Accepted Manuscript Novel Sesquiterpenes from Schisandra grandiflora: Isolation, Cytotoxic activity and Synthesis of their triazole derivatives using “Click” reaction B. Poornima, Bandi Siva, G. Shankaraiah, A. Venkanna, Lakshma Nayak, Sistla Ramakrishna, C. Venkat Rao, K.Suresh Babu PII:

S0223-5234(14)01149-0

DOI:

10.1016/j.ejmech.2014.12.040

Reference:

EJMECH 7600

To appear in:

European Journal of Medicinal Chemistry

Received Date: 18 August 2014 Revised Date:

22 December 2014

Accepted Date: 23 December 2014

Please cite this article as: B. Poornima, B. Siva, G. Shankaraiah, A. Venkanna, L. Nayak, S. Ramakrishna, C. Venkat Rao, K.S. Babu, Novel Sesquiterpenes from Schisandra grandiflora: Isolation, Cytotoxic activity and Synthesis of their triazole derivatives using “Click” reaction, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2014.12.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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Novel Sesquiterpenes from Schisandra grandiflora: Isolation, Cytotoxic activity and Synthesis of their triazole derivatives using “Click” reaction. B. Poornima, Bandi Siva, G. Shankaraiah, A.Venkanna, Lakshma Nayak, C. Venkata Rao,

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K. Suresh Babu.*

Phytochemical investigation of fruits of schisandra grandiflora afforded three novel sesquiterpenes (1-3) along with the three known compounds (4-6). Further, a series of

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triazole analogues of 3 and 4 were prepared using “Click” reaction protocol.

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Graphical Abstract

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Novel Sesquiterpenes from Schisandra grandiflora: Isolation, Cytotoxic activity and Synthesis of their triazole derivatives using “Click” reaction. B. Poornima, Bandi Siva, G. Shankaraiah, A.Venkanna, Lakshma Nayak, Sistla

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Ramakrishna, C. Venkata Rao, K. Suresh Babu.*

Phytochemical investigation of fruits of schisandra grandiflora afforded three novel

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sesquiterpenes (1-3) along with the three known compounds (4-6). Further, a series of triazole analogues of 3 and 4 were prepared using “Click” reaction protocol.

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Novel Sesquiterpenes from Schisandra grandiflora: Isolation, cytotoxic activity and Synthesis of their triazole derivatives using “Click” reaction. B. Poornima,a Bandi Siva,a G. Shankaraiah,a A.Venkanna,a Lakshma Nayak,b Sistla

a

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Ramakrishna, b C. Venkat Rao,c and K. Suresh Babu.a* Natural Products Laboratory, Division of Natural Product Chemistry,

CSIR-Indian Institute of Chemical Technology, Hyderabad 500 607, India b

Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical

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Technology, Hyderabad 500 007, India

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c Department of Chemistry, Sri Venkateswara University, Tirupati-517502, India

Abstract: Phytochemical investigation of hexane extract from the fruits of schisandra grandiflora afforded three novel sesquiterpenes (1-3) along with the three known compounds (4-6). The structures of these isolates were determined by extensive analysis of spectroscopic

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data (1D, 2D NMR). Further, a series of triazole analogues of 3 and 4 were prepared using “Click” reaction protocol. The reaction scheme involving one-carbon homologation of 3 and 4 using the Bestmann-Ohira reagent followed by regioselective Huisgen 1,3-dipolar

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cycloaddition reaction of various azides leading to the formation of triazole analogs (20a-20k & 21a-21c) which is being reported for the first time. All the triazole products were

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characterised using spectral data analysis. The anti-proliferative activity of the isolates and the synthetic analogues were studied against Hela (Cervical cancer), A549 (Lung cancer), DU-145 (Prostate cancer), MCF-7 (Breast cancer) and B-16 (Mouse melanoma) cancer cell lines.

Keywords: Schisandra grandiflora, sesquiterpenes, Bestmann-Ohira homologation, Click reaction, triazole derivatives and anti-proliferative activity. * Corresponding authors: E-mail: [email protected] (Dr. K. S. Babu) Tel: +91-40-27191881, Fax: +91-40-27160512.

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ACCEPTED MANUSCRIPT 1. Introduction The genus schisandra of schisandraceae family is medicinally important and well-known resource for biosynthesizing structurally diverse skeletons with highly oxygenated, fused heterocyclic skeletons [1]. Several plants of this genus are used in folk medicine for treatment

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of various ailments like diabetes, palpitation, insomnia, nocturnal enuresis, dysentery, cough, asthma, phlegm, and jaundice [2]. The dibenzocyclooctadiene lignans (C18) are by far the most abundant metabolites of this plant genus which are known to possess wide range of

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biological activities [3]. Schisandra grandiflora is a small tree belongs to schisandraceae family and distributed widely in the north-western part of mainland China, Bhutan, India, and

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Myanmar [4]. The fruits of this plant are edible and have been used therapeutically for the treatment of various ailments by the local community [5]. There are only two reports on the chemical constituents of this plant species, in which a series of tetranortriterpenoids and lignans have been reported [4, 6]. However, there has been relatively little information (or)

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no reports pertaining to its sesquiterpenes constituents and their cytotoxic activities. As part of our continuous endeavours of phytochemical–pharmacological integrated studies on the Indian medicinal plants [7], we were focused on the investigation of sesquiterpenes

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constituents from the plants of schisandracae family, as part, we have recently reported the

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isolation and characterization of four novel sesquiterpenoids from hexane extract of S.chinensis [8]. In a continuing study, we have procured the fruits of Schisandra grandiflora, and conducted detailed phytochemical study into its chemical constituents. Our current studies led to the isolation three new compounds (1-3), along with three known (4-6) compounds. The new compounds (1-3) featured a novel type of skeletons, particularly, compound 1 possess a unique peroxide linkage at C-7 & C-3. The structures of these novel metabolites were established by spectroscopic methods, especially, 2D NMR techniques and mass spectral data. Further, a series of triazole derivatives were prepared from the 3 and 4

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ACCEPTED MANUSCRIPT using Bestmann-Ohira reagent [9] and followed by regioselective Huisgen 1,3-dipolar cycloaddition reaction of various azides under “Click” reaction conditions [10]. All the isolates and analogues were tested for their cytotoxic activity against the panel of human cancer cell lines and healthy cell lines. In this paper, we describe the isolation, structural

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elucidation and biological activities of these novel compounds along with synthesis of analogues. (Figure 1)

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2. Results and Discussion 2.1. Isolation and characterization

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The concentrated hexane extract was chromatographed on silica gel, and the resultant fractions were subjected to bioassay for cytotoxic activity against cancer cell lines. Repeated column chromatography of the bioactive fractions resulted in the isolation of six compounds, out of which compounds 1-3 were new compounds and structures were established using IR,

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MS, 1D and 2D NMR (HSQC HMBC COSY and NOESY) spectroscopic techniques. The known compounds were identified as, widdaranal A (4) [8], widdaranal B (5) [8], isocuparenal (6) [8] from 1H and

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C NMR spectral data, which were compared with those

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reported in literature. To the best of our knowledge, this is the first report on chemical analysis of 4, 5 and 6 from schisandra grandiflora.

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Compound 1 was isolated as yellow liquid with optical rotation [α]D25 -10.25 (c 0.4,

CHCl3). The HRESIMS analysis of 1 revealed molecular ion peak at m/z 237.1848 [M+H]+ (calcd.237.1849), which corresponds to the molecular formula C15H25O2 indicating four degrees of unsaturation. The IR spectrum of 1 was found to exhibit absorptions of carbon– carbon double bond at 1460 cm-1. The 1H and

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C NMR spectra (Table 1) provided signals

that were characteristic of sesquiterpenes. The 1H NMR spectrum of 1 ( recorded in CDCl3) showed signals (Table 1) attributed to a di-substituted double bond protons at δH 6.80 (1H, d,

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ACCEPTED MANUSCRIPT J = 8.8 Hz), 6.35 (1H, d, J = 8.8 Hz), and four methyl groups at δH 1.36 (3H,s) 1.07 (3H,s), 1.04 (3H, s) 1.06 (3H, s) together with overlapped multiplets due to aliphatic methylenes between at δH 2.28 to 1.64. The

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C NMR (Table 1) data for compound 1 indicated the

presence of 15 carbons, and were further classified by DEPT and HSQC experiments into

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categories of four methyl at δC (25.9, 28.1, 21.4, 21.4), five methylene at δC (29.5, 34.1, 26.1, 19.3, 42.0), two methyne at δC (134.9, 134.5), and quaternary carbons at δC (73.5, 49.9, 82.2, 45.0). A careful analysis and comparison of the 1D NMR spectroscopic data of 1 with those

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of known sesquiterpene, widdarol, indicated that compound 1 is a sesquiterpene with the structure similar to that of widdarol [11]. With four degrees of unsaturation accounted by the

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molecular formula, the structure of 1 was suggested to contain three rings, in association with one double bond.

(Figure-2)

Two-dimensional NMR studies (COSY, HMBC and HSQC) permitted the assignment of the H NMR and 13C NMR spectra and establishment of the final structure. Thus, 1H-1H COSY

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1

spectrum revealed three discrete spin systems, including -CH2-CH2-CH2- (from H-8 to H-10 via H-9), -CH-CH- (from H-1 to H-2) and –CH2-CH2- (from H-5 to H-4), as drawn with bold

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lines in Figure 2. The HMBC study (Fig. 2) established the connectivity of these three

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fragments, methyl groups, and quaternary carbons. In the HMBC spectrum (Fig 2), two methyl groups resonating at δH 1.04 (3H, s) and 1.36 (3H, s) were assigned to C-14 and C-15, based on the correlations from H3-14 (21.4) to C-5 (δc 34.1), C-11 (δC 45.0 ) and C-7 (δC 82.2), H3-15 (δH 1.36) to C-3 (δc 73.5), C-4 (δC 29.5), and C-2 (at δC134.5), respectively. Further, a geminal pair of methyl groups (Me-13 and Me-12) exhibited a correlation to a quaternary carbon, C-11 (δC 45.0), as well as correlations to C-6 (δC 49.9) and C-10 (δC 42.0), a methylene carbon. The HMBC cross peaks of H-1/C-7, H-2/C-1, and H-1/C-2 were consistent with the occurrence of a double bond between C-1 and C-2. On the basis of the

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ACCEPTED MANUSCRIPT molecular formula C15H25O2 revealed by HRESIMS, there were two oxygen atoms in the molecule. From the

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C NMR spectrum, the carbon signals at δC 82.2 (C-7) and 73.5 (C-3)

should be connected to the oxygen atoms on the basis of the HMBC spectrum (Fig. 2). Since there was two oxygen atoms left, these two carbons were concluded to be attached to the two

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oxygen atoms to form peroxide bridge, which is rare feature that was found in sesquiterpenes [12]. Analysis of HMBC and HSQC spectra also supported this structure (Fig. 2). Further, we also performed the reductive cleavage of peroxide ring with 10% w/w of 5% palladium on

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carbon in MeOH [13] to afford diol 7, which was further confirmed the cyclic peroxide linkage between C-7 and C-3. Combination of the above observations led to the

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establishment of the planar structure of compound 1.

