16, 112.87, 106.11, 101.66, and 101.37.

2-(benzo[d][1,3]dioxol-5-yl)pent-4-enal (100) The mixture (E/Z) of allyl vinyl ether 99 (1 g), as such was dissolved in toluene and solution was refluxed for 10-12 h. After the completion of reaction (TLC check), toluene was removed under reduced pressure. The crude product was purified through silica gel column using petether;ethyl acetate (90:10) as solvent system to afford pure 4- pentenal 100 as colourless viscous liquid. The product in hand was the mixture of two inseparable isomers (0.98 g, 98%).

166

IR (Neat, cm‒1): 2938, 1725, 1641, 1101, 910. 1 H NMR (400 MHz, CDCl3): δ 9.52 (s, 1H), 6.82‒6.42 (m, 3H), 6.02‒5.75 (m, 2H), 5.75‒5.50 (m, 1H), 5.10‒4.80 (m, 2H), 3.50‒3.38 (m, 1H), 2.80‒2.50 (m, 1H), 13 2.45‒2.25 (m, 1H); C NMR (100 MHz, CDCl3): δ 199.27, 149.00, 147.04, 136.23, 133.88, 122.31, 115.32, 110.77, 109.19, 101.51, 54.71, 35.72.

5-(benzo[d][1,3]dioxol-5-yl)octa-1,7-dien-4-ol (101) Mixture of 4-Pentenal 100 (3.1g, 15.19 mmol) was dissolved in aq. THF (10%

H2O), was added allyl bromide (3.68g, 30.39 mmol) and Zn-dust (2 g, 30 mmol). The reaction mixture was refluxed for 4-5 h at 60 oC. After completion of reaction (checked by TLC), reaction mixture was quenched with saturated solution of ammonium chloride and was filtered through celite bed. The filtrate was concentrated under reduced pressure and extracted with ethyl acetate (3x15 ml). The organic layer was washed with dil. HCl solution, followed by water. Then organic layer was dried over sodium sulphate and concentrated under reduced pressure. Crude product was further purified by column chromatography with pet ether:ethyl acetat ( 80:20) as elute to give dienol 101 (2.61 g, 70%).

IR (Neat, cm‒1): 3470, 2937, 1639, 1448, 1103, 910. 1 H NMR (400 MHz, CDCl3): δ 6.88‒6.65 (m, 3H), 6.10‒5.70 (m, 4H), 5.28‒5.08 (m, 4H), 3.90‒3.78 3.50‒3.35 (m, 1H), 2.80‒2.70 (m, 1H), 2.70‒2.57 (m, 1H), 2.53‒2.42 13 (m, 2H), 2.41‒2.27 (m, 1H), 2.27‒2.17 (m, 1H); C NMR (100 MHz, CDCl3): δ 143.01, 142.17, 133.32, 129.92, 129.69, 127.35, 117.54, 113.49, 104.97, 103.29, 68.50, 39.05, 38.21, 36.37.

6-(benzo[d][1,3]dioxol-5-yl)cyclohex-3-enol (102) The enantiomeric mixture of dienol 101 (2 g) was stirred with the catalytic amount (5 mol%) of Grubb’s second generation catalyst in dry DCM, at rt under nitrogen atmosphere for 4 hours. After completion of reaction, DCM was removed and the crude product was purified by column chromatography using petroleum ether:ethyl acetate (90:10) mobile phase to give cyclohexenol 102 as white solid (1.6 g, 90%).

167

IR (Neat, cm‒1): 3412, 2930, 1948, 1442, 1118, 912. 1 H NMR (400 MHz, CDCl3): δ 6.85‒6.58 (m, 3H), 6.02‒5.90 (m, 2H), 5.90‒5.75 (m, 1H), 5.75‒5.57 (m, 1H), 4.17‒4.02 (m, 1H), 3.00‒2.89 (m, 1H); 13C NMR (100 MHz,

CDCl3): δ 147.61, 146.20, 137.00, 135.00, 121.45, 117.52, 115.92, 108.10, 100.08, 74.03, 51.35, 39.72, 36.04.

