Recent insights into synthetic β-carbolines with anti-cancer activities

Accepted Manuscript Recent insights into synthetic β-carbolines with anti-cancer activities Sumit Kumar, Amandeep Singh, Kewal Kumar, Vipan Kumar PII:

S0223-5234(17)30430-0

DOI:

10.1016/j.ejmech.2017.05.059

Reference:

EJMECH 9489

To appear in:

European Journal of Medicinal Chemistry

Received Date: 16 February 2017 Revised Date:

22 May 2017

Accepted Date: 28 May 2017

Please cite this article as: S. Kumar, A. Singh, K. Kumar, V. Kumar, Recent insights into synthetic βcarbolines with anti-cancer activities, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.05.059. 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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Recent Insights into Synthetic β-carbolines with Anti-Cancer activities Sumit Kumar,a Amandeep Singh,a Kewal Kumar,b and Vipan Kumar,a,* Department of Chemistry, Guru Nanak Dev University, Amritsar-143005, India

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a

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β-carboline-derivatives

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Department of Applied Chemistry, Giani Zail Singh Campus College of Engineering & Technology, MRSPTU, Dabwali Road, Bathinda-151001.

β-carboline-hybrids

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Recent developments (2011-16) in synthesis and anticancer activities of β-carbolines with an emphasis on structure-activity relationship, in vivo profiles and mechanism of action.

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Recent Insights into Synthetic β-carbolines with Anti-Cancer activities Sumit Kumar,a Amandeep Singh,a Kewal Kumar,b and Vipan Kumar,a,* a

Department of Chemistry, Guru Nanak Dev University, Amritsar-143005, Punjab, India Department of Applied Chemistry, Giani Zail Singh Campus College of Engineering & Technology, MRSPTU Dabwali Road, Bathinda-151001.

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b

Abstract: Cancer, an uncontrolled and rapid proliferation of abnormal cells, has become one of the leading cause of death worldwide. The development of resistance among the numerous drugs in clinical use has provided strong impetus for the identification and development of novel

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cancer therapeutics. β-carbolines constitute an important class of pharmacologically active scaffolds known to exert their anticancer activities via diverse mechanisms. The purpose of

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present review article is to update the readers on the current developments in β-carbolines with an emphasis on synthetic strategies, structure- activity relationships, mechanism of action and in vivo studies wherever possible.

Keywords: β-carbolines, Anti-cancer activity, Structure Activity Relationship, Synthetic Strategies. 1. Introduction

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Cancer is a major health burden in both developed and under developed countries. According to the World health organization (WHO) report, 8.8 million people died of cancer all over the world in 2015 [1]. Most cancers are identified by uninhibited growth of cells without differentiation due to de-regulation of critical enzymes and proteins

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controlling cell division and proliferation [2,3]. The biological process responsible for the transformation of normal cells into malignant cancer cells has been the focus of

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scientific endeavours in the biomedical sciences. Although much progress has been made from the identification to the treatment of cancer, the factors like poor patient compliance, drug resistance and drug induced toxicities has provided strong stimulus for the discovery and development of novel cancer chemotherapeutics of clinical significance [4-6]. Anticancer drugs have been categorized in consonance with their mechanism of action as molecular targeting agents, antimetabolites, anti-tubulin and DNA-interactive agents, monoclonal antibodies and hormones [7,8].

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β-carboline alkaloids constitute a group of alkaloids with a common tricyclic pyrido-[3,4-

b]indole ring [9]. β-carbolines were originally isolated from the seeds of Peganum harmala and has been traditionally used for the treatment of alimentary tract cancers

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and malaria [10, 11]. A recent upsurge in the chemistry of β-carbolines has been observed because of their reported anticancer potential with some of the distinguished members include harmane, harmine, harmaline, harmol and callophycin (Figure 1) [12,13,14]. Anticancer activity of β-carbolines is linked through diverse mechanisms,

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such as intercalation into DNA, inhibition of topoisomerase I and II, blocking cell mitosis or targeting specific cancer signalling pathways etc. Keeping in view, the present review will update the readers on the recent developments (2011-16) in synthesis and

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anticancer activities of this particular framework. For the sake of convenience, the article has been divided into three sub-divisions viz. β-carboline and their derivatives, βcarboline-hybrids and β-carboline based metal complexes. (Insert Figure 1 here) 2. β-carboline and their derivatives

3-benzylamino-β-carbolines:

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2.1

Konakahara and co-workers have reported the synthesis and anti-tumor activity of βcarboline derivatives substituted at 6-,8- or 6,8-positions. The basic skeleton of βcarboline 3-methyl ester 4 was prepared using Pictet-Spengler reaction of L-tryptophan

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1, with subsequent esterification and aromatization (Scheme 1) [15]. The treatment of 4 with N-chloro succinimide resulted in the introduction of chloro-substitutent at C-6

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which upon a sequence of reactions was converted to the corresponding acyl azide 7. Curtius rearrangement of 7 led to the formation of 3-amino-6-chloro-β-carboline 8. 3amino-group of 8 was then protected via acetylation while C-8 position was nitrated using sodium nitrite in trifluoroacetic acid to afford 10. De-protection of amino group of 10 followed by its benzylation via reductive amination and subsequent reduction of nitro substituent at C-8 position afforded the desired 8-amino-3-benzylamino-6-chloro-βcarboline 13. Transformation of 8-amino substituent to 8-amido was also carried out using a variety of methods as shown in Scheme 1 to afford 14. Treatment of 14 with

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hydrazine hydrate and subsequently with palladium on carbon afforded 15, which upon reaction with aldehyde and sodium cyanoborohydride gave 16. (Insert Scheme 1 here)

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The synthesized compounds were evaluated for their anti-tumor profiles against Hela S3, Sarcoma 180 and 293T cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay. All the hybrids were found considerably potent with IC50 values ranging between 0.5-16.0 µM, with the most potent compound, 14a

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exhibiting IC50 values of 0.50, 0.53 and 0.99 µM against 293T, Sarcoma 180 and Hela S-3 cell lines, respectively. Structure Activity Relationship (SAR) studies revealed a slight decrease in antitumor activity with the introduction of pyridyl ring as substituent against

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all three cell lines used, while the introduction of alkyl group resulted in an increase in hydrophobicity which consequently enhanced the cell membrane permeability and antitumor potential (Figure 2).

(Insert Figure 2 here)

Further, the above approach was extended towards the synthesis of 8-amino-β-

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carbolines 17 as depicted in Scheme 2.

(Insert Scheme 2 here)

SAR studies revealed that these compounds exhibited improved antitumor activities compared to 14 when evaluated against HeLa S-3, Sarcoma 180, and 293T cancer cell

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lines. The IC50 values were found to be ranging between 0.20-9.80 µM, with the most potent compound 17a displaying IC50 values of 0.20, 0.25 and 0.35 µM against HeLa, S-3,

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Sarcoma 180, and 293T cell lines, respectively (Figure 3). The antitumor potential improved with the introduction of alkyl substituents while reduction in antitumor activities was observed with the introduction of aryl/hetero aryl substituents. (Insert Figure 3 here) (Insert Scheme 3 here)

Two pair of compounds viz. 19b, 18 and 14b, 16a (Figure 4) were selected for determining their apoptotic induction potential. Fluoresence analysis and DNA fragmentation assay confirmed that both the pair of compounds has potential to cause

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apoptotic cell death. The average G2/M population in the control cells and in 19b-, 18, 14b-, and 16a treated cells for 9-48h were 16.21, 57.00, 46.93, 49.09, and 65.56%, respectively. Also, the population had a tendency to shift from the G2/M phase to the

(Insert Figure 4 here) 2.2

β-carbolines as potential haspine kinase inhibitors:

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mean apoptotic population sub-G1 phase after 24 and 48 h.