(Scheme-1)

The relative configuration of 1 was established based on the biogenetic pathway of sesquiterpenes, as these are thought to be derived from fernasyl pyrophosphate (Scheme-2).

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The configuration of the basic portion of compound 1 was assumed to be the same as that of known sesquiterpenes bearing the same skeleton such as widdarol [14]. Biogenetically, it is suggested that the methyl group at C-15 position was β-orientation as in widdarol. Further,

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The NOESY spectra exhibited weak correlations between Me-13 and Me-15, indicating that

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they are β-oriented and confirming a disposition to be identical to that of widdarol skeletons. Therefore, based on these spectral data, the structure was conformed as shown in figure-1, and trivially named as Widdarol-peroxide. Compound 2 was obtained as pale yellow oil, and its molecular formula C15H23O was

established by molecular ion peak in HRESIMS spectrum at m/z 219.1742 [M + H]+ (calcd.219.1743), indicating the five degrees of unsaturation. Its IR spectrum showed absorption bands for α,β-unsaturated carbonyl (1671 cm-1) and olefinic functional groups. The 1H NMR spectrum of 2 (300 MHz, CDCl3) showed signals (Table 1) attributed to two a

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ACCEPTED MANUSCRIPT tri-substituted double bond protons at δH 6.99 (1H,s), 5.44 (1H,s) and three methyl singlets at δH 1.58 (3H, s), 0.88 (3H, s), and 0.91 (3H, s), respectively. Further, NMR spectrum also showed the signal at δH 9.41 (1H, s) attributed to aldehyde group together with signals with complex coupling patterns overlapped multiplets in the aliphatic range. The

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C NMR and

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DEPT spectra (Table 1) showed 15 carbon resonances including three methyls, five methylenes, two methines (two olefinic), and four quaternary carbons (one carbonyl and two olefinic). These spectral features indicated 2 to be a widdarol type sesquiterpene derivative

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possessing α,β-unsaturated aldehyde and olefinic functional groups. Comparison of its 1H NMR and 13C NMR data with those of widdaranal C [8], isolated from Schisandra chinensis,

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indicated an overall similarity, except for the shift of double bond from C1/C7 to C7/C8. Detailed analysis of 2D NMR spectra of 2 refined the proposed structure. Specifically, three discrete spin systems observed from its 1H-1H COSY spectrum [ -CH-CH2- (from H-1 to H2), -CH-CH2-CH2- (from H-8 to H-10 via H-9) and –CH-CH2- (from H-4 to H-5)] coupled

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with the HMBC correlation of H-2 (δH 6.99) with C-7 (δC 35.9), C-1 (δC 30.6) and C-3 (δc 141.9) ( Figure 3) confirmed position of double bond at C7/C8. Thus, the structure of 2 was

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established as shown in figure- 1 and trivially named as widdaranal C. (Figure- 3)

Compound 3 was obtained as yellow oil. The molecular formula C15H23O was derived from

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the HRESIMS molecular ion peak at m/z 219.1743 [M+ H]+ (calcd. 219.1744), indicating the five degrees of unsaturation. The IR spectrum afforded absorption bands typical for olefinic (1452 cm−1) and α,β- unsaturated carbonyl (1678 cm−1) groups, respectively. Analysis of the 1

H NMR,

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C NMR and 2D NMR (CDCl3) spectroscopic data (Table 1) indicated the

presence of a 5-membered ring bounding to a germacradiene system. The

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C NMR and

DEPT/13C NMR spectra revealed the presence of 15 carbons: quaternary carbons, C-5 (δC 47.1), C-4 (δC 159.1), C-8 (δC 141.5), methylenes C-2(δC 24.4), C-3 (δC 29.7), C-6 (δC 23.5),

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ACCEPTED MANUSCRIPT C-7(δC 18.3), C-10 (δC 37.2), C-14 (104.2), methines C-1(δC 54.2), C-9 (δC 151.4), C-11 (δC 29.7) and methyl groups C-12 (δC 21.2), C-13 (δC 23.2), and carbonyl group at C-15 (δC 193.5). The 1H NMR spectrum (recorded in CDCl3) displayed three olefinic hydrogens, two of them at δH 4.76 (1H, s) and 4.55 (1H, s) were attributed to exo-CH2 group (H2-14) by

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correlation with an SP2 carbon (C-4) in HSQC while the third proton appeared downfield at δH 6.85 (1H, bs) was a characteristic tri-substituted double bond. Further, it also displayed characteristic one proton multiplet [δH 2.30 (1H, overlapped, H-11)], two methyl doublets[ δH

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0.89 (3H, d, J = 6.7Hz) and 0.96 (3H, d, J = 6.7Hz)] indicating the presence of a isopropyl group and an aldehyde group at δH 9.41(1H, s), respectively. Combined analysis of 1D and

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2D NMR spectra data of 3 demonstrated that its structure was closely related to that of schisansphenin B [15] except for the replacement of acid with aldehyde group. This was supported by its 1H NMR and

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C NMR spectra, which clearly indicated the signal

corresponds to aldehyde group at δH 9.41(1H, s) in 1H NMR and δC 193.6 in

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C NMR

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spectra. Further, the HMBC correlation of H-15 with C-7 (δC 18.3), C-9 ( δC 151.4), ), C-8 ( δC 141.5), confirmed position of aldehyde group at C-15 (Fig. 4). Thus, structure of 3 was

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established as shown in figure-1 and trivially named as Schisanspheninal A. (Figure 4)

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2.2. Biogenetic considerations

A plausible biogenetic pathway is shown in Scheme-2. As suggested by Young et al [16] biosynthetic connection of these structurally complex sesquiterpenes involved a series of oxidative or reductive cyclizations and hydride shifts of the bisabolyl carbocation (9), which serves as a biosynthetic precursor to the array of the sesquiterpenes skeletons including the widdarol types and this precursor was presumed to be originated from (E, E)-farnesyl diphosphate through sequential isomerisation, ionization and ring closure reactions. The new compounds 1-3 represent the novel class of sesquiterpenes, which were confirmed to exist in

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ACCEPTED MANUSCRIPT the hexane crude extract by the LC-MS and HPLC techniques. Particularly, compound 1 is interesting because of existence of peroxide linkage at juncture (C- 3 and C- 7), which, to the best of our knowledge, it is a rare feature among the reported sesquiterpenes. We proposed a biosynthetic hypothesis for the compounds 1-3 from the common intermediate, homo

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bisabolyl cation 9, and arised via path a and b. As shown in scheme-2, in path a, cyclisation of homo bisabolyl carbocation [11, 6-closure] and followed by hydride shift leading to give the spiro intermediate ion 10. Subsequent rearrangement of intermediate ion 10 by shift of C-

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1, leading to the formation of the widdarane ion 11, which further undergo sequential transformation of 14-methyl shift, deprotonation at C-8, and followed by oxidation of methyl

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group may lead to the formation of 2. The biogenetic-hypothesis for the formation of 1 envisioned from 13. Thus, intermediate 13 was reacted with O2/hv to give 14 [17], which undergo for a rearrangement to form the unusual peroxide 1, to the best of our knowledge, unprecedented feature among the reported sesquiterpenes. Alternatively, path b involved the

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initial 10,6-ring closure of homo-bisabolyl cation (9) and followed by sequential rearrangement (Wagner-Meerwein), deprotonation and oxidation, for the formation of

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compound 3.

(Scheme 2)

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2.3. Synthesis of triazole derivatives of 3 and 4. The widdarol-type sesquiterpenes/motifs are well represented in many pharmaceutical drugs and manifesting diverse biological activities [18]. In this perspective, we have planned to design and synthesize new class of derivatives and studied their cytotoxic activity. Thus, we prepared the triazole derivatives of 3 and 4 by using “Click” reaction protocol. As shown in scheme-3, initially, the aldehydes 3 and 4 were treated with Bestmann–Ohira reagent in MeOH to yield corresponding terminal alkynes in good yields [9]. This one-step transformation proceeded smoothly and efficiently under mild conditions to get the targeted

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ACCEPTED MANUSCRIPT products, which are the templates for the next step. Formation of terminal alkyne products (18 & 19) could easily be confirmed by the disappearance of aldehyde signal at δH 9.41 in the 1

H NMR spectra and the presence of the alkyne proton at δH 2.78 (1H, s). Subsequently, these

terminal acetylenes were allowed to undergo 1, 3-dipolar cycloaddition reaction typically

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called Huisgen cycloaddition with the aromatic azides under “Click” chemistry conditions (CuI, THF) to afford regioselectively 1,4-disubstituted-1,2,3-triazoles in good to excellent yields. [10] (Scheme 3, Table 2). On the other hand, aromatic azides were prepared from their

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corresponding aromatic amines by diazotisation with sodium nitrite in acidic conditions followed by displacement with sodium azide. Under these conditions a series of triazole

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analogs from 3 and 4 were synthesized to look for structure–activity relationship studies. Formation of the final products could easily confirmed by presence of characteristic H-16 proton at δH 6.5 -8.0 in 1H NMR spectra. All the triazole products (20a-20k & 21a-21c) were characterised using spectral data analysis including 1H NMR, 13C NMR and HRMS as well as

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ESI-MS spectral data.

(Scheme 3)

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2.4. Biological activity

In view of the broad spectrum cytotoxic potentials of sesquiterpenes, new compounds (1-3)

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and their synthetic triazole derivatives (20a-20k & 21a-21c) were screened for their cytotoxicity against Hela (Cervical cancer), A549 (Lung cancer), DU-145 (Prostate cancer) MCF-7 (Breast cancer) and B-16 (Mouse melanoma) cell lines in vitro using MTT assay[19]. The clinically applied anticancer agent, doxorubicin, was used as positive control for cytotoxicity assays at concentrations of 100 µM and 10 µM in each 96-well plate format. The values represent averages of three independent experiments, each with duplicate samples. The results are summarized in Table-3 and expressed as IC50 values (drug concentration causing 50 % inhibition; µM). The results revealed that some of the synthetic analogues were

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ACCEPTED MANUSCRIPT exhibited promising anticancer activity when compared their parent isolated compounds. Among the tested triazole derivatives, compound 20g manifested potent activity against lung cancer cell line (A549) with an IC50 value of 11.2± 0.03 µM, while compounds 20h, 21b and 20c showed moderate activities with IC50 values of 20.1± 0.98, 20.5±1.41 and 24.68±1.11

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µM respectively. Similarly, compounds 20b (15.10± 1.12) and 20h (18.8±1.31) showed moderate activities against Hela cell line. It is important to mention that all the tested compounds were not active/moderate active against DU-145, MCF-7 and B-16 Cell lines.