6-(benzo[d][1,3]dioxol-5-yl)cyclohex-3-en-1-yl-4-methylbenzenesulfonate (103) Tosyl chloride (1.16 g, 6.09 mmol) was added in the reaction mixture of alcohol 102 (1 g, 4 mmol) dissolved in DCM, triethyl amine (1.23 g, 12 mmol) and catalytic amount of 1-methyl imidazole (0.166 g, 2.03 mmol) at rt. The reaction mixture was stirred at rt for 4 h. After completion of reaction (TLC check), aqueous solution of sodium bicarbonate was added, and extracted with DCM (2x10 ml). Combined organic layer dried over sodium sulphate and concentrated under reduced pressure. The crude product obtained was purified by column chromatography (hexane:ethyl acetate, 95:5) to give tosyl ether 103 (1.58 g, 80%).

1 H NMR (400 MHz, CDCl3): δ 7.63‒7.49 (m, 2H), 7.27 (s, 1H), 7.22‒7.10 (m, 2H), 6.66‒6.50 (m, 3H), 5.97‒5.75 (m, 4H), 5.70‒5.51 (m, 1H), 3.05‒2.91 (m, 1H), 2.64‒2.50 (m, 2H), 2.50‒2.33 (m, 4H), 2.33‒2.20 (m, 1H); 13C NMR (100 MHz,

CDCl3): δ 147.37, 146.30, 144.07, 134.58, 129.38, 127.59, 126.65, 122.39, 121.13, 108.41, 107.94, 100.81, 81.07, 42.34, 31.38, 29.70, 21.53.

6-(6-(hydroxymethyl)benzo[d][1,3]dioxol-5-yl)cyclohex-3-en-1-yl-4-methylbenzene sulfonate (104) o POCl3 (0.217g, 1.4 mmol) was added in the DMF solvent at 0 C and stirred for 10 minutes. Tosylated compound 103 (0.350g, 0.93 mmol) dissolved in DMF was o added in the reaction mixture containing POCl3, at 0 C drop wise over a period of 10 minutes. The reaction mixture was stirred for 2 h and quenched with water. Reaction mixture was extracted with ethyl acetate (3x15 ml), combined organic layer was dried over sodium sulphate and concentrated under reduced pressure go get intermediate o aldehyde. Aldehyde was dissolved in methanol and NaBH4 was added at 0 C. After completion of reaction, methanol was removed under reduced pressure and extraction was done with ethyl acetate (3x10 ml). The combined organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was

168 further purified by column chromatography. The product in hand is alcohol 104 (0.220g, 85%).

IR (Neat, cm‒1): 3413, 2930, 1648, 1438, 1115, 911. 1 H NMR (400 MHz, CDCl3): δ 7.56 (m, 1H), 7.18 (m, 1H), 6.86 (s, 1H), 6.81 (s, 1H), 5.92 (s, 2H), 5.91‒5.78 (m, 2H), 5.64‒5.05 (m, 1H), 4.86 (s, 2H), 3.07‒2.92 (m, 1H), 2.66‒2.50 (m, 2H), 2.50‒2.30 (m, 5H); 13C NMR (100 MHz,

CDCl3): δ 147.40, 146.32, 144.02, 135.02, 134.62, 129.40, 127.56, 126.64, 122.35, 108.38, 107.93, 100.71, 81.05, 58.53, 42.32,, 31.40, 29.65, 21.50.

(6-(6-(methylamino)cyclohex-3-en-1-yl)benzo[d][1,3]dioxol-5-yl)methanol (105) Reaction mixture containing alcohol 104 (0.2 g) dissolved in DCM and excess of methyl amine was heated at 40 oC for 10h. After completion of reaction, DCM was removed under reduced pressure and crude product amine 105 was purified by column chromatography. Pet ether:ethyl acetate (96:4) is used as mobile phase for column chromatography to give substituted methyl amine (0.097 g, 75%)

IR (Neat, cm‒1): 3435, 2932, 1645, 1441, 1110, 914. 1 H NMR (400 MHz, CDCl3): δ 6.96 (s, 1H), 6.94 (s, 1H), 5.93 (s, 2H), 5.85‒5.50 (m, 2H), 4.83 (s, 2H), 3.31 (s, 3H), 3.10‒3.00 (m, 2H), 2.60‒2.50 (m, 1H), 2.50‒5.39 (m, 2H), 2.39‒2.27 (m, 1H); 13C NMR (100 MHz,

CDCl3): δ 147.37, 146.30, 134.58, 129.38, 127.59, 126.65, 122.39, 108.41, 107.94, 100.81, 68.60, 62.51, 42.34, 31.38, 29.70, 21.53.