Haspine kinase inhibitors are considered as useful probes for elucidating the cellular

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roles of proteins and have therapeutic utility in treating cancer. Harmine, a β-carboline has been identified as an inhibitor of DYRK family of kinases with IC50 values ranging between 0.03 and 0.35 µM against DYRK1A, with 50-folds lower potency towards

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DYRK2. Based on these observations, Cuny and co-workers have reported the design, synthesis and evaluation of β-carboline as potential haspine kinase inhibitors [16]. The synthetic methodology involved an initial alkylation of Harmol 20 with N-Boc-protected alkyl amines in the presence of cesium carbonate with subsequent deprotection under acidic conditions to afford the corresponding free amines 22. Harmine 23, on the other

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hand was initially N-alkylated using phthalimide protected alkyl-amines to afford 24 with subsequent de-protection using hydrazine hydrate to yield 25 (Scheme 4). (Insert Scheme 4 here)

All the synthesized compounds viz.22 and 25 were evaluated for their inhibitory activity

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against haspin kinases. Activity data revealed that although the compounds 22a and 22b were inactive haspin inhibitors, the compounds 25a and 25b inhibited haspin kinases

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with IC50 values of 0.46 and 0.17 µM, respectively. Based on the promising activity results of the above synthesized scaffolds, the work was further extended towards the synthesis of additional β-carboline as haspin kinase inhibitors. Thus, the treatment of indole 26 with N,N-dimethyl-2-nitroethenamine resulted in the isolation of 27 which upon reduction with lithium aluminium hydride (LAH) afforded 28. Pictet-Spengler reaction of 28 with various aldehydes followed by oxidation yielded the corresponding β-carbolines, 30. N-alkylation of 30 with

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phthalimide-protected alkylamines gave 31 which upon deprotection with hydrazinehydrate led to the isolation of 32. (Insert Scheme 5 here)

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Other β-carbolines were prepared using 24 as an intermediate. In one such route, 24 was initially demethylated using HBr in acetic acid to yield 33 followed by its conversion to the corresponding triflate 34. Pd-catalyzed coupling of 34 with methyl sulfonamide resulted in the formation of 35 with subsequent deprotection to yield 36. In another

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route, compound 24 was initially converted to tert-butyl ether by reacting with Me2NCH(O-t-Bu)2 with subsequent deprotection using hydrazine hydrate to afford the corresponding amine 38. Similarly, compound 24 was also de-protected to yield 39

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which was converted either to tertiary amine 40 via reductive amination or it was converted to secondary amine 42 via the formamide 41 (Scheme 6). (Insert Scheme 6 here)

Haspase-kinase inhibitory activity of the synthesized β-carboline derivatives was carried out utilizing the High Throughput Screening (HTS) at different concentrations of the compounds.

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Compounds 25a and 25b proved to be the most potent compounds exhibiting IC50 values of 0.46 and 0.17 µM, respectively. The synthesized derivatives were also evaluated for inhibition of Human DYRK2 using previously reported protocol [17]. The evaluation studies revealed the reduction in potency for DYRK2 inhibition with the introduction of amine at N-9-position.

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Further, haspine inhibitor 43 was assayed against a panel of 292 kinases at concentration of 10 µM. Most of these enzymes belongs to CMGC group of kinases,

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unlike haspine which is a divergent member of ePK family. The compound 43 in addition to haspine, inhibited thirteen of these kinases (≥90%).

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(Insert Figure 5 here)

3-phenyl-4-substitiuted-β-carbolines:

Xu et.al. has reported the synthesis of 3-phenyl-4-substituted β-carbolines with cyclization as the key step along with their in vitro evaluation against three tumor cell lines [18]. The synthetic approach encompassed an initial base promoted iodination of 2-methyl indole 44 in the presence of KOH to yield the corresponding 3-iodoindole 45.

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Because of stability issues, 3-iodoindole was immediately treated with benzene sulfonyl chloride to yield the corresponding N-sulfonyl-derivatives 46. Coupling of 46 with phenyl acetylene in the presence of Pd(PPh3)2Cl2 and Et3N led to the formation of 47 which on

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treatment with NBS and AIBN afforded 48. Treatment of 48 with sodium azide led to the formation of corresponding azide 49 which on subsequent reaction with iodine in nitromethane resulted compound 50. Coupling reaction of 50 with phenyl boronic acid or cyclopropyl boronic acid using Pd(PPh3)2Cl2 as catalyst in toluene resulted in the

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formation of 51. Hydrolysis of 51 with NaOH in aq. dioxane led to the formation of 52. SAR analysis revealed that the introduction of electron withdrawing substituents at C-4 position of phenyl ring resulted in poor anti-tumor activities against Hela and MCF cell

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lines while improvement in cytotoxicities were observed with the introduction of electron donating substituents at C-4 position of phenyl ring (Scheme 7). (Insert Scheme 7 here) 2.4

1-carboxamide and 6-sulfonamide-substituted β-carbolines as B-Raf inhibitors:

In order to explore the anti-cancer potential of β-carbolines, Lu and co-workers have

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designed a series of 1-carboxamide and 6-sulfonamide-substituted β-carbolines as B-Raf inhibitors, which serves as a critical component of MAPK signal-transduction pathway regulating cell proliferation, differentiation and survival [19]. Synthetic methodology for the preparation of 1-carboxamide-substituted β-carbolines 59 involved an initial Pictet-

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Spengler reaction of tryptamine 53 with glyoxylic acid in the presence of conc. hydrochloric acid and 30% potassium hydroxide to afford the corresponding β-carboline

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54 with subsequent esterification to yield 55. Dehydrogenation of 55 followed by ammonolysis yielded the corresponding carboxamide 57. Treatment of 57 with chlorosulfonic acid with subsequent treatment with amines afforded 1-carboxamide-6sulfonamide β-carbolines 59 (Scheme 8). (Insert Scheme 8 here)

For the synthesis of 6-sulfonamide-β-carbolines 62, the methodology involved a sequence of synthetic steps consisting of an initial Pictet-Spengler reaction of tryptamine

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1 followed by a sequence of dehydrogenation-decarboxylation-chloro-sulfonylationammonolysis as shown in Scheme 9. (Insert Scheme 9 here)

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The synthesized compounds viz. 59 and 62 were evaluated for their inhibitory activities against wild type B-Raf. All the compounds exhibited significant inhibitory activities with their IC50 values ranging from 1.62 to 91.55 µM. The most active compound 59e, bearing two dimethyl-aminopropyl groups, displayed IC50 value of 1.62µM. Replacement of both

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dimethyl-aminopropyl with diethylaminopropyl groups resulted in slight decrease in activity as evidenced by compound 59b exhibiting an IC50 value of 2.81µM. The comparable activities observed between the compounds 59p and 62a evidenced that

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the carboxamide group is not essential for inhibitory potency (Figure 6). (Insert Figure 6 here)

The observed activity profile was further confirmed via docking 59e into the wild type BRaf kinases. It was concluded that the NH group of carboxamide and N-atom of pyridine formed two hydrogen bonding with carbonyl oxygen and amide of CYS531, respectively. Harmine derivatives or N2-benzylated-β-carbolines:

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Cao et. al. synthesized a series of harmine derivatives and evaluated their cytotoxic potential against a panel of cancer cell lines viz. cervical carcinoma (Hela), liver carcinoma

(Bel-7402 and HepG2), gastric carcinoma (769-P, 786-O and OS-RC-2);

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epidermoid carcinoma of the nasopharytix (KB), non-small lung carcinoma (A-549), malignant melanorma (A375), colon carcinoma (HT-29), bladder squamous carcinoma

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(5CaBER), malignant bladder carcinoma (Blu-87) and malignant guoina (U251) [20]. Synthetic pathway included sodium hydride promoted N-alkylation of Harmine 63 to yield the corresponding 2,7,9-trisubstituted-β-carbolines 64. Further demethylation at C7 position of 64 using acetic acid/hydrobromic acid resulted in the formation of 65. NaH promoted O-alkylation of 65 in dry DMF afforded 66, which was converted to the corresponding N2-benzylated β-carbolinium bromates 67 via refluxing with benzyl bromide in ethyl acetate (Scheme 10). (Insert Scheme 10 here)

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The cytotoxic activities of the synthesized harmine derivatives was evaluated using cisplatin as the reference drug. The cytotoxic potency of 66 showed no distinct difference with IC50 values ranging from 10-100 µM. However, the benzylated series of compounds

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i.e. 67 exhibited interesting cytotoxic potencies with IC50 lower than 10µM against all the tested cancer cell lines. Promising compounds from preliminary cytotoxic studies were chosen for in vivo anti-tumor evaluation in mice with Lewis Lung Cancer and Sarcoma 180. Compounds viz. 67a, 67b, 67c, 67d and 67e displayed acute cytotoxicity with LD50

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values of 12.5, 12.5, 15.0, 5.0 and 6.25 mg/kg, respectively (Figure 7). Most of the compounds exhibited notable antitiumor activities with over 40 % tumor inhibition rate compared to harmine 1 which has inhibition rate of 30.8 % and 33.7% against Sarcoma

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180 and lewis lung cancer respectively. 67c and 67d, proved to be the most potent of the tested compounds with tumor inhibitory rate of 53 % and 52.6 % respectively against Sarcoma 180.

(Insert Figure 7 here) 2.6

C-1 aryl substituted β-carbolines:

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Zhu and co-workers have reported the synthesis of a series of C-1 aryl substituted βcarbolines along with their cytotoxic evaluation against five tumor cell lines [21]. The desired scaffolds were obtained from L-tryptophan methyl ester 68 via Pictet-Spengler condensation, affording tetrahydro-β-carboline, 69 followed by its DDQ-promoted

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oxidation to yield 70. N-alkylation of 70 using alkyl halide in the presence of K2CO3 resulted in the formation of 71. The reduction of 70 and 71 with lithium aluminium

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hydride or reaction with different Grignard reagents afforded the desired compounds 72 and 73 (Scheme 11).

(Insert Scheme 11 here)

The synthesized compounds, having alcoholic group at C-3 position exhibited good cytotoxicity with more than half of the compounds displaying IC50 values lower than 20 µM against most of the human cancer cell lines investigated. Compounds 73c and 73d exhibited remarkable cytotoxic activity, especially 72a displaying IC50 values of 0.75,

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0.91, 1.00, 1.13, 2.54 µM against the HL-60, SMMC-7721, A-549, MCF-7 and SW480 cell lines, respectively (Figure 8). (Insert Figure 8 here) 2.7

Eudistomin U derivatives:

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Eudistomins, a family of β-carboline natural products, are structurally diverse and show wide range of biological properties (Figure 9). A synthetic derivative of eudistomin K was found to be extremely potent against LI210, Molt-4F, MT-4 and P-388 leukemic cells.

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Based on this literature, Mulcahy and co-workers has reported efficient-total synthesis of Eudistomin U along with its cytotoxic evaluation [22]. (Insert Figure 9 here)

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The synthetic strategy involved an initial Bischler-Napieralski cyclization of tryptamine 53 to afford 74, which was oxidized to 75 using DDQ followed by its triflation using trifluoromethanesulfonic anhydride to afford 76. A number of pre-catalysts and solvents combination were tested to optimize the reaction condition for the Suzuki cross coupling reaction between β-carboline and indole. High yields of 77 were observed using

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Tris(dibenzylideneacetone) dipalladium(0) as a pre-catalyst in the presence of triethylamine and triphenylphosphine, which in the presence of base gave the desired 78 (Scheme 12). The synthesized compound was then tested for its cancer cell cytotoxicity using MTT-cell viability assay. The compound exhibited an IC50 of 15.6 µg/mL

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against C19 leukemia cells.

(Insert Scheme 12 here)

Tripeptide linked β -carboline derivatives:

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Zhang and co-workers has synthesized a new class of β-carboline derivatives and bioevaluated against different cancer cell lines using MTT assay [23]. Synthetic methodology involved an initial formation of the tripeptide benzyl esters with a combination of different amino acids under different reaction conditions as shown in the Scheme 13. (Insert Scheme 13 here)

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On the other hand, β-carboline-3-carboxylic acid 86 was obtained via Pictet-Spengler reaction of tryptophan with vaniline 85 in acetic acid yielding 1-(4-hydroxy-3methoxyphenyl) tetrahydro-β-carboline-3-carboxylic acid as mixture of two isomer viz. 1S, 3R and 1R, 3S. Esterification of precursor, 86 with methanol in the presence of SOCl2

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resulted in the isolation of corresponding methyl 1-(4-hydroxy-3-methhoxyphenyl)-βcarboline-3-carboxylate 87, which on further oxidation with SeO2 in acetic acid yielded 88. Base catalysed hydrolysis of 88 gave 89 which upon DCC-promoted amide coupling

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with different tripeptide units gave the corresponding β-carboline derivatives 90 (Scheme 14).

(Insert Scheme 14 here)

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The synthesized β-carboline derivatives were evaluated against human colon cancer (HT29), human lung adenocarcinoma (A549), chronic myeloid leukemia (K562) and human immature granulocyte leukemia (HL-60) cell lines using MTT assay. The results of this study suggested that the synthesized β-carboline derivatives exhibited moderate cytotoxic activity. Compounds, 90a and 90b displayed highest efficacy in the in vivo

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antitumor assay with S180 mice, exhibiting 63.4 % and 62.9 % tumour inhibition, respectively which is equipotent with the positive control, Adriyamycin (61.1 %) (Figure 10). In vivo results suggested change in antitumor activities with the slight changes in size and shape of the alkyl side chains of amino acids linked to β-carbolines, implying the

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importance of spacial orientation and characteristics of pendant group in DNA recognition and binding. The UV and fluorescence study of synthesized derivatives with

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calf thymus DNA confirmed their interactions to be intercalative. (Insert Figure 10 here)

β-carboline hydroxamic acid derivatives as HDAC inhibitors:

Ling et. al. recently reported the synthesis of a series of β-carboline hydroxamic acids which have shown significant anti-proliferation against human colorectal cell line and exhibited in vitro histone deacetylase inhibitory effects [24]. Substituted β-carbolines were prepared in a two-step sequence, involving an initial conversion of L-tryptophan 1 to 1-substituted 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid 91 via Pictet−Spengler

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reaction with subsequent oxidation with KMnO4 in DMF to yield the corresponding βcarbolines 92. This was then reacted with NH2(CH2)nCOOMe 94 in the presence of ethyl chloroformate 93 to give the corresponding esters 95 with subsequent treatment with

(Insert Scheme 15 here)

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NH4O-K+ to yield the target compounds 96 (Scheme 15). For the second series of β-carbolines, acid 97 was initially esterified to give methyl ester 98, which upon reaction with iodomethane in the presence of NaH afforded 99. The

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intermediate 99 was treated with hydrazine hydrate to form the corresponding hydrazide derivative 100 with subsequent conversion to the acyl azide in the presence of NaNO2. The treatment of acyl azide 101 with 94 gave 102 via Curtius rearrangement

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which was finally converted to hydroxamic acid 103 as shown in Scheme 16. (Insert Scheme 16 here)

The synthesized compounds were tested for their inhibitory activity against HeLa cell nuclear extract which is a rich source of HDACs. Common HDAC inhibitors consist of three domains: zinc binding group; a cap group consisting of hydrophobic aromatic

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substituent and a saturated or unsaturated linker domain. In the present case, βcarboline ring acted as a cap group while hydroxamic acid fragments acted as a zinc binder. Compound having one (103a) or two methylene (103b) units exhibited weak enzymatic inhibition while 103c displayed increased inhibitory efficacy (Figure 11).