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Different substitutions on the aromatic ring of the triazole derivatives affect the activity. Though it is difficult to discuss the structure activity relationship criteria responsible for

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cytotoxic activities in this set of compounds, from these results, it can be concluded that methoxyl group at 3rd position & chloro substitution at 4th position of aromatic ring significantly enhanced the activity against A-549, Hela cell lines when compared to their parent compound 1. However, tri-methoxyl substitution on the aromatic ring for 20k, activity drastically

decreased

or

completely

lost.

For

the

other

compounds,

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was

remarkable/significant enhancement in the activity was not observed irrespective of their groups and their position on the aromatic ring. Further, we also studied the toxicity of the

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compounds against the human embryotic kidney cell (HEK-293), which clearly demonstrated

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that all the tested compounds were not toxic towards the healthy cell. This information clearly indicated that all the tested compounds were toxic particularly against cancer cell lines and not toxic against the normal human cells (table-3). An exhaustive literature survey revealed that triazole formation has not been reported on any sesquiterpenes so far using click chemistry approach and represents the first of its kind on any widdarol type sesquiterpenes and thus constitutes an initial structure–activity relationship of triazole derivatives of sesquiterpenes class of natural products.

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ACCEPTED MANUSCRIPT 3. Conclusion In summary, we have isolated three novel sesquiterpenes from the hexane extract of schisandra grandiflora along with the three known compounds and their structures were established based on the spectroscopic methods. In an attempt to search for a potent

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sesquiterpenes, we also prepared triazole derivatives from 3 and 4 using the Bestmann-Ohira reagent followed by region selective Huisgen 1, 3-dipolar cycloaddition reaction of various azides. Further, we assayed the ability of natural sesquiterpenes and synthetic triazole

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derivatives against the panel of cancer cell lines. Among the tested derivatioves, 20g & 20b displayed potent activity against A-549 & Hela cell lines. This has laid a solid foundation for

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further lead optimization of this class of compounds by a systematic chemical modification including the synthesis of water-soluble compounds to improve their overall pharmaceutical properties. 4. Experimental

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4.1 General

Optical rotations were measured using a JASCO DIP 300 digital polarimeter and at 1ml cell at 25oC. IR spectra were recorded on a Nicolet-740 spectrometer with KBr pellets.

and 75 MHz for

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The NMR spectra were recorded on a Bruker FT-300 MHz spectrometer at 300 MHz for 1H 13

C respectively, using TMS as internal standard. The chemical shifts are

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expressed as δ values in parts per million (ppm) and the coupling constants (J) are given in hertz (Hz). Mass spectra were performed on a LC-MS/MS (Agilent Technologies 6510) QTOF Mass spectrometer. The 2D experiments (1H–1H COSY, HSQC, HMBC, NOESY) were performed using standard Bruker microprograms. Column chromatography was performed with silica gel (100–200 mesh, Qing-dao Marine Chemical, Inc., Qingdao, China). Semipreparative HPLC was performed on an Agilent 1100 series LC/MSD Trap SL, with a Phenomenex Luna C18 (250x10 mm 10µ) column. Preparative HPLC was performed on a

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ACCEPTED MANUSCRIPT Dionex P680 equipped with PDA detector and Shimadzu PRC-ODS (K) column: Zorbax SB (C18, 9.4 x 50 mm, 5 µ), Analytical TLC was performed on precoated Merck plates (60 F254, 0.2mm) with the solvent system EtOAc-hexane (50:50), and compounds were viewed under a UV lamp and sprayed with 10% H2SO4, followed by heating.

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4.2. Plant material

The fruits of Schisandra grandiflora were procured from M/S Kuber Impex Limited, Indore, India and were authenticated by Dr. K. Madhava Chetty, and a voucher specimen was

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deposited in the herbarium of the Botany department, Sri Venkateswara University, Tirupati,

4.3. Extraction and isolation

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Andhra Pradesh, India.

The fruits of Schisandra grandiflora (2 kg) were shade dried, powdered, and extracted with hexane by using soxhlet apparatus. The resulting hexane extract was evaporated to dryness under reduced pressure, affording syrup residue (10g) which was subjected to column

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chromatography (silica gel, 100–200 mesh) eluting with hexane/EtOAc mixture of increasing polarity to give 60 column fractions. All these column fractions were analyzed by TLC (silica gel 60F254, hexane: EtOAc (85:15), and fractions with similar TLC patterns were combined

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to give four major fractions (F1, F2, F3 and F4). Fraction F1 did not show any prominent

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spots and waxy nature and hence it was not examined further. Fraction F2 was subjected to column chromatography (CC) on silica gel (100–200 mesh) eluted using a hexane–EtOAc (10:0–9.5:0.5) to yield sub fractions C1, C2 . Sub fraction C1 showed only waxy nature and Sub fraction C2 was then purified by preparative TLC with EtOAc : hexane (9.5:0.5) to get compound 2 (2 mg) and compound 5 (8 mg). From fraction F3 chromatography (CC) on silica gel (100–200 mesh) eluted using a hexane–EtOAc (90:10) to get compound 1 (40mg) and compound 3 (300mg). Fraction F4 was subjected to column chromatography(CC) on silica

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ACCEPTED MANUSCRIPT gel (100–200 mesh) eluted using a hexane–EtOAc (10:0–9.5:0.5) to yield compound 4 (600mg) and compound 6 (10mg). 4.4. Spectral data 4.4.1. Compound 1: Yellow liquid; [α]D25 -10.25 (c 0.4, CHCl3); IR (KBr) νmax 2924, 13

C NMR: see Table1.HRMS (ESI+): m/z

237.1848 ( [M+H]+, C15H25O2; calcd.237.1849).

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2877,1460,1379,1099 cm-1; For 1H NMR and

4.4.2. compound 2 :Yellow oil; [α]D25 + 130.25 (c 0.4, CHCl3); IR (KBr) νmax 2980, 1676, 1460, 1099 cm-1; 1H and

13

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C NMR: see Table 1.HRMS (ESI+): m/z 219.1742 ([M+H]+,

C15H23O; calcd.219.1743).

1460, 1099 cm-1; For 1H NMR and [M+H]+, C15H23O; calcd.219.1743).

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4.4.2. Compound 3: Yellow oil; [α]D25 -15.25 (c 0.8, CHCl3); IR (KBr) νmax 2980, 1676, 13

C NMR: see Table 1.HRMS (ESI+) m/z 219.1744

4.5. Experimental procedure for the synthesis of 7.

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To a solution of compound 1 (0.0423mol) in methanol (2ml) was added to this 10%w/w of 5%palladium on carbon was added. Thus the mixture was stirred under a hydrogen atmosphere. The reaction mixture was monitor until the reaction was completed. Reaction

EP

mixture was filtered through celite with methanol, filtrate was evaporated under rota

AC C

evaporator crude residue left over in round bottomed flask, and further flash column chromatography was done by using mesh (60-120) silica final product obtained as a white solid.

4.5.1.(9aS)-1,1,7,9a-tetramethyidecahydro-1H-benzo[7] annulene- 4a,7-diol,(7) . White gummy; 1H NMR (300 MHz, CDCl3) δH 2.26 (1H, m),1.81(3H, m), 1.74(2H, m), 1.48(2H, m), 1.34(2H,m), 1.22(3H,s), 1.15(3H, s), 1.05(3H, s), 0.95(3H, s), 0.88 (3H, s); 13C NMR (75 MHz, CDCl3) δC 76.0, 70.2, 52.3, 44.9, 43.1, 36.0, 35.6, 32.9, 32.1, 30.1, 28.2, 25.8, 24.5, 21.8, 18.5 ppm; HRMS (ESI+) m/z: 263.1984 ([M+Na]+, C15H28O2; calcd. 263.1981).

15

ACCEPTED MANUSCRIPT 4.6. General Experimental procedures for preparation of compound 18 and 19: To a solution of aldehyde (3 & 4) 500mg (2.29mol), 132g (0.93mol) of K2CO3was added followed by Bestmann–Ohira reagent (0.32 mol) at room temperature under nitrogen atmosphere. The reaction mixture was allowed for stirring for 6h at room temperature. After

RI PT

completion of the reaction (monitored by TLC), reaction mixture was filtered through celite, evaporated to dryness. To dried compound 10 ml of water and 15ml ethyl estate was added, proceed for work up, washed with brine solution and then dried over sodium sulphate. The

SC

solvent was evaporated under reduced pressure to yield crude product which was further

homologated compounds 18 and 19. 4.6.1

M AN U

purified by column chromatography eluting with hexane: ethyl acetate (90:10) to get pure

(4aR,9aS)-7-ethynyl-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1H-

benzo[7]annulene) (18). Dark yellow liquid (84.7%); [α]D25 -44.67(c 0.6,CHCl3); 1H NMR (300 MHz, CDCl3) δH 6.17 (1H, dd, J = 2.4,5.0Hz), 4.92 ( 1H, s), 4.47 (1H, s), 2.78 (1H, s),

TE D

2.26 (2H, m), 2.22 (2H, m), 2.02(2H, m), 1.87 (2H, m), 1.75(2H, m), 1.49(2H, m), 0.88 (3H, s), 0.83(3H, s); 13C NMR (75 MHz, CDCl3) δC 148.2, 135.7, 118.7, 110.9, 85.4, 74.3 , 44.5, 37.2, 36.8, 32.0 , 29.5, 27.0, 25.5, 24.9, 23.6, 22.9 ppm; IR (v/cm-1) :3448, 2925, 1637, 1217 ;

EP

HRMS (ESI+) m/z: 215.1790 ([M+H]+, C16H23; calcd. 215.1794). 4.6.2.(1S,5S)-8-ethynyl-1-isopropyl-4-methylenespiro[4.5]dec-7-ene (19). Yellow viscous

AC C

liquid (80%yield); [α]D25 -40.00(c 0.7,CHCl3); 1H NMR (300 MHz, CDCl3) δH 6.37 (1H, s), 4.69 (1H, s), 4.56 (1H, s), 2.83 (1H, s), 2.40 (2H, m), 2.31 (2H,m)), 2.11 (2H, m), 2.04 (1H, m), 1.85 (1H, m), 1.64 (2H, m), 1.17 (2H, m), 0.95 (3H, s), 0.87 (3H, s); 13C NMR (75 MHz, CDCl3) δC 152.5, 137.5, 125.7, 120.5, 104.0, 85.7, 75.0, 46.4, 45.6, 43.4, 36.2, 30.5, 26.6, 26.4, 25.2, 21.6 ppm; IR(v/cm-1): 3459, 2920, 1738, 1462, 1260; HRMS (ESI) m/z 215.1794[M+H]+, C16H23; calcd. 215.1796). 4.7. General Experimental procedure for preparation of triazole derivatives (20a-