(6-(2-(methylamino)phenyl)benzo[d][1,3]dioxol-5-yl)methanol (9) Secondary amine 105 (0.100 g) was dissolved in DCM and catalytic amount of DDQ was added. The reaction mixture was stirred for 8h at rt. After completion of reaction, solvent was removed under reduced pressure and crude product was purified by column chromatography using ethyl acetate:pet ether (5:95) as mobile phase. Product in hand was Ismine 9, a white solid (0.079 g, 81%).

IR (Neat, cm‒1): 3430, 2930, 1605, 1460, 1160, 1084, 951, 817.

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1 H NMR (400 MHz, CDCl3): δ 7.22‒7.10 (m, 1H), 6.86‒6.77 (m, 2H), 6.59 (s, 1H), 13 6.12 (s, 1H), 5.97 (s, 2H), 4.17 (s, 2H), 2.78 (s, 3H); C NMR (100 MHz, CDCl3): δ 147.53, 147.38, 147.23, 134.89, 130.51, 129.66, 128.92, 127.90, 117.85, 111.09, 110.39, 109.89, 109.19, 63.17, 32.14.

6-(6-((1,3-dioxoisoindolin-2-yl)methyl)benzo[d][1,3]dioxol-5-yl)cyclohex-3-en-1-yl- 4-methylbenzenesulfonate (106) To a stirred solution of alcohol 104 ( 1 g, 5.08 mmol), triphenyl phosphine ( 2.66 g, 10.16 mmol) and phthalimide (1.5 g, 10.16 mmol) in dry THF (25 mL) at 0 °C was added diethyl azo- dicarboxylate (DEAD, 1.6 mL, 10.16 mmol) and reaction mixture was allowed to stir at room temperature for 2 h. After completion of reaction water was added to the reaction mixture. Organic layer was separated, collected in a conical flask and aqueous layer was extracted with ethyl acetate (3x15 ml). Combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography using ethyl acetate/pet ether (5:95) mobile phase to give phthalimide 107 as solid compound (1.14 g, 87%)

IR (Neat, cm‒1): 2962, 1633, 1455, 931, 832. 1 H NMR (400 MHz, CDCl3): δ 7.92‒7.67 (m, 4H), 7.62‒7.50 (m, 2H), 7.27‒7.18 (m, 2H), 6.92‒6.78 (m, 2H), 5.93 (s, 2H), 5.90‒5.80 (m, 2H), 5.66‒5.55 (m, 1H), 4.80 (s, 2H), 3.12‒3.00 (m, 1H), 2.64‒2.39 (m, 2H), 2.39‒2.25 (m, 13 5H); C NMR (100 MHz, CDCl3): δ 168.02, 147.45, 146.07, 139.92, 139.39, 129.02, 127.93, 127.12, 123.00, 122.85, 121.89, 121.05, 114.13, 111.07, 101.02, 42.06, 31.90.

6-(6-(aminomethyl)benzo[d][1,3]dioxol-5-yl)cyclohex-3-en-1-yl-4-methylbenzene sulfonate (107) To a stirred solution of phthalimide 107 (600 mg, 1.69 mmol) in ethanol (5 mL) was added 80% aqueous hydrazine hydrate (0.20 mL, 13.38 mmol) and stirred at room temperature for 30 min. After completion of reaction the mixture was diluted with

CH2Cl2 and washed with water. Aqueous layer was exteacted with CH2Cl2 (3x5 mL).

Combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. Crude residue was filtered through short bed of silica gel using

MeOH/CHCl3 (5:95) as mobile phase to get corresponding amine 107 (0.410 g, 90%).