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(Insert Figure 11 here)

Further, the synthesized compounds were also evaluated against four colon cancer cell

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lines viz.HCT116, SW620, SW480 and LOVO. These compounds inhibited cell proliferation and had higher potency than harmine while the activity was comparable to SAHA. The anti-proliferative inhibitory potency depended upon the length of spacer with compounds having three to four methylene units being potent. Structural modification on the β-carboline ring also altered the activity profiles with an increase in activity being observed via replacing the amide group at C-3 position with the urea functionality. The effect of 103d on cell growth and death was examined using shRNA control and shRNA p53 HCT 116 CRC cells in parallel. Results revealed not only an increase in p53

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activation but also p53 acetylation for cancer cell growth inhibition. In vivo analysis revealed that mice injected with 240 mg/kg dose of 103d survived and did not suffer from abnormality. The tumor weight (0.53 ± 0.12 g), in mice treated with oral dose of

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103d at 90 mg/kg, was reduced by 61.6% compared to that of the vehicle-treated controls (1.38 ± 0.35 mg), while the tumor weight was 0.62 ± 0.13 g in SAHA-treated group at the same dosing. 2.10

3,9-substituted -β-carbolines:

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Huang and co-workers have synthesized a series of 3,9-substituted-β-carboline derivatives 106 and 107 along with evaluation of their cytotoxic potential [25]. The methodology involved an initial synthesis of ethyl-1,2,3,4-tetrahydro-β-carboline-3-

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carboxylate which was oxidized to the corresponding β-carboline 104 in the presence of manganese dioxide. The target compounds were synthesized by benzylation of 104 with various aryl/methyl halides 105 to yield 106 and 107. Alkaline hydrolysis as well as hydride promoted reduction was also carried out to yield the corresponding β-carboline3-carboxylate 108 and carbinols 109, respectively (Scheme 17).

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(Insert Scheme 17 here)

All the synthesized β-carboline ester, carboxylic acid and alcohol derivatives viz. 106, 107 and 109 were examined for their anticancer potential against seven cancer cell lines and the results were compared with previously reported 1-Benzyl-3-(5-hydroxymethyl-2-

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furyl)indazole (YC-1). The synthesized compounds displayed anticancer activities in the range of 1.2 to >100 µM, with ester and acid derivatives exhibiting comparable activities

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while reduction of acid into corresponding alcohol resulted in the complete loss of anticancer potential. Compound 106b displayed the best anticancer activity among the ester series with IC50 values of 3.9 and 8.9 µM against HL-60 and Hep-3B cancer cell lines respectively. Acid derivatives, 107a and 107b exhibited IC50 values below 15 µM, which were better than YC-1. Hybrid 107j with 4-chlorophenyl as substituent displayed IC50 value of 1.2 µM against Hep-3B cell line. Flow cytometric analysis of compound 107b against HL-60 cells at various concentrations was done so as to investigate its effect on cell cycle distribution using propidium iodide staining. Compound 107b induced selective

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activation of caspase cascades. Western blot assay indicated that the compound 107b upregulated caspase-3 and caspase-9 expression and induced PARP cleavage. (Insert Figure 12 here) 2.11

β--carbolines dithiacarbamate derivatives as topoisomerase-II inhibitors:

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Kamal et.al. synthesized β-carboline derivatives containing dithiacarbamate unit and characterized them as DNA topoisomerase-II inhibitors with apoptosis inducing ability and DNA binding properties [26]. Synthetic pathway included Pictet-Spengler condensation of initially esterified L-Tryptophan with various substituted benzaldehydes

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resulting into tetrahydro-β-carbolines-3-carboxylates with subsequent oxidation with trichloroisocyanuric acid (TCC) to afford 110. Sodium hydride promoted methylation of

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110 led to the synthesis of 115. LiBH4 promoted reduction of 110 yielded the corresponding alcohols 111 which were subsequently converted into corresponding azides 112 in presence of diphenylphosphorylazide (DPPA). Staudinger reduction of 112 gave the corresponding amine 113 which upon treatment with carbon disulfide and alkyl halide yielded the desired β-carboline based dithiacarbamate 114. Similar sequence of

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synthetic events on 115 led to the isolation of 116 (Scheme 18). (Insert Scheme 18 here) (Insert Figure 13 here)

Cytotoxic studies against panel of cancer cell lines viz. A-549, MCF-7, DU-145 and HeLa

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revealed that the two pharmacophoric units namely dithiacarbamate and β-carboline greatly influenced their cytotoxicity. β-carboline-dithiacarbamate derivatives 114a and

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114b found to be the most potent compounds of the series displaying strong DNA binding and inhibitory activity against topoisomerase-II which were further explained on the basis of molecular docking studies. In silico computational studies were carried out to determine Lipinski parameters, topological surface area, and percentage of absorption. Most of the compounds satisfied Lipinski parameters and displayed 29.8%68.4% absorption.

2.12

1,3,6-trisubstituted β-carbolines:

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Bhutani et. al. synthesized a series of 1,3,6-trisubstituted β-carbolines 120 along with their cytotoxic potential against four cancer cell lines viz. A-549,HeLa, Hep G2 and MCF-7 [27]. Pictet-Spengler iminium ion cyclization was implemented for the synthesis of

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tetrahydro-β-carboline from tryptophan methyl ester and acetaldehyde. KMNO4 promoted aromatization with subsequent nitration yielded methyl-1-methyl-6-nitro-9Hpyrido[3,4-b]indole-3-carboxylate 118. Reduction of 118 with sodium borohydride in the presence of catalytic amount of Pd/C afforded the corresponding amine 119 which was

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acylated via reaction with various acyl halides (Scheme 19). (Insert Scheme 19 here)

The synthesized scaffolds, 120 were assayed for their cytotoxic potential against a panel

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of cancer cell lines at concentrations of 10 µM with Docetaxel as the positive control. The compound 120b, proved to be the most cytotoxic compound with IC50s of 4.72, 3.59, 3.65 and 4.17 µM against A-549, Hela, Hep G2 and MCF-7 cancer cell lines while 120a exhibited IC50s of 15.47, 5.30, 6.15 and 13.39 µM against the same panel of cancer cell lines. Further 120a and 120b were found to be apoptotic against A-549 and MCF-7 cell

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lines (Figure 14).