16

ACCEPTED MANUSCRIPT 20k.and 21a-21c) To a solution of alkynes 0.010 mg (0.0458 mol) in 2ml dry THF was added azide (0.0688 mol) followed by addition of CuI (catalytic amount) at room temperature. The reaction mixture was then refluxed for approximately12-15h and the reaction progress was monitored

RI PT

by TLC. After the complete consumption of alkyne, reaction mixture was filtered by using celite. Filtrate was portioned between ethyl acetate (20mL) and water(10mL). The organic layer was then washed with brine solution. The combined organic layers were dried over

SC

sodium sulphate and evaporated under reduced pressure using rotary evaporator to afford the crude residue, which was purified by column chromatography using silica gel (60-120 mesh)

M AN U

by eluting with hexane/ ethylacetate (70:30) to yield the product in pure form. 4.7.1.4-((4aR,9aS)-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1Hbenzo[7]annulen-7-yl)-1-(4-methoxyphenyl)-1H-1,2,3-triazole (20a). Brownliquid; [α]D25 22.86 (c 0.7,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.78 (1H, s), 7.61 (2H, d, J = 9.0),

TE D

7.0(2H, d, J = 9.0Hz, ), 6.56 (1H, m), 4.89 (1H, s), 4.57 (1H, s), 3.87(3H, s), 2.42 (2H, m), 2.30 (2H, m), 2.12 (2H, m), 1.82 (2H, m), 1.66 (2H, m), 1.52 (2H, m), 0.93 (3H, s), 0.89(3H, s);

13

C NMR (75 MHz, CDCl3) δC 161.1, 152.3, 151.6, 148.6, 141.7, 137.0, 125.9, 124.5,

): 3143, 2926, 2857, 1537, 1495,1453, 1348, 1217; HRMS (ESI+) m/z: 364.2383([M+H]+,

AC C

1

EP

122.0, 114.6, 110.9 ,55.6, 44.9 , 36.9, 32.1, 29.1, 25.6, 25.0, 24.3, 23.7, 23.0 ppm; IR (v/cm-

C23H30ON3; calcd. 364.2385). 4.7.21-(4-(4-((4aR,9aS)-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1Hbenzo[7]annulen-7-yl)-1H-1,2,3-triazol-1-yl)phenyl)ethanone(20b).Yellow liquid; [α]D25 82.3(c 0.3,CHCl3); 1H NMR (300 MHz, CDCl3) δH 8.11(2H, d, J = 9.0Hz), 7.86(2H, d, J = 9.0Hz), 7.84 (1H, s), 6.63 (1H, m), 4.89 (1H, s), 4.55 (1H, s), 2.66 (3H, s), 2.40 (2H, m), 2.30 (2H, m), 2.14 (2H, m), 1.83 (2H, m), 1.66(2H, m, ), 1.56 (2H, m), 0.94 (3H, s), 0.89(3H, s); 13

C NMR (75 MHz, CDCl3) δC 196.6,150.0, 148.6, 140.2, 136.4, 130.0, 125.5, 119.7, 110.9,

17

ACCEPTED MANUSCRIPT 45.1, 37.3, 36.8,32.1,31.9, 26.6, 25.6, 25.0, 24.3, 23.6, 23.0 ppm; HRMS (ESI+) m/z: 376.2387 ([M+H]+, C24H30ON3; calcd. 376.2387). 4.7.34-((4aR,9aS)-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1Hbenzo[7]annulen-7-yl)-1-(3-nitrophenyl)-1H-1,2,3-triazole (20c). Pale yellow liquid; [α]D25 -

RI PT

180.0(c 0.2,CHCl3); 1H NMR (300 MHz, CDCl3) δH 8.55(1H, t, J =4.1,2.0Hz), 8.28 (1H, dd, J = 8.1,2.0Hz), 8.19 (1H, dd, J = 8.1,2.0Hz), 7.87 (1H, s), 7.73 (1H, t, J = 8.1,8.3Hz), 6.65 (1H, m), 4.90 (1H, s), 4.56 (1H, s), 2.39 (2H, m), 2.30 (2H, m), 2.16 (2H, m), 1.84 (2H, 13

C NMR (75 MHz, CDCl3) δC

SC

m), 1.66 (2H, m), 1.56 (2H, m), 0.94 (3H, s), 0.89 (3H, s);

150.1, 148.8 148.6, 137.9, 130.8, 125.8, 125.7, 125.4, 122.7, 115.7, 114.7, 110.9, 45.1, 37.3,

C22H27O 2N4; calcd. 379.2128).

M AN U

36.9, 32.1, 29.2, 25.6, 25.0, 24.3, 23.7, 23.0 ppm; HRMS (ESI+) m/z: 379.2130 ([M+H]+,

4.7.4.1-(4-iodophenyl)-4-((9aR)-9a-methyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1Hbenzo[7]annulen-7-yl)-1H-1,2,3-triazole (20d). Light brown gummy [α]D25 -46.67 (c

TE D

0.15,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.83(2H, d, J = 8.8Hz), 7.74 (1H, s), 7.49 (2H, d, J = 8.8Hz), 6.59 (1H, m), 4.89 (1H, s), 4.55 (1H, s), 2.40 (2H, m), 2.31 (2H, m), 2.14 (2H, m), 1.82 (2H, m), 1.67 (2H, m), 1.55 (2H, m), 0.93 (3H, s), 0.88 (3H,s); 13C NMR (75 MHz,

EP

CDCl3) δC 148.6, 138.7, 136.8, 130.2, 125.6, 125.1, 121.8, 115.7, 110.9, 93.0, 45.1, 37.3,

AC C

36.8, 32.1, 29.4, 25.6, 25.0, 24.3, 23.7, 23.0 ppm; HRMS (ESI+) m/z: 460.1241 ([M+H]+, C22H27IN3; calcd.460.1244). 4.7.5.1-(2-(4-((4aR,9aS)-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1Hbenzo[7]annulen-7-yl)-1H-1,2,3-triazol-1-yl)phenyl)ethanone (20e). Yellow oil; [α]D25

-

46.67(c 0.15,CHCl3) 1H NMR (300 MHz, CDCl3) δH 8.24(2H, t, J = 9.0,7.9Hz), 8.01 (2H, t, J = 7.9, 2.0Hz), 7.84 (1H, s), 7.63 (1H, t, J = 7.9,2.0Hz) 6.62 (1H, m), 4.90 (1H, s), 4.56 (1H, s), 2.68 (3H, s), 2.40 (2H, m), 2.30(2H, m), 2.14 (2H, m), 1.83 (2H, m), 1.70 (2H, m), 1.56 (2H, m), 0.94 (3H, s), 0.89 (3H, s);

13

C NMR (75 MHz, CDCl3) δC 196.7, 150.0, 148.6,

18

ACCEPTED MANUSCRIPT 138.3, 137.5,130.3, 120.8, 125.6,125.2, 124.6, 119.3, 116.0, 110.8, 45.1, 37.3, 36.6, 32.1., 29.1, 26.7, 25.6, 25.0, 24.3, 23.7, 23.0 ppm; HRMS (ESI+) m/z: 376.2385 ([M+H]+, C24H30ON3; calcd.376.2383). 4.7.62-(4-((4aR,9aS)-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1H-

RI PT

benzo[7]annulen-7-yl)-1H-1,2,3-triazol-1-yl)-N-(p-tolyl)acetamide (20f). Yellow liquid;

-

[α]D25 37.67(c 0.6,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.54 (1H, s), 7.32(2H, d, J = 8.3Hz), 7.12 (2H, d, J = 8.2Hz), 6.53 (1H, m), 5.12 (2H, s), 4.88 (1H, s), 4.53 (1H, s), 2.37

SC

(2H, m), 2.30 (3H, s), 2.13 (2H, m), 1.82 (2H, m), 1.65 (2H, m), 1.58 (2H, m), 0.92 (3H, s), 0.87 (3H, s); 13C NMR (75 MHz, CDCl3) δC 163.1, 149.9, 148.5, 134.9, 134.1, 133.4, 129.5,

M AN U

127.8, 125.6, 125.2, 120.3, 120.0, 110.0, 53.7, 45.3, 37.3, 36.8, 32.1., 29.7, 25.6, 25.1, 24.2, 23.7,23.0, 20.8 ppm; HRMS(ESI+) m/z: 405.2643 ([M+H]+, C25H33ON4; calcd.405.2649). 4.7.7.4-((4aR,9aS)-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1Hbenzo[7]annulen-7-yl)-1-(3-methoxyphenyl)-1H-1,2,3-triazole (20g).Yellow oil; [α]D25 -

TE D

72.80(c 1,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.75 (1H, s), 7.39 (2H, t, J =8.3, 7.5Hz), 7.32 (2H, t, J = 4.5,2.2 Hz), 7.23 (1H, dd , J = 8.3,6.7Hz), 6.94 (1H, dd, J = 7.5,2.2Hz), 6.59 (1H, m), 4.89 (1H, s), 4.56 (1H, s), 3.87 (3H, s), 2.41 (2H, m), 2.31 (2H, m), 2.14 (2H, m),

EP

1.83 (2H, m), 1.65 (2H, m), 1.56 (2H, m), 0.94 (3H, s), 0.88 (3H, s);

13

C NMR (75 MHz,

AC C

CDCl3) δC 160.5, 149.6, 148.6, 132.9, 130.4, 133.4, 125.8, 124.8, 116.2, 114.2, 112.2, 110.0, 106.1, 55.6, 45.1, 36.9, 32.1, 31.9, 29.2, 25.6, 25.0, 24.3, 23.7, 23.0 ppm; HRMS (ESI+) m/z: 364.2381([M+H]+, C23H30ON3; calcd. 364.2385). 4.7.81-(4-chlorophenyl)-4-((4aR,9aS)-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9aoctahydro-1H-benzo[7]annulen-7-yl)-1H-1,2,3-triazole (20h). Yellow liquid; [α]D25 -65.67(c 0.3,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.74 (1H, s), 7.65 (2H, d, J =8.1Hz), 7.47 (2H, d, J =8.8Hz), 6.59 (1H, s), 4.87 (1H, s), 4.56 (1H, s), 2.41 (2H, m), 2.31 (2H, m), 2.12 (2H, m), 1.80 (2H, m), 1.53 (2H, m), 0.93 (3H, s), 0.88 (3H, s); 13C NMR (75 MHz, CDCl3) δC 149.8,

19

ACCEPTED MANUSCRIPT 148.6, 136.9, 135.6, 129.8, 125.6, 125.1, 115.9, 110.0, 45.1, 37.3, 36.8, 32.1, 29.1, 25.6, 25.0, 24.3, 23.7, 23.0 ppm; HRMS (ESI+) m/z: 368.1888 ([M+H]+, C22H27ClN3; calcd.368.1888). 4.7.9.4-((4aR,9aS)-4a,9a-dimethyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1Hbenzo[7]annulen-7-yl)-1-(p-tolyl)-1H-1,2,3-triazole,(20i).