170

1 H NMR (400 MHz, CDCl3): δ 7.60 (m, 2H), 7.26 (m, 2H), 6.59 (s, 1H), 6.57 (s, 1H), 5.78‒5.99 (m, 3H), 5.65‒5.52 (m, 1H), 3.82 (s, 2H), 3.05‒2.92 (m, 1H), 2.63‒2.50 (m, 1H),2.50‒2.32 (m, 5H), 2.32‒2.19 (m, 1H).

1,4,4a,5,6,11b-hexahydro-[1,3]dioxolo[4,5-j]phenanthridine (108) Amine 108 (0.2 g, 4.98 mmol) dissolved in THF was added in the suspension of NaH (0.114g, 4.98 mmol) in THF at 0oC and reaction mixture was heated at 60 oC for 1h. After completion of reaction (TLC check), water was added drop wise to quench the reaction mixture. The reaction mixture was extracted with ethyl acetate (3x15 ml) and combined organic layer was washed with water, dried over sodium sulphate and concentrated under reduced pressure to give tricyclic phenanthridine 109. The crude product was purified by column chromatography using petroleum ether: ethyl acetate (90:20) as column solvent system (0.088 g, 77%).

1 H NMR (400 MHz, CDCl3): δ 6.90 (s, 1H), 6.89 (s, 1H), 6.58 (s, 2H), 5.72‒5.57 (m, 2H), 3.95‒3.79 (m, 2H), 3.25‒3.12 (m, 2H), 2.42‒2.29 (m, 1H), 2.29‒2.17 (m, 2H), 2.17‒2.02 (m, 1H).

[1,3]dioxolo[4,5-j]phenanthridine (44) Tricyclic phenanthridine 109 was dissolved in dry DCM and catalytic amount of DDQ was added. The reaction mixture (0.100 g) was refluxed for 1h. After completion of reaction (checked by TLC), solvent was removed under reduced pressure and extracted with ethyl acetate (3x15 ml). The combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to give solid compound, Trisphaeridine 44 (0.068 g, 70%).

IR (Neat, cm‒1): 3006, 2918, 1660, 1494, 1466, 1260, 1027, 758. 1 H NMR (400 MHz, CDCl3): δ 9.06 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.85 (s, 1H), 7.67 (td, J = 8.4, 1.2 Hz, 1H), 7.60 (td, J = 7.8, 0.6 Hz, 1H), 7.28 (s, 1H), 13 6.13 (s, 2H); C NMR (100 MHz, CDCl3): δ 151.7, 151.4, 148.1, 144.1, 130.1, 130.0, 127.9, 126.6, 124.2, 123.0, 121.9, 105.4, 101.9, 99.8.

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5-methyl-[1,3]dioxolo[4,5-j]phenanthridin-5-ium (45) Trisphaeridine 44 (67 mg, 0.3 mmol, 1.0 equiv) and MeI (methyliodide, 852 mg, 6.0 mmol, 20 equiv) were dissolved in toluene (3 mL) in a screw-capped vial (10-mL), sealed with cap and allowed to stir at room temperature for 18 h. The crude reaction mixture was concentrated in vacuum. The residue was then added AgCl (172 mg, 1.2 mmol, 4 equiv) in H2O (3 mL) and allowed to stir at room temperature for 12 h. The reaction mixture was filtered by funnel, and the solid residue was washed by EtOH and

CH2Cl2 to afford the pure product as a yellow solid (70 mg, 86%). Mp: 252–254 °C.

IR (Neat, cm‒1): 3437, 2132, 1655, 1377, 1029, 994, 822, 736. 1 H NMR (400 MHz, CDCl3): δ 9.95 (s, 1H), 9.03 (d, J = 8.4 Hz, 1H), 8.63 (s, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.08 (t, J = 8.4 Hz, 1H), 8.01 (t, J = 7.8 Hz, 1H), 7.90 (s, 1H), 6.48 13 (s, 2H), 4.57 (s, 3H); C NMR (100 MHz, CDCl3): δ 157.2, 152.4, 150.3, 134.3, 133.7, 131.9, 129.5, 125.5, 125.1, 120.3, 119.4, 107.9, 104.2, 101.45, 46.42.

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