(Insert Figure 14 here)

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1-Indolyl-substituted β-carboline:

Liew and co-workers have reported the synthesis of 1-indolyl-substituted β-carboline

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natural products viz. Pityriacitrin 124, pityriacitrin B 125 and hyrtiosulawesine 126 via Pictet-Spengler condensation-oxidation strategy and evaluated their cytotoxic potential

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against a panel of 60 human tumor cell lines [28]. For the preparation of pityriacitrin, Pictet-Spengler condensation of substituted tryptamine 121a was carried out with the N-Boc-indole-3-acetaldeyhyde 122 under acidic conditions to yield the corresponding 1,2,3,4-tertahydro β-carboline 123. A variety of conditions were attempted to selectively oxidize the tetrahydro-pyridine ring without concomitant oxidation of the benzylic methylene. However, the reaction inevitably resulted in the isolation of dihydro-βcarboline along with the pityriacitrin 124. Similarly, for the preparation of pityriacitrin B 125, L-tryptophan methyl ester 121b was treated with 122 under acidic conditions to

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yield the corresponding 1,2,3,4-tetrahydro-β-carboline 123b. Subsequent oxidation of 123b using DDQ in CH2Cl2 followed by LiOH promoted hydrolysis gave pityriacitrin B 125 in 83% yields. For the preparation of third natural product, hyrtiosulawesine 126, 5-

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methoxy indole, through a sequence of synthetic reactions was converted to aldehyde 122b which upon Pictet-Spengler coupling with 5-methoxy tryptamine 121c afforded the corresponding tetrahydro β-carboline 123c. DDQ promoted oxidation of 123c gave 124c which upon de-methylation using BBr3 in CH2Cl2 resulted in the isolation of

SC

hyrtiosulawesine 126 in 81% yield (Scheme 20). (Insert Scheme 20 here)

The cytotoxic studies of synthesized analogous as well as natural products against a

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panel of 60 human tumor cell lines revealed a general lack of cytotoxicity for most of the compounds, 127 being an exception exhibiting modest anti-leukemic activity towards HL-60 (TB) cell line with an LC50 of 4.2 µM (Figure 15).

(Insert Figure 15 here) 3

β-Carboline Hybrids:

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Hybrid anticancer drug approach is an innovative synthetic strategy that involves either the merging or blending of hepatophoric moieties of different drugs in a new molecular structure or combining two or several drugs directly through cleavable/non-cleavable linkages. Hybrid anticancer drugs are of great therapeutic interests as they can

EP

potentially overcome most of the pharmacokinetic drawbacks encountered with conventional anticancer drugs. It is believed that the presence of two or more

AC C

pharmacophores in single unit not only synergises their biological effect but also increases their ability to inhibit more than one biological target. Major advantages of hybrid anticancer drugs involved increased specificity, better patient compliance, and lower side effects along with reduction in chemo-resistance. The successful utilization of this technique in design and synthesis of novel anticancer hybrids has been well illustrated and documented in the literature [29]. This portion of the review will give an emphasis on the recent developments in the synthesis and anticancer evaluation of βcarboline-hybrids along with relevant discussion on their SAR.

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3.1

β-carboline-4-benzylidene-4H-oxazol-5-one hybrids:

Sarragiotto and co-workers have reported the synthesis and in vitro anticancer evaluation of a series of β-carboline-4-benzylidene-4H-oxazol-5-one hybrids 131 [30].

RI PT

The synthetic protocol involved an initial preparation of 1-substituted-β-carboline-3carboxylic acid 128 via well-established Pictet-Spengler condensation of L-tryptophan. DCC/DMAP promoted amide coupling between β-carboline-3-carboxylic acid 128 and

SC

glycine ethyl ester afforded 129 which was hydrolysed with sodium carbonate in refluxing methanol to yield 130. Erlenmeyer-plöchi reaction of 130 with aryl aldehydes having electron withdrawing groups gave good yields of corresponding oxazolones 131

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(Scheme 21).

(Insert Scheme 21 here)

The synthesized β-carboline-oxazolone hybrids 131 were evaluated for their cytotoxic potential against a panel of cancer cell lines with Doxorubcin as the positive control. SAR studies revealed the preference of an electron donating substituents at C-1 position of β-carboline-oxazolone

TE D

hybrids for good cytotoxic activities. The hybrid 131a (Figure 16) with an optimum combination of 4-methoxyphenyl ring at C-1 position and a phenyl ring on oxazolone was the most potent of the synthesized hybrids exhibiting IC50 values of 0.48, 1.50 and 1.07 µM against glioma (U251),

EP

prostate (PC-3) and ovarian (OVCAR-O3) cancer cell lines, respectively. In silico studies of the synthesized hybrids were also carried out so as to determine their Lipinski parameters, topological surface area and percentage absorption. Most of the hybrids

AC C

were found to comply with Lipinski parameters while their percentage absorption range from 59.35-81.05 % indicating good membrane permeability.

3.2

(Insert Figure 16 here)

Bivalent β-carbolines:

Song et.al. has synthesized a series of bivalent β-carbolines having spacer length of three to ten methylene units between two indole nitrogens and evaluated their cytotoxic potential against a panel of cancer cell lines [31]. Synthetic methodology involved an initial formation of

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monovalent β-carboline 132 via Pictet-Spengler reaction of L-tryptophan in acetic acid with different aldehydes. Product obtained was further aromatized to β-carbolines 133 either by reacting with Sulphur in xylene or by treatment with sodium bithiosulphate in acetic acid. Esterification of 132 with ethanol in the presence of thionyl chloride yielded the corresponding

RI PT

1,2,3,4-tetrahydro-β-carboline-3-carboxylate 134 followed by dehydrogenation with Sulfur in refluxing xylene to afford 135. The desired bivalent β-carbolines 136 were obtained by sodium hydride promoted alkylation of various monovalent-β-carbolines, 135 and 133 using

SC

dibromoalkanes in dry DMF (Scheme 22). (Insert Scheme 22 here)

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Synthesized series of bivalent β-carbolines were screened against a panel of cancer cell lines for assaying their anti-tumor potential. SAR studies showed that appropriate replacement of substituent at C-1 position of β-carboline improved the antitumour profiles. Variation in the length of alkyl chain introduced as linker affected the antitumor potencies with the preference for four to six methylene units. Compound 136a

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exhibited good cytotoxicity against four cancer cell line viz. BGC, A375, KB and SK-OV-3 with IC50 lower than 20 µM. The promising compounds were further evaluated in animal model studies. Acute toxic assays of the selected bivalent β-carbolines indicated no obvious neurotoxic effects like tremor, twitch or jumping. Preliminary SAR indicated the

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antitumor activities being dependent upon the length of alkyl chain along with the nature of substituent at C-1 position. Nine β-carbolines were selected for in vivo

AC C

evaluation against mice bearing CT-26 colon cancer, Lewis lung cancer sarcoma 180, H22 liver cancer and B16 melanoma and the results are compared with the reference drug, cyclophosphamide (CTX). At a dose of 30 and 40 mg/kg, compounds viz. 136e and 136d exhibited tumor inhibition rate of over 40 % against mice bearing CT-26 colon cancer, Lewis lung cancer, Sarcoma 180 and H22 liver cancer respectively. Compound 136d (Figure 17) was found to be the most potent antitumor agent with the tumor inhibition rate of 50.8 and 56.2% against mice bearing CT-26 colon cancer and Sarcoma 180, respectively. It is interesting to note that CT-26 colon cancer was more susceptible to all

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tested compounds than Lewis lung cancer, Sarcoma 180, H22 liver cancer and B16 melanoma. (Insert Figure 17 here)

RI PT

Cao et. al. has synthesized a series of bivalent β-carbolines and evaluated against different cancer cell lines viz. BGC823, A375, 769-P, KB and SK-OV-3.[32] Synthetic methodology involved base promoted alkylation of monovalent β-carbolines 137 in dry DMF. Most of the synthesized bivalent β-carbolines 138 with a spacer of three to ten

SC

methylene units exhibited selective cytotoxic potential against 769-P and KB cell lines. Acute toxicities and antitumor efficacy of the promising compounds were also evaluated in mice. Compound 138a (Figure 18) was found to be the most potent among the

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synthesized hybrids displaying tumor inhibition rate of 64.2 % against Lewis lung cancer in mice. SAR studies revealed the introduction of six methylene unit to be more efficient while the presence of substituents at C-1 position to be detrimental for in vivo activities in mouse model.