Brown

liquid;

[α]D25-83.50(c

RI PT

0.2,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.73 (1H, s), 7.58(2H, d, J = 8.5Hz), 7.29 (2H, d, J = 8.0Hz ), 6.74 (1H, s), 4.89 (1H, s), 4.56 (1H, s), 2.45 (2H, m), 2.41 (3H, s), 2.30 (3H, m), 2.14 (2H, m), 1.83 (1H, m), 1.65 (2H, m), 1.53 (2H, m), 0.94 (3H, s, ), 0.88 (3H, s); 13C

SC

NMR (75 MHz, CDCl3) δC 149.5, 148.1, 138.4, 134.9, 130.0, 125.9, 124.5, 120.3, 116.2, 110.9, 45.1, 37.3, 36.9, 32.1, 29.1, 25.6, 25.0, 24.3, 23.7, 23.0, 29.6, 21.0 ppm; HRMS

M AN U

(ESI+) m/z: 348.2431([M+H]+, C23H30N3; calcd.348.2434).

4.7.10.2-(4-((9aR)-9a-methyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1H-benzo[7]annulen7-yl)-1H-1,2,3-triazol-1-yl)-N-phenylacetamide (20j). Yellow liquid; [α]D25 -149.60(c 0.2,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.63 (1H, bs), 7.45(2H, d, J = 9.0Hz), 7.32 (2H,

TE D

d ,J = 9.0Hz), 7.30 (1H, s), 7.14 (1H, t, J = 7.4, 2.2Hz), 6.41 (1H, m), 4.60 (1H, s), 4.44 (1H, s), 2.36 (2H,m), 2.30 (2H, m), 2.14 (2H, m), 1.83 (2H, m), 1.63 (2H, m, ), 1.54, 0.99(3H, s), 0.88(3H, s); 13C NMR (75 MHz, CDCl3) δC 162.5, 151.8, 148.4, 136.4, 129.0, 128.6, 122.2,

EP

120.3, 11.9, 53.9, 44.7, 36.8, 37.2, 31.9, 29.7, 25.7, 25.1, 24.9, 23.6, 23.1, 22.7 ppm; HRMS

AC C

(ESI+) m/z: 391.2490 ([M+H]+, C24H31ON3; calcd.391.2492). 4.7.11.4-((9aR)-9a-methyl-1-methylene-2,3,4,4a,5,6,9,9a-octahydro-1H-benzo[7]annulen-7yl)-1-(2,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (20k).Yellow liquid; [α]D25 -71.33 (c 0.9,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.72 (1H, s), 6.92 (2H, s), 6.58 (1H, s), 4.90 (1H, s), 4.56 (1H, s), 3.91 (3H, s), 3.89 (3H, s), 2.40 (2H, m), 2.31 (2H, m), 2.12 (2H, m), 1.81 (2H, m), 1.67 (2H, m), 1.55 (2H, m), 0.95 (3H, s), 0.87 (3H, s);

13

C NMR (75 MHz,

CDCl3) δC 153.8,149.6,148.6, 138.0, 133.0, 125.8, 124.8, 116.4, 110.9, 98.3, 61.0, 56.3,

20

ACCEPTED MANUSCRIPT 45.1, 37.1, 36.8, 32.1, 31.9, 29.1, 25.6, 25.0, 23.7, 23.0, 22.6 ppm; HRMS (ESI+) m/z: 424.2598 ([M+H]+, C25H34O 3N3; calcd.424.2596). 4.7.12.2-(4-((1S,5S)-1-isopropyl-4-methylenespiro[4.5]dec-7-en-8-yl)-1H-1,2,3-triazol-1-yl)N-(p-tolyl)acetamide (21a). Yellow oil; [α]D25-101.67(c 0.3,CHCl3); 1H NMR (300 MHz,

RI PT

CDCl3) δH 8.32 (1H, s), 7.69 (2H, d, J = 6.7Hz ), 7.35 (2H, d, J = 6.7Hz ), 7.10 (2H, d, J = 6.7Hz), 6.76 (1H, s), 5.20 (2H, s) , 4.73 (1H, s), 4.61 (1H, s), 2.48 (2H, m), 2.30 (3H, s), 2.12 (2H, m), 2.08 (1H, m), 1.88 (2H, m) 1.82 (1H, m), 1.63 (2H, m), 1.35 (2H, m) , 0.96 (3H, d, J

SC

= 6.7Hz), 0.78 (3H, d, J = 7.5Hz); 13C NMR (75 MHz, CDCl3) δC 162.8, 152.2, 149.6, 135.8, 134.6, 129.2, 126.8, 126.5, 120.2, 120.0, 103.4, 53,4, 46.2, 44.8, 43.6, 35.8, 29.4, 26.5,26.2,

M AN U

26.0, 24.8, 21.2, 20.6 ppm; IR (v/cm-1) :3413, 3102, 2857, 1718, 1593, 1370, 1217; HRMS(ESI+) m/z: 405.2646 ([M+H]+, C25H33ON4; calcd.405.2649). 4.7.13.

4-((1R,5S)-1-ethyl-4-methylenespiro[4.5]dec-7-en-8-yl)-1-(2,4,5-trimethoxyphenyl)-

1H-1,2,3-triazole (21b). Brown liquid [α]D25 -84.56(c 0.9,CHCl3); 1H NMR (300 MHz,

TE D

CDCl3) δH 7.80 (1H, s), 6.94 (1H, s), 6.82 (1H, s), 4.73 (1H, s), 4.62 (1H, s), 3.94 (3H, s, ), 2.53 (2H, m), 2.35 (2H, m), 2.11 (2H, m), 2.05 (1H, m), 1.85 (1H, m), 1.64 (2H, m), 1.17 (2H, m), 0.98 (3H,d, J = 6.8Hz), 0.82 (3H, d, J = 6.8Hz); 13C NMR (75 MHz, CDCl3) δC

EP

153.8, 152.6, 149.7, 138.0, 133.0, 127.2, 126.5, 124.9, 116.9, 103.6, 98.3, 61.0, 56.4, 46.5,

AC C

45.1, 43.9, 36.1, 29.7, 26.4, 26.2, 25.2, 21.2, ppm; HRMS (ESI+) m/z: 424.2594 ([M+H]+, C25H34O 3N3; calcd.424.2596). 4.7.14.4-((1S, 5S)-1-isopropyl-4-methylenespiro[4.5]dec-7-en-8-yl)-1-(4-methoxyphenyl)-1H1,2,3-triazole,(21c). Yellow liquid [α]D25 -46.67(c 0.3,CHCl3); 1H NMR (300 MHz, CDCl3) δH 7.78 (1H, s), 7.64 (2H, d, J = 9.0Hz ), 7.01 (2H, d ,J = 9.0Hz), 6.81 (1H,s), 4.73 (1H,s), 4.62 (1H, s), 3.87 (3H, s), 2.59 (2H, m), 2.41 (2H, m), 2.13 (2H, m), 2.03 (1H, m), 1.88 (1H, m), 1.60 (2H,m), 1.54 (2H, m), 0.93 (3H, d, J = 6.7Hz), 0.81 (3H, d, J = 7.5Hz);

13

C NMR

(75 MHz, CDCl3) δC 152.7, 149.5, 142.2, 134.7, 129.7, 122.1, 116.9, 114.6, 103.6, 55.6, 46.6,

21

ACCEPTED MANUSCRIPT 45.1, 43.9, 36.1, 29.7, 26.9, 26.5, 25.2, 21.5 ppm; HRMS(ESI+) m/z: 364.2387[M+H]+, C23H30ON3; calcd. 364.2385). 5. Cytotoxicity Assay All the isolates and its derivatives were tested for in vitro cytotoxicity on different

RI PT

cancer cell lines by MTT assay. The cell lines used in this study were HELA (cervical cancer), MCF-7 (breast cancer), HEP G2 (liver cancer) and HT-29, COLO-205 (colon cancer). All the cells were obtained from National Center for cellular Sciences (NCCS), Pune,

SC

India. DMEM (Dulbecco’s modified Eagles medium), MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide], trypsin, EDTA were purchased from sigma chemicals Co.

M AN U

(St. Louis, MO) and Fetal bovine serum were purchased from Gibco. Cells were plated at a density of 10,000 cells per well in 100µl media in 96 well plates and grown for 24 hours. The cells were then exposed to a series of concentrations of the test compounds (10 to 200µg/ml) for 48 hrs and the viability of the cells was measured by using MTT method. Briefly, a

TE D

volume of 10µl of MTT solution (5 mg/ml in PBS) was added to each well containing 90µl of the media. The plates were then incubated for 4 hrs at 370C. After incubation, a volume of 200µl of DMSO was added to each well for 10 minutes at room temperature. Absorbance was

EP

measured at 570 nm using multidetection reader (Synergy 4, Biotek, USA). The mean % of

AC C

cell viability relative to that of untreated cells was estimated from data of three individual experiments. The IC50 value of each compound was calculated by curve fitting method. Acknowledgements

This work was financially supported by NaPAHA project grant CSC-0130 from the Council of Scientific and Industrial Research, New Delhi (India) under CSIR-Network program. BS, BP, and AV and thank to UGC and CSIR for financial support.

22

ACCEPTED MANUSCRIPT Supporting information Supporting information (spectral data) related to this article is available. References [1] (a) W. L. Xiao, R. T. Li, S. X. Huang, J. X. Pu, H. D. Sun, Nat. Prod. Rep. 25(2008)

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871–891

[2] A. Panossian, G. Wikman, J. Ethnopharmacol. 118 (2008) 183-212.

[3] J.L.Hancke, R.A. Burgos, F. Ahumada, Fitoterapia 99(1999) 451-471.

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[4] B. Talapatra, A. Basak, S.K. Talapatra. Indian. J.Chem.21B (1982), 76-78. [5] J.D. Hooker, Flora of British India, Vol I ( L.Reeves), 1875, pp 44.