Four bivalent β-carbolines were selected for in vivo evaluation against CT-26 colon cancer and

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Lewis lung cancer in mice and their results were compared with monovalent β-carboline-3carboxylic acids and cyclophosphamide (CTX). Synthesized compounds viz. 138a and 138b exhibited better antitumor activities against Lewis lung cancer in mice than their monovalent β carboline-3-carboxylic acids. Particularly, compound 138a was found to be the most potent

EP

with the tumor inhibition rate of 64.2% against Lewis lung cancer in mice. Compound 138b having a 3-phenylpropyl group at C-9 of β-carboline ring displayed tumor inhibition rate of

AC C

53.5% against Lewis lung cancer in mice at a dose of 26.9 mmol kg-1. These results were suggestive of the fact that the introduction of substituents at C-1 of β-carboline ring could be detrimental to antitumor potency. (Insert Scheme 23 here) (Insert Figure 18 here)

Inspired by unique structure and interesting biological activities of neokauluamine, Winkler and co-workers have reported the synthesis of simple dimeric β-carbolines along with their cytotoxic evaluation against a panel of cancer cell lines [33].

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(Insert Figure 19 here) Dimers of β-carboline were prepared via an initial condensation reaction between 1formyl-β-carboline 139 with linkers 140 and 143 as elucidated in the Scheme 24 followed by reduction using sodium cyanoborohydride to yield 141 and 144. Partial conversion of

RI PT

the dimers led to the simultaneous formation of monomeric β-carbolines 142 and 145 which served as control compounds for testing the importance of dimeric structures for biological activities.

SC

(Insert Scheme 24 here)

Both the dimers viz. 141 and 144 exhibited IC50 values in the range of 1.6-200.6 µM against H1299, A375 and IMR90 cancer cell lines, and the results were compared with

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Neokauluamine. Biological data revealed that the dimers were more potent than the monomers, for example, 141 and 144 exhibited IC50 values of 1.6 and 3.0 µM against H1299 and A375 cancer cell lines, which is comparable to that of Neokauluamine. Dimers were found to be eight times more selective and ten times more potent than monomers against most of cancer cell lines.

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In order to circumvent the poor solubility profiles exhibited by bivalent β-carboline, Cao and co-workers have reported the synthesis of piperazine linked bivalent β-carbolines 147 along with their antitumor evaluation [34]. The synthetic methodology involved the condensation of formyl-β-carboline 146 with piperazine in anhydrous dichloroethane at

EP

60 0C followed by reduction with NaBH3CN to afford the desired hybrids 147 (Scheme 25). The synthesized bivalent β-carbolines were assayed for their cytotoxic potential

AC C

against a panel of cancer cell lines along with Lewis Lung Carcinoma (LLC), with cis-platin as the reference drug. SAR of the synthesized β-carbolines revealed the significant role played by substituents at C-1 and C-9 position of β-carboline in modulating the antitumor activity. The presence of alkyl groups at C-9 position greatly enhanced the cytotoxic potencies while the introduction of tert-butyl substituent was considered optimal. Compound 147a and 147b proved to be the most potent among the series with 147a exhibiting IC50 values viz. 7.62, 8.95, 5.32, 3.02, 8.35, 5.51, 7.62 and 5.15 µM against MCF-7, HepG2, 22RV1, 769-P, A-375, SK-OV-3, BCG-823 and LLC tumor cell lines

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respectively. Compound 147b was also found to be exhibit angiogenic inhibitory effects in the CAM assay with its anti-angiogenic potency comparable to that of reference drug, Combretastatin A-4 phosphate(CA4P) (Figure 20).

(Insert Figure 20 here) 3.4

β-carboline-chalcone hybrids:

RI PT

(Insert Scheme 25 here)

Chalcones represent an interesting class of anti-cancer therapeutics with ease of preparation, high therapeutic index and safety [35]. Chauhan et.al. has recently reported

SC

the synthesis of β-carboline-chalcone hybrids where one aryl group of the chalcone is replaced by β-carboline ring along with their evaluation as anti-breast cancer agents.[36]

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The synthetic strategy involved an initial Pictet-Spengler reaction of tryptamine 53 with dimethoxyglyoxal to afford tetrahydro-β-carboline 148 which was further oxidized with KMnO4 to yield the corresponding acetal 149. Deprotection of acetal gave the corresponding aldehyde 152 while base-promoted alkylation of 149 followed by deprotection gave the aldehyde 151. Both the aldehydes viz. 151 and 152 were then

TE D

employed for Claisen-Schmidt condensation with substituted ketones to yield the desired β--carboline-chalcone hybrids 153 and 154, respectively (Scheme 26). (Insert Scheme 26 here)

Anticancer activities of the synthesized hybrids were determined against panel of cancer

EP

cell lines viz. DLD-1 (Colorectal adenocarcinoma), MCF-7 (Breast adenocarcinoma), PLC/PRF/5 (Liver hepatoma), A549 (Lung carcinoma), SKOV-3 (Ovarian adenocarcinoma),

AC C

MiaPaca-2 (Pancreatic adenocarcinoma), DU145 (Prostate carcinoma), A-172 (Brain glioblastoma). A range of substituents (both electron donating and withdrawing) on the chalcone counterpart as well as alkylation (methylation and ethylation) on β-carboline were explored in order to study their influence on anti-cancer profiles. SAR studies of the synthesized hybrids revealed that the compound having electron donating substituent at the phenyl ring of the chalcone exhibited good activity. N-alkylation of the hybrids did not improve the activity which unmasked the importance of free -NH group for anti-cancer activities. Dose response curve indicated that hybrids 154a and 154b

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were potentially active against all three breast cancer cell lines in a dose dependent manner. However, the hybrid 154a was more effective in killing breast cancer cells than 154b at the same concentration (Figure 21).