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[6] W-L. Xiao, Y-Q, Gong, R-R, Wang, Z-Y, Weng, X.Luo, Xi-N, Li, G-Y, Yang, F.He, J-X, Pu, Li-M, Yang, Y-T, Zheng, Y.Lu, H-D. Sun, J. Nat.Prod. 72 (2009), 1678-1681; (b). [7] (a). M. S.A. Rao, G. Suresh, P.A.Yadav, K.R.Prasad, V.L. Naik, S. Ramakrishna, C.V.Rao, K.S. Babu, Tetrahedron Lett. 53 (2012) 6241-6244; (b). B. Siva, B.Poornima,

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A.Venkanna, K. R. Prasad, B. Sridhar, V.L.Nayak, S. Ramakrishna, K.S. Babu, Phytochemistry 98(2014) 174-182;(c). B.Siva, G. Suresh, B. Poornima, A. Venkanna, K.S. Babu, K.R. Prasad, L.P. Reddy, A.S. Sreedhar, C.V. Rao, Tetrahedron Lett.

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54(2013) 2934-2937.

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[8] A. Venkanna, B. Siva, B. Poornima, P. R. Rao Vadaparthi, K.R.Prasad, K. A. Reddy, G. B.P. Reddy, K. S. Babu, Fitoterapia 95 (2014) 102-108. [9]. R. Kumar, D.Varma, S. M. Mobin, I. N. N. Namboothiri, Org. Lett. 14(2012) 4070-4073. [10].(a) F. Colombo, C.Tintori, A.Furlan, S.Borrelli, M.S. Christodoulou, R. Dono, F. Maina, M. Botta, M. Amat, J. Bosch, D. Passarella, Bioorg. Med. Chem. Lett. 22 (2012) 40934096; (b).D. Luvino, C. Amalric, M.Smietana, J-J.Vasseur, Synlett (2007) 3037-3041. [11]. Y.O. Nunez, I.S. Salabarria, I.G. Collado, H.R. Galan, Phytochemistry 68 ( 2007) 24092414.

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ACCEPTED MANUSCRIPT [12] (a). H.Li, H.Huang, Ch. Shao, Hu. Huang, J.Jiang, X.Zhu, Y.Liu, L.Liu, Y.Lu, Y. Lin, Z.She, J. Nat.Prod. 74(2011) 1230-1235; (b).L. Zhang, W-S. Zhou, X-X. Xu, J.Chem.Soc.Chem.Commun. (1988) 523. [13] P.Valente, T.D.Avery, D.K.Taylor, E.R.T.Tiekink, J.Org.Chem.74(2009) 274-282.

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[14] (a). Y-O-Nunez, I.S.Salabaria, I-G. Collado, R-H. Gallon, Phytochemistry 68 (2007) 2409–2414; (b). W.G.Dauben, E.I. Aoyagi, Tetrahedron 26 (1970) 1249-1253.

[15] K. Mendhayar, I-W. Lo, Ch-Ch. Liaw, Yu-Chi, Lin, A.E. Fazary, Y-Ch. Kuo, H-J.

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Wang, B.H.Chiang, S-S. Liou, Y-C. Shen, Helv.Chim.Acta 94(2011) 2295-2302. [16] Y. J. Hong, D. J.Tantillo, J. Am.Chem. Soc. 131(2009) 7999-8015.

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[17] G.D.Brown, Molecules 15(2010) 7603-7698.

[18] H. J. Kwon, Y. K. Hong, C. Park, Y. H. Choi, H. J. Yun, E. W. Lee,B. W. Kim, Cancer Letters 290 (2010) 96-103.

[19]. V.R.S.Rao, G. Suresh, K.S. Babu, S.S.N.Raju, M.V.P.S. VishnuVardhan, S.

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Ramakrishna, J.M. Rao, Tetrahedron 67(2011) 1885-1892.

Figure captions:

Figure-1: Isolated compounds from Schisandra grandiflora Figure-2: Key HMBC and COSY correlation of compound 1 Figure-3: Key HMBC and COSY correlations of compound 2 Figure-4: Key HMBC and COSY correlations of compound 3

24

ACCEPTED MANUSCRIPT Table 1: 1H NMR data of compound 1-3 in CDCl3(300 MHz, δ in ppm) Position

Compound1 δ(1H)

δ(13C)

Compound 2 δ(1H)

Compound 3 δ(1H)

δ(13C)

δ(13C)

6.80(1H,d, J = 8.8Hz)

134.9

2.48(1H, m) & 2.25(1H, m)

30.6

1.48(1H, m)

54.2

2

6.35(1H,d, J = 8.6Hz)

134.5

6.99(1H, s)

153.8

1.86(1H, m) & 1.83(1H, m)

24.4

3

----

73.5

----

141.9

4

2.02(1H,m) & 1.47(1H,m)

29.5

2.49(1H, m) & 2.12(1H, m)

20.1

5

2.25(1H,t,overlapped) & 1.44(1H,t J = 4.5,3.9Hz)

34.1

1.72(1H, m) & 1.24(1H, m)

29.6

6

---

49.9

---

41.9

7

---

82.2

---

8

2.28(1H, m) & 1.50(1H, m)

26.1

1.71(1H, m) & 1.22(1H, m)

9

1.64(1H,t, overlapped) & 0.88(1H, m)

19.3

10

1.58(1H,m) & 1.39(1H, m)

42

11

---

45

12

1.06(3H, s)

25.9

13

1.07(3H, s)

14

1.04(3H, s)

15

1.36(3H,s)

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1

29.7

----

159.1

----

47.1

1.85(1H, m) & 1.52(1H, m) 23.5

---

35.9

2.45(1H, m) & 2.1(1H, m) 18.3

---

32.6

---

141.5

2.0(2H, m)

22.8

6.85(1H, s)

151.4

5.44(1H, s)

123.5

2.75(1H, m) & 2.15(1H, m)

37.2

---

138.4

2.3(1H, overlapped)

29.7

1.58(3H, s)

23.2

0.89(3H,d,J = 6.7Hz)

21.2

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2.48(1H, m)&2.30(1H, m)

28.1

0.88(3H, s)

22.8

0.96(3H,d,J = 6.7Hz)

23.2

21.4

0.91(3H, s)

24.8

4.76(1H,s) & 4.55(1H,s)

104.2

21.4

9.41(1H, s)

193.5

9.41(1H,s)

193.6

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Assignments were based on 2D NMR including DQF-COSY, HSQC, HMBC and NOESY. Well-resolved couplings are expressed with coupling patterns and coupling constants in Hz in parentheses. For overlapped signals, only chemical shift values are given

25

ACCEPTED MANUSCRIPT Table-2: Preparation of different triazole derivatives of 3 and 4. Entry

Stating material

Azide partners

20a

Product N N N

N3

Yield (%)

Time

70

12h

80

14h

O

20b

N3

N3

N N N

O

O

O2N

20c

N3

20d

NO2

N N N

I

20e

N N N

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20f

O

74

12h

78

16h

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I

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O N N N

O

60

14h

67

15h

75

12h

77

15.5h

60

13h

67

14h

80

12.5h

73

11h

69

12.5h

78

13h

NH

N3

N H

N3

20g

N N N

O

N3

20h

O

N N N

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Cl

N3

20i

21a

N3

N N N

O NH

O

N3

O

O

N N N

O

O O

O

N3

21b

N N N

N H

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20k

EP

O

20j

Cl

N N N

N H

O NH

O

N3

O

O

N N N

O

O O

N3

21c

N N N

O O

26

ACCEPTED MANUSCRIPT

Table-3: Cytotoxicity of the compounds -IC50 in µM Hela

A549

DU-145

MCF-7

B-16

1

122.2±2.43

94.83±1.22

114.2±1.01

129.11±2.51

342.6±5.32

479.8±2.65

3

269.3±6.43

170.4±3.04

165±1.06

209.63±2.64

428.25±2.06

550.18±2.11

4

190.5±0.97

145.0±9.55

151.8±0.94

319.49±4.64

5

162.7±3.54

138.48±4.76

191.1±2.83

166.5±1.01

6

252.3±4.06

216.5±7.08

260.3±3.02

202.08±2.03

18

158.3±1.11

147.7±3.76

386.5±6.45

574.9±8.65

506.9±0.99

524.2±2.85

19

97.6±3.37

102.9±2.45

121.4±3.21

134.7±2.11

122.8±1.31

740.5±4.03

20a

43.63±5.03

34.65±2.12

58.87±2.45

117.76±2.56

208.8±5.03

410.33±7.55

20b

15.10±1.12

46.42±2.65

51.73±1.14

65.44±2.54

61.00±2.11

520.3±4.09

20c

31.93±2.51

24.68±1.11

62.51±1.24

40.31±1.11

31.7±1.01

698.86±5.01

20d

143.9±7.62

137.0±2.87

144.59±8.76

161.50±3.42

143.9±5.43

214.57±2.16

20e

55.70±1.11

37.65±0.95

48.50±3.03

29.9±1.96

42.24±1.32

523.52±2.84

20f

131.1±1.07

20g

27.9±0.97

20h

18.8±1.31

20i

69.10±2.06

20j

62.9±1.99

229.8±1.87

733.94±2.84

145.0±1.61

696.33±3.76

257.40±2.04

770.69±5.52

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HEK-293

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Compound

254.45±4.73

80.07±0.074

210.3±3.01

621.7±1.99

11.2±0.03

38.89±0.06

56.2±1.01

34.65±0.01

734.07±1.31

20.1±0.98

25.99±1.11

35.09±0.98

27.2±1.01

608.99±1.15

58.81±3.14

131.7±7.54

54.89±1.11

57.6±1.03

524.40±4.61

72.25±4.72

146.35±7.06

147.5±3.25

148.5±0.72

491.0±2.56

AC C

EP

269.9±6.82

20k

45.03±1.09

32.62±1.11

49.38±2.43

41.06±0.04

47.16±1.02

333.9±7.43

21a

54.1±5.82

31.7±1.11

44.50±3.76

141.90±1.76

194.8±4.53

302.97±3.67

21b

37.44±3.92

20.5±1.14

29.73±1.25

44.01±3.54

29.07±0.35

509.45±8.92

21c

54.9±8.53

43.63±1.04

65.09±2.03

63.08±2.06

107.16±2.66

388.42±4.28

Doxorubicin

3.57±0.25

3.03±0.73

5.30±1.01

2.24±0.02

2.78±0.11

NT

27

ACCEPTED MANUSCRIPT 12 8

1 7 6

10 12

O O

14 13

13

1

8

O

7

3

3 15

6

10

5 12 13

14

15

6

1 5

H

5

H 8

3 10

O

O

H

H

15

O

14

H 2

3

4

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Figure-1: Isolated compounds from Schisandra grandiflora

5

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1

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Figure-2: HMBC and COSY correlation of compound 1

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Figure -3: HMBC and COSY correlation of compound 2

Figure -4: Key HMBC and COSY correlation of compound 3

O 6

28

ACCEPTED MANUSCRIPT

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Scheme 1:-Conversion of peroxide (1) to diol(7)

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Scheme 2: Hypothetical biosynthetic pathway for the compounds 1-3.

29

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ACCEPTED MANUSCRIPT

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Scheme - 3: Preparation of triazole derivatives from 3& 4

ACCEPTED MANUSCRIPT Table 1: 1H NMR data of compound 1-3 in CDCl3(300 MHz, δ in ppm) Position

Compound1 δ(1H)

δ(13C)

Compound 2 δ(1H)

Compound 3 δ(1H)

δ(13C)

δ(13C)

6.80(1H,d, J = 8.8Hz)

134.9

2.48(1H, m) & 2.25(1H, m)

30.6

1.48(1H, m)

54.2

2

6.35(1H,d, J = 8.6Hz)

134.5

6.99(1H, s)

153.8

1.86(1H, m) & 1.83(1H, m)

24.4

3

----

73.5

----

141.9

2.48(1H, m)&2.30(1H, m)

29.7

4

2.02(1H,m) & 1.47(1H,m)

29.5

2.49(1H, m) & 2.12(1H, m)

20.1

5

2.25(1H,t,overlapped) & 1.44(1H,t J = 4.5,3.9Hz)

34.1

1.72(1H, m) & 1.24(1H, m)

29.6

6

---

49.9

---

41.9

7

---

82.2

---

35.9

8

2.28(1H, m) & 1.50(1H, m)

26.1

1.71(1H, m) & 1.22(1H, m)

9

1.64(1H,t, overlapped) & 0.88(1H, m)

19.3

2.0(2H, m)

10

1.58(1H,m) & 1.39(1H, m)

42

11

---

45

12

1.06(3H, s)

25.9

13

1.07(3H, s)

28.1

14

1.04(3H, s)

15

1.36(3H,s)

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1

159.1

----

47.1

1.85(1H, m) & 1.52(1H, m) 23.5

---

2.45(1H, m) & 2.1(1H, m) 18.3

---

32.6

---

141.5

22.8

6.85(1H, s)

151.4

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----

123.5

2.75(1H, m) & 2.15(1H, m)

37.2

---

138.4

2.3(1H, overlapped)

29.7

1.58(3H, s)

23.2

0.89(3H,d,J = 6.7Hz)

21.2

0.88(3H, s)

22.8

0.96(3H,d,J = 6.7Hz)

23.2

21.4

0.91(3H, s)

24.8

4.76(1H,s) & 4.55(1H,s)

104.2

21.4

9.41(1H, s)

193.5

9.41(1H,s)

193.6

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5.44(1H, s)

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Assignments were based on 2D NMR including DQF-COSY, HSQC, HMBC and NOESY. Well-resolved couplings are expressed with coupling patterns and coupling constants in Hz in parentheses. For overlapped signals, only chemical shift values are given

ACCEPTED MANUSCRIPT Table-2: Preparation of different triazole derivatives of 3 and 4. Stating material

Azide partners

Product

Yield (%)

Time

20a

70

12h

20b

80

14h

RI PT

Entry

20c

12h

78

16h

SC

20d

74

60

14h

67

15h

75

12h

77

15.5h

60

13h

67

14h

80

12.5h

73

11h

21b

69

12.5h

21c

78

13h

20e

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20f

20g

20i

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20h

21a

N3

AC C

20k

EP

O

20j

N H

ACCEPTED MANUSCRIPT

Table-3: Cytotoxicity of the compounds -IC50 in µM Hela

A549

DU-145

MCF-7

B-16

1

122.2±2.43

94.83±1.22

114.2±1.01

129.11±2.51

342.6±5.32

479.8±2.65

3

269.3±6.43

170.4±3.04

165±1.06

209.63±2.64

428.25±2.06

550.18±2.11

4

190.5±0.97

145.0±9.55

151.8±0.94

319.49±4.64

5

162.7±3.54

138.48±4.76

191.1±2.83

166.5±1.01

6

252.3±4.06

216.5±7.08

260.3±3.02

202.08±2.03

18

158.3±1.11

147.7±3.76

386.5±6.45

574.9±8.65

506.9±0.99

524.2±2.85

19

97.6±3.37

102.9±2.45

121.4±3.21

134.7±2.11

122.8±1.31

740.5±4.03

20a

43.63±5.03

34.65±2.12

58.87±2.45

117.76±2.56

208.8±5.03

410.33±7.55

20b

15.10±1.12

46.42±2.65

51.73±1.14

65.44±2.54

61.00±2.11

520.3±4.09

20c

31.93±2.51

24.68±1.11

62.51±1.24

40.31±1.11

31.7±1.01

698.86±5.01

20d

143.9±7.62

137.0±2.87

144.59±8.76

161.50±3.42

143.9±5.43

214.57±2.16

20e

55.70±1.11

37.65±0.95

48.50±3.03

29.9±1.96

42.24±1.32

523.52±2.84

20f

131.1±1.07

20g

27.9±0.97

20h

18.8±1.31

20i

69.10±2.06

20j

733.94±2.84

145.0±1.61

696.33±3.76

257.40±2.04

770.69±5.52

M AN U

SC

229.8±1.87

254.45±4.73

80.07±0.074

210.3±3.01

621.7±1.99

11.2±0.03

38.89±0.06

56.2±1.01

34.65±0.01

734.07±1.31

20.1±0.98

25.99±1.11

35.09±0.98

27.2±1.01

608.99±1.15

58.81±3.14

131.7±7.54

54.89±1.11

57.6±1.03

524.40±4.61

62.9±1.99

72.25±4.72

146.35±7.06

147.5±3.25

148.5±0.72

491.0±2.56

20k

45.03±1.09

32.62±1.11

49.38±2.43

41.06±0.04

47.16±1.02

333.9±7.43

21a

54.1±5.82

31.7±1.11

44.50±3.76

141.90±1.76

194.8±4.53

302.97±3.67

21b

37.44±3.92

20.5±1.14

29.73±1.25

44.01±3.54

29.07±0.35

509.45±8.92

21c

54.9±8.53

43.63±1.04

65.09±2.03

63.08±2.06

107.16±2.66

388.42±4.28

Doxorubicin

3.57±0.25

3.03±0.73

5.30±1.01

2.24±0.02

2.78±0.11

NT

AC C

269.9±6.82

EP

TE D

HEK-293

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Compound

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Figure-1: Isolated compounds from Schisandra grandiflora

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ACCEPTED MANUSCRIPT

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Figure-2 : HMBC and COSY correlation of compound 1

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Figure -3: HMBC and COSY correlation of compound 2

Figure -4: Key HMBC and COSY correlation of compound 3

ACCEPTED MANUSCRIPT Highlights


Three novel compounds were isolated from Schisandra grandiflora.
Structures were characterized by spectroscopic and chemical methods.

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Triazolyl derivatives were synthesized using “Click” chemistry Protocol.
Isolates and their derivatives were screened for anti-cancer activities.

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Among the tested, compound 20g showed potent activity with IC50 11.2 ± 0.03 µM

ACCEPTED MANUSCRIPT

Novel Sesquiterpenes from Schisandra grandiflora: Isolation, cytotoxic activity and Synthesis of their triazole derivatives using “Click” reaction.

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B. Poornima,a Bandi Siva,a G. Shankaraiah,a A.Venkanna,a Lakshma Nayak,b Sistla Ramakrishna ,b C. Venkat Rao,c and K. Suresh Babu.a* a

Natural Products Laboratory, Division of Natural Product Chemistry,

CSIR-Indian Institute of Chemical Technology, Hyderabad 500 607, India

Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical

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b

Technology, Hyderabad 500 007, India

Table of Contents 1) HRESIMS SPECTRUM OF COMPOUND 1 2) IR SPECTRUM OF COMPOUND 1

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3) 1HNMR SPECTRUM OF COMPOUND 1

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c Department of Chemistry, Sri Venkateswara University, Tirupati-517502, India

16) HMBC SPECTRUM OF COMPOUND 2

17) COSY SPECTRUM OF COMPOUND 2

18) NOESY SPECTRUM OF COMPOUND 2

19) HRESIMS SPECTRUM OF COMPOUND 3

5) DEPT SPECTRUM OF COMPOUND 1

20) IR SPECTRUM OF COMPOUND 3

6) HSQC SPECTRUM OF COMPOUND 1

21)1HNMR SPECTRUM OF COMPOUND 3

7) HMBC SPECTRUM OF COMPOUND 1

22)13C NMR SPECTRUM OF COMPOUND 3

8) COSY SPECTRUM OF COMPOUND 1

23) DEPT SPECTRUM OF COMPOUND 3

9) NOESY SPECTRUM OF COMPOUND 1

24) HSQC SPECTRUM OF COMPOUND 3

10) HRESIMS SPECTRUM OF COMPOUND 2

25) HMBC SPECTRUM OF COMPOUND 3

11) IR SPECTRUM OF COMPOUND 2

26) COSY SPECTRUM OF COMPOUND 3

12)1HNMR SPECTRUM OF COMPOUND 2

27) NOESY SPECTRUM OF COMPOUND 3

13)13C NMR SPECTRUM OF COMPOUND 2

28) HRESIMS SPECTRUM OF COMPOUND 7

14) DEPT SPECTRUM OF COMPOUND 2

29)1HNMR SPECTRUM OF COMPOUND 7

15) HSQC SPECTRUM OF COMPOUND 2

30)13C NMR SPECTRUM OF COMPOUND 7

AC C

EP

4)13C NMR SPECTRUM OF COMPOUND 1

31)1HNMR SPECTRUM OF COMPOUND 18

ACCEPTED MANUSCRIPT

50)13C NMR SPECTRUM OF COMPOUND 20i

33)1HNMR SPECTRUM OF COMPOUND 20a

51)1HNMR SPECTRUM OF COMPOUND 20j

34)13C NMR SPECTRUM OF COMPOUND 20a

52)13C NMR SPECTRUM OF COMPOUND 20j

35)1HNMR SPECTRUM OF COMPOUND 20b

53)1HNMR SPECTRUM OF COMPOUND 20k

36)13C NMR SPECTRUM OF COMPOUND 20b

54)13C NMR SPECTRUM OF COMPOUND 20k

37)1HNMR SPECTRUM OF COMPOUND 20c

55)1HNMR SPECTRUM OF COMPOUND 19

38)13C NMR SPECTRUM OF COMPOUND 20c

56)13C NMR SPECTRUM OF COMPOUND 19

39)1HNMR SPECTRUM OF COMPOUND 20d

57)1HNMR SPECTRUM OF COMPOUND 21a

40)13C NMR SPECTRUM OF COMPOUND 20d

58)13C NMR SPECTRUM OF COMPOUND 21a

41)1HNMR SPECTRUM OF COMPOUND 20e

59)1HNMR SPECTRUM OF COMPOUND 21b

M AN U

SC

RI PT

32)13C NMR SPECTRUM OF COMPOUND 18

42)13C NMR SPECTRUM OF COMPOUND 20e

60)13C NMR SPECTRUM OF COMPOUND 21b

43)1HNMR SPECTRUM OF COMPOUND 20f

61)1HNMR SPECTRUM OF COMPOUND 21c

44)13C NMR SPECTRUM OF COMPOUND 20f 45)1HNMR SPECTRUM OF COMPOUND 20g

46)13C NMR SPECTRUM OF COMPOUND 20g

TE D

47)1HNMR SPECTRUM OF COMPOUND 20h

48)13C NMR SPECTRUM OF COMPOUND 20h

AC C

EP

49)1HNMR SPECTRUM OF COMPOUND 20i

62)13C NMR SPECTRUM OF COMPOUND 21c

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

O

AC C

EP

O

1) HRESIMS SPECTRUM OF COMPOUND 1

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

2) IR SPECTRUM OF COMPOUND 1

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

O

7.5

7.0

6.5

6.0

TE D

O

5.5

5.0

4.5

4.0

3.5

3.0

2.5

AC C

EP

3)1HNMR SPECTRUM OF COMPOUND 1

2.0

1.5

1.0

0.5

0.0

ACCEPTED MANUSCRIPT

RI PT

O

140

130

120

110

100

M AN U

SC

O

90

80

70

60

50

40

30

20

10

0

TE D

4) 13C NMR SPECTRUM OF COMPOUND 1

O

140

130

EP

O

120

110

100

90

80

70

60

50

AC C

5) DEPT SPECTURM OF COMPOUND 1

40

30

20

ACCEPTED MANUSCRIPT

O

M AN U

SC

RI PT

O

AC C

EP

TE D

6) HSQC SPECTRUM Of COMPOUND 1

O

O

7) HMBC SPECTRUM OF COMPOUND 1

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

RI PT

1

AC C

EP

8) COSY SPECTRUM OF COMPOUND 1

ACCEPTED MANUSCRIPT

O

M AN U

SC

RI PT

O

AC C

EP

TE D

9) NOESY SPECTRUM OF COMPOUND 1

ACCEPTED MANUSCRIPT

RI PT

O

M AN U

SC

H

10) HRESIMS SPECTRUM OF COMPOUND 2

TE D

O

AC C

EP

H

11) IR SPECTRUM OF COMPOUND 2

ACCEPTED MANUSCRIPT

RI PT

O

9

8

7

M AN U

SC

H

6

5

4

3

2

1

0

TE D

12) 1HNMR SPECTRUM OF COMPOUND 2

O

200

AC C

EP

H

175

150

125

100

75

13) 13CNMR SPECTRUM OF COMPOUND 2

50

25

0

RI PT

ACCEPTED MANUSCRIPT

O

150

125

100

75

M AN U

175

TE D

14) DEPT SPECTRUM OF COMPOUND 2

O H

EP

200

AC C

2

SC

H

15) HSQC SPECTRUM OF COMPOUND 2

50

25

RI PT

ACCEPTED MANUSCRIPT

O

M AN U

SC

H

AC C

EP

TE D

16) HMBC SPECTRUM OF COMPOUND 2

ACCEPTED MANUSCRIPT

RI PT

O

M AN U

SC

H

AC C

EP

TE D

17) COSY SPECTRUM OF COMPOUND 2

ACCEPTED MANUSCRIPT

RI PT

O

M AN U

SC

H

EP

TE D

18) NOESY SPECTRUM OF COMPOUND 2

AC C

O

H

19) HRESIMS SPECTRUM OF COMPOUND 3

RI PT

ACCEPTED MANUSCRIPT

O

M AN U

SC

H

AC C

EP

TE D

20) IR SPECTRUM OF COMPOUND 3

9

8

7

O H

6

5

4

3

21) 1HNMR SPECTRUM OF COMPOUND 3

2

1

0

RI PT

ACCEPTED MANUSCRIPT

SC

O

175

150

125

100

75

EP

TE D

22)13C NMR SPECTRUM OF COMPOUND 3

AC C

200

M AN U

H

50

25

RI PT

ACCEPTED MANUSCRIPT

O

150

125

100

75

M AN U

175

TE D

23) DEPT SPECTRUM OF COMPOUND 3

O

EP

H

AC C

200

SC

H

24) HSQC SPECTRUM OF COMPOUND 3

50

25

RI PT

ACCEPTED MANUSCRIPT

SC

O

TE D

M AN U

H

AC C

EP

25) HMBC SPECTRUM OF COMPOUND 3

TE D

O

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

H

26) COSY SPECTRUM OF COMPOUND 3

RI PT

ACCEPTED MANUSCRIPT

SC

O

TE D

M AN U

H

AC C

EP

27) NOESY SPECTRUM OF COMPOUND 3

ACCEPTED MANUSCRIPT

RI PT

OH

M AN U

SC

OH

TE D

28) HRESIMS SPECTRUM OF COMPOUND 7

OH

7.5

AC C

EP

OH

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

29) 1H NMR SPECTRUM OF COMPOUND 7

1.5

1.0

0.5

0.0

OH

RI PT

ACCEPTED MANUSCRIPT

200

175

150

125

M AN U

SC

OH

100

75

50

AC C

EP

TE D

30) 13C NMR SPECTRUM OF COMPOUND 7

25

0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

200

AC C

EP

TE D

31) 1H NMR SPECTRUM OF COMPOUND 18

175

150

125

100

75

32) 13C NMR SPECTRUM OF COMPOUND 18

50

25

0

RI PT

ACCEPTED MANUSCRIPT

SC

N N N

7.5

7.0

6.5

6.0

5.5

5.0

4.5

M AN U

O

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

N N N

TE D

33) 1H NMR SPECTRUM OF COMPOUND 20a

200

AC C

EP

O

175

150

125

100

75

34) 13C NMR SPECTRUM OF COMPOUND 20a

50

25

0

RI PT

ACCEPTED MANUSCRIPT

SC

N N N

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

M AN U

O

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

-0.5

EP

N N N

TE D

35) 1H NMR SPECTRUM OF COMPOUND 21b

200

AC C

O

175

150

125

100

75

50

36) 13C NMR SPECTRUM OF COMPOUND 20b

25

0

N N N

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

SC

8.0

NO 2

3.0

2.5

2.0

1.5

1.0

0.5

0.0

M AN U

8.5

RI PT

ACCEPTED MANUSCRIPT

EP

N N N

TE D

37) 1H NMR SPECTRUM OF COMPOUND 20c

AC C

NO 2

200

175

150

125

100

75

50

38) 13C NMR SPECTRUM OF COMPOUND 20c

25

0

RI PT

ACCEPTED MANUSCRIPT

I

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

-0.5

M AN U

8.0

SC

N N N

TE D

39) 1H NMR SPECTRUM OF COMPOUND 20d

I

AC C

EP

N N N

200

175

150

125

100

75

50

40) 13C NMR SPECTRUM OF COMPOUND 20d

25

0

RI PT

ACCEPTED MANUSCRIPT

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

M AN U

SC

N N O N

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

TE D

41) 1H NMR SPECTRUM OF COMPOUND 20e

AC C

EP

N N O N

200

175

150

125

100

75

50

42) 13C NMR SPECTRUM OF COMPOUND 20e

25

0

N N N

O

5.0

4.5

RI PT

ACCEPTED MANUSCRIPT

8.5

8.0

7.5

7.0

6.5

6.0

5.5

M AN U

SC

NH

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

-0.5

TE D

43) 1H NMR SPECTRUM OF COMPOUND 20f

N N N

O

200

AC C

EP

NH

175

150

125

100

75

44) 13C NMR SPECTRUM OF COMPOUND 20f

50

25

0

N N N

7.5

7.0

6.5

O

6.0

5.5

5.0

M AN U

8.0

SC

RI PT

ACCEPTED MANUSCRIPT

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

EP

TE D

45) 1H NMR SPECTRUM OF COMPOUND 20g

N N N

AC C

200

O

175

150

125

100

75

46) 13C NMR SPECTURM OF COMPOUND 20g

50

25

0

RI PT

ACCEPTED MANUSCRIPT

SC

N N N

7.5

7.0

6.5

6.0

5.5

M AN U

Cl

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

EP

TE D

47) 1H NMR SPECTRUM OF COMPOUND 20h

AC C

N N N

200

175

150

Cl

125

100

75

48) 13C NMR SPECTRUM OF COMPOUND 20h

50

25

0

RI PT

ACCEPTED MANUSCRIPT

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

M AN U

SC

N N N

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

EP

TE D

49) 1H NMR SPECTRUM OF COMPOUND 20i

AC C

N N N

200

175

150

125

100

75

50

50) 13C NMR SPECTRUM OF COMPOUND 20i

25

0

-0.5

N N N

RI PT

ACCEPTED MANUSCRIPT

O

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

M AN U

8.0

SC

NH

1.5

1.0

0.5

0.0

TE D

51) 1H NMR SPECTRUM OF COMPOUND 20j

N N N

O

AC C

EP

NH

200

175

150

125

100

75

50

52) 13C NMR SPECTRUM OF COMPOUND 20j

25

0

ACCEPTED MANUSCRIPT

N N N

O O

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

SC

8.0

RI PT

O

1.0

0.5

0.0

TE D

M AN U

53) 1H NMR SPECTRUM OF COMPOUND 20k

N N N

O

O

200

AC C

EP

O

175

150

125

100

75

54) 13C NMR SPECTRUM OF COMPOUND 20k

50

25

0

7.0

6.5

6.0

5.5

5.0

4.5

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

AC C

EP

TE D

55) 1H NMR SPECTRUM OF COMPOUND 19

200

175

150

125

100

75

56) 13C NMR SPECTRUM OF COMPOUND 19

50

25

0

N N N

O

RI PT

ACCEPTED MANUSCRIPT

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

M AN U

SC

NH

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

EP

N N N

TE D

57) 1H NMR SPECTRUM OF COMPOUND 21a

O

AC C

NH

200

175

150

125

100

75

50

58) 13C NMR SPECTRUM OF COMPOUND 21a

25

0

ACCEPTED MANUSCRIPT

N N N

RI PT

O

O

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

M AN U

8.0

SC

O

O

EP

N N N

TE D

59) 1H NMR SPECTRUM OF COMPOUND 21b

O

200

AC C

O

175

150

125

100

75

50

60)13CNMR SPECTRUM OF COMPOUND 21b

25

0

RI PT

ACCEPTED MANUSCRIPT

N N N

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

M AN U

8.0

SC

O

TE D

61) 1H NMR SPECTRUM OF COMPOUND 21c

O

AC C

EP

N N N

200

175

150

125

100

75

62) 13C NMR SPECTRUM OF COMPOUND 21c

50

25

0