RI PT

(Insert Figure 21 here)

Compound 154c being the most potent, was selected to determine its apoptotic potential using 7-ADD/Annexin-V staining while its role in DNA fragmentation was

SC

ascertained using APoBrdU TUNNEL assay. Results demonstrated that MCF-7 cells treated with 154c showed enhanced green fluorescence compared to vehicle (DMSO) treated cells, confirming the capability of 154c to induce apoptosis in breast cancer cells. β-carboline-chalcone and β-carboline (N-acetyl)-dihydropyrazole hybrids as

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3.5

topo-I Inhibitors:

Kamal and co-workers synthesized a series of β-carboline-chalcone and β-carboline (Nacetyl)-dihydropyrazole hybrids along with their anticancer evaluation.[37] Synthetic approach included an initial Pictet-Spengler condensation of L-tryptophan methyl ester

TE D

with variedly substituted benzaldehydes to yield 1,2,3,4-tetrahydro-β-carboline derivatives which were then aromatized using Sulfur in refluxing xylene. LAH-promoted reduction of methyl ester to corresponding alcohol with subsequent-oxidation via Dess Martin periodinane afforded 3-carboxaldehyde β-carbolines 155. Barium hydroxide

EP

promoted Claisen-Schmidt condensation with a variety of aromatic/heteroaromatic ketones afforded the corresponding β-carboline-chalcone hybrids, 156. This upon

AC C

treatment with hydrazine monohydrate in acetic acid, led to the formation of βcarboline-(N-acetyl)-dihydropyrazole hybrids 157 (Scheme 27). The synthesized hybrids were evaluated against a panel of cancer cell lines by employing MTT assay. Most of the synthesized hybrids exhibited significant cytotoxicity against MCF-7 breast cancer cell line with IC50 of most potent hybrids ranging from 1.56-5.29 µM. SAR studies revealed the

presence

of

trifluoro-substituent

at

phenyl

ring

and

furan

containing

chalcone/pyrazole moieties at C-3 position for potent cytotoxic profiles. Further apoptotic induction was confirmed by Anneixin V-FITC, Hoechst staining and DNA

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fragmentation analysis. DNA photo-cleavage studies further revealed that the hybrids viz. 156a and 157a (Figure 22) could effectively cleave PBR322 plasmid DNA upon irradiation with UV light.

(Insert Figure 22 here)

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(Insert Scheme 27 here)

Hybrids 156a and 157a displayed significant apoptosis against MCF-cells by using Annexin-VFTIC dual staining assay. Results indicated that the hybrids 156a and 157a

SC

exhibited 58.29 % and 62.64 % apoptosis, respectively compared with the control having 0.03 % of apoptosis induction. Further, spectroscopic studies were carried out in order to understand the nature of interactions of synthesized hybrids with DNA. Higher

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fluorescence quenching has observed in case of 157a indicating combilexin type binding with DNA. 3.6

Lanthanum-promoted β-carbolines-benzimidazole hybrids:

Nagesh et.al. reported the Lanthanum-promoted synthesis of β-carboline-benzmidazole hybrids with substituted aryl ring at C-1 position. Synthetic chemistry involved an initial

TE D

ring closure of L-tryptophan with variedly substituted aryl-aldehydes in toluene with subsequent oxidation using Sulphur in xylene [38]. Further reduction of the free ester with LAH in dry THF followed by oxidation with Dess-Martin periodinane afforded the corresponding aldehyde 158. Lanthanum nitrate promoted condensation of β-carboline

EP

aldehydes 158 with substituted o-phenylene-diamine or pyridine 2,3-diamine under aerobic conditions afforded the desired β-carboline-benzmidazole hybrids 159 and 160

AC C

respectively (Scheme 28).

(Insert Scheme 28 here)

Synthesized β-carboline-benzmidazole hybrids were evaluated against a panel of cancer cell lines viz. HeLa, DU145, A549, BHK21 and L929. SAR studies revealed the presence of 4-methoxyphenyl, 3,4-difluorophenyl or 4-fluorophenyl substituents at C-1 position for good cytotoxic profiles. Hybrids viz. 160a, 160b and 160c exhibited GI50 values ranging from 0.36 to 7.1µM against most of the cancer cell lines along with DNA topoisomerase1 inhibition (Figure 23). DNA photo-cleavage study showed that the hybrid 160a was

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able to generate singlet oxygen that can cleave DNA by converting super coiled form to relaxed form. (Insert Figure 23 here)

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Various spectroscopic techniques were employed to determine the interactions of hybrids with DNA. The results obtained indicated the stabilization of DNA at lower temperature while unfolding of CT-DNA was observed at higher temperature. UV-Visible spectroscopy indicated the binding between hybrids and DNA to be intercalative. Interestingly, the synthesized β-carboline-benzmidazole hybrids have also exhibited

SC

topo-I inhibition at 100 as well as 200 µM conc. which could be attributed to their observed low IC50 values.

DNA interactive β-carboline-chalcone hybrids:

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3.7

Kamal et.al. has reported a series of DNA interactive β-carboline-chalcone hybrids and evaluated their in vitro cytotoxicity against a panel of cancer cell lines along with DNA binding studies [39]. The synthetic approach involved an initial esterification of commercially available L-tryptophan followed by Pictet-Spengler condensation with

TE D

different aldehydes in acidic medium to yield tetrahydro-β-carbolines. Aromatization of tetrahydro-β-carbolines with sulphur in refluxing xylene afforded the corresponding βcarbolines. Selective reduction of ester group using LAH gave the corresponding alcohol which was oxidized using Dess-Martin Periodinane to yield 3-carboxaldehyde derivatives

EP

161. Claisen-Schmidt condensation of 161 with different acetopheneones in the presence of Ba(OH)2 afforded the corresponding β-carboline-chalcone hybrids 162

AC C

(Scheme 29). The synthesized hybrids were evaluated for their in vitro cytotoxic activities

against

HeLa

(cervical

carcinoma),

HT-29

(colon

cancer),

A-549

(adenocarcinoma), PC-3 (prostate cancer) and B-16 (mouse malonoma) cell lines using MTT method. Most of the synthesized hybrids showed excellent cytotoxicity on the selected human cancer cell lines. The hybrid, 163 proved to be most active on the selected anticancer cell lines with IC50s of 23.08 µM against HT-29, 5.3 µM against A-549, 19.59 µM against prostate cancer and 6.37 µM against B-16 cell lines (Figure 24). SAR studies confirmed the presence of fluoro and methoxy substituents on phenyl ring at C-1

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position of β-carboline for good cytotoxicity. In addition, the hybrids exhibited significant elevation in ΔTm of DNA in comparison to standard drug Adriamycin, suggesting significant interaction and DNA stabilization.

(Insert Figure 24 here) 3.10

1H-1,2,3-triazole-tethered β-carboline hybrids:

RI PT

(Insert Scheme 29 here)

Salehi and co-workers in a recent communication has reported the synthesis of 1H-1,2,3-

SC

triazole-tethered β-carboline hybrids along with their anti-cancer evaluation against two cancer cell lines viz. Hela and HepG2 using MTT assay.[40] The synthetic protocol included an initial Pictet-Spengler reaction of L-tryptophan methyl ester with O-

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propagylated benzaldehydes to yield 1,2,3,4-tetrahydro-β-carbolines as a mixture of two diastereoisomers which upon refluxing with 5 equivalents of sulphur gave the corresponding β-carbolines 164. The synthesized O-propargylated β-carbolines 164 were then subjected to Cu-promoted azide-alkyne cycloaddition reaction with various alkyl/aryl azides to afford the desired 1H-1,2,3-triazole-tethered β-carboline hybrids 165

TE D

(Scheme 30). Cytotoxic studies of synthesized hybrids against cancer cell lines using paclitaxel as the positive control, revealed that compounds viz. 165a, 165b, 165c and 165d displayed good cytotoxic activity with IC50<100 µM. Compound 165e having 3,4dichloro-phenyl as the azide fragment, displayed highest cytotoxicity with IC50 values of

EP

46and 32 µM against Hela and HepG2 cell lines, respectively (Figure 25). (Insert Scheme 30 here)

3.11

AC C

(Insert Figure 25 here)

C-3 functionalised-1H-1,2,3-triazole tetheredβ-carboline hybrids:

Kamal et.al. further extended the above study and synthesized C-3 tethered-1H-1,2,3triazoleβ-carboline hybrids and evaluated against different cancer cell lines viz. PC-3, MCF-7, HeLa, HT-29 and HGC-27 [41]. Synthetic methodology for the preparation of desired hybrids involved an initial synthesis of β-carboline methyl azide 166 via an initial LAH-promoted reduction of β-carboline methyl ester to yield the corresponding alcohol which was mesylated and reacted with sodium azide. Cu-promoted azide-alkyne

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cycloaddition reaction of β-carboline methyl azide with variedly substituted alkynes gave the desired 1H-1,2,3-triazole-tethered hybrids 167 as shown in Scheme 31. (Insert Scheme 31 here)

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SAR analysis confirmed the presence of napthalimide and phthalimide functionality for good cytotoxic activity. The most potent among the synthesized hybrids viz. 167a and 167b displayed strong cytotoxic activities against all the cancer cell lines tested (Figure 26). Molecular modeling studies further corroborated the observed SAR establishing hydrogen bonding and π-π stacking

SC

interactions between phthalimide ring and DNA base pairs. (Insert Figure 26 here) 4

β-Carbolines-metal Complex:

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Over the past few years, bio-organometallic chemistry has been established as a rapidly emerging area which connects classical organometallic chemistry to biology, medicine, and molecular biotechnology. Organometallic analogues of biologically active compounds have emerged as an important strategy since metallopharmaceuticals offer potential advantages which include the preparation of stable transition metal complexes

TE D

with predictable structures, the ability to tune ligand affinities, substitution rates and reduction potentials along with efficient biological targeting [42]. Advances in the rational design of metal-based therapeutic agents have increased after the discovery of cis-platin, which has been the main stimulus for the expansion of metal complexes in

metal complexes.

β-carboline based Ruthenium(II) complexes:

AC C

4.1

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cancer and other pathologies. This portion of the review is dedicated to the β-carboline-

Abbas et.al. have reported the synthesis of β-carboline based Ruthenium(II) complex 168 along with its anticancer potential against various cancer cell lines.[43] The desired complex was prepared by initially reacting [Ru(tpy)]Cl3 with AgSO3CF3 followed by the addition of β-carboline in refluxing ethanol. The synthesized complex, 168 was evaluated for its anti-cancer potential against cancer cell lines viz. MCF-7, MCF-10A, NCl-H460, A549, HepG2, Bel-7402, L-02, HeLa, HCT-116 and HEK-293.The complex exhibited IC50 values in the range of 0.68 to 57.2 µM with most of the IC50 values lying below 5 µM and

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the results were compared with standard drug, Cisplatin. Evaluation studies revealed that the complex 168 was more potent than Cisplatin against all cancer cell lines with the most significant IC50 values displayed against HeLa, HepG2 and Bel-7402 cell lines

(Insert Figure 27 here)

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(Figure 27).

The methodology was further extended towards the synthesis of a series of β-carboline based Ru(II) complexes,169a-d as shown in Figure 28 and evaluated against cancer cell

SC

linesviz. MCF-7, MCF-10A, NCl-H460, A549, HepG2, Bel-7402, L-02, HeLa, HCT-116 and HEK-293. The synthesized complexes exhibited IC50 values in the range of 15.1 to 61.2 µM, with the most potent complex 169d showed an IC50 value of 15.1 µM. A

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comparative study among the complexes 168 and 169 showed the decrease in anticancer potential with the decrease in number of β-carboline moieties. (Insert Figure 28 here)

4.2

Cyclometalated Ir(III) complexes of β-carbolines:

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As an alternate to Cisplatin, Iridium complexes have recently gained increasing attention because of their anti-cancer efficacy through induction of apoptosis and interaction with DNA or protein kinase [44]. Mao and co-workers have recently reported the synthesis of two cyclometalated Ir(III) complexes with 2-(2-thienyl)pyridine (thp) as an auxiliary

EP

and β-carboline as functional ligands.[45] The complexes were synthesized by refluxing two equivalents of β-carboline and the corresponding cyclometalated Ir(III) dimer. The

AC C

synthesized complexes viz. 170a and 170b along with their ligands and Ir2(thp)4Cl2 were evaluated for their anticancer potential against a panel of human cancer cell lines viz. A549, A549cisR, Cisplatin-resistant A549; HepG2, hepatocellular liver carcinoma; HeLa, cervical cancer and human hepatic cell line LO2. The ligands were inactive at the highest tested concentration, probably because of their poor solubility profiles while both the complexes displayed much higher antiproliferative activity than Cisplatin, especially against Cisplatin-resistant A549cisR cell line. Mechanistically, the complexes induced

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ROS-mediated and caspase-independent cell death through an autophagic pathway while Cisplatin induced apoptotic cell death. (Insert Figure 29 here) 4.3

3-(1H-benzo[d]imidazole-2yl)-β-carboline metal complexes:

RI PT

Lu et.al. synthesized various metal complexes of 3-(1H-benzo[d]imidazole-2yl)-βcarboline 172 and evaluated their cytotoxic activity with Cisplatin as the reference drug [46]. β-carboline-3-carboxylic acid 171 was synthesized via an initial Pictet-Spengler reaction of L-tryptophan with formaldehyde followed by oxidation using KMnO4.

SC

Condensation reaction between O-phenylenediamine and 171 afforded β-carbolineimidazole hybrids 172 which upon treatment with various metal salts yielded the desired

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metal-β-carboline complexes, 173-176. Bio-evaluation results obtained for the synthesized complexes against different cancer cell line were not superior to that of reference drug, Cisplatin. Complexes 174 and 176 proved to be significantly active against different cancer cell line and exhibited strong DNA binding and DNA cleavage properties comparative to that of complexes 173 and 175.

Conclusions:

TE D

(Insert Scheme 32 here)

β-carbolines represent an importance class of pharmacologically active scaffolds with

broad spectrum of anticancer activities exerting via diverse mechanisms. Past few years

variedly

EP

have witnessed an upsurge in the development of synthetic strategies for acquiring functionalized β-carboline

derivatives, β-carboline-hybrids

as

well

as β-

AC C

carboline-metal complexes along with their anticancer evaluation. The principle objective in the development of novel β-carbolines as potential chemotherapeutics include enhancing potency, specificity along with improvement in pharmacokinetic profiles, metabolic stability while minimizing the undesirable side effects and incidence of resistance. The pre-requisite to such design include their affinity not only towards the targeted receptors but also their prospective for in vitro and in vivo selectivity. Refrences 1 World Health Organization report-2016 http://www.who.int/cancer/en/

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2 M. Mareel, A. Leroy, Physiol. Rev.,83 (2003) 337. 3 J. Wesche, K.Haglund, E.M. Haugsten, Biochem. J., 437 (2011) 199. 4 S.K. Grant, Cell. Mol. Life. Sci., 66 (2009) 1163.

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5 G.I. Solyanik, Exp. Oncol., 32 (2011) 181. 6 S. Vijayaraghavalu, C. Peetla, S. Lu, V. Labhasetwar, Mol. Pharm., 9 (2012), 2730. 7 M.M. Gottesman, T. Fojo, S.E. Bates, Nat. Rev. Cancer, 2 (2002) 48.

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SC

Y. Cao, P. Nordlund, Science, 341 (2013) 84.

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C. Zhang, J. Ju, J. Nat. Prod.,74 (2011) 2122.

11 A. Laine, C. Lood, A. Koskinen, Molecules,19 (2014) 1544.

12 (a) K. Patel, M. Gadewar, R. Tripathi, S. Prasad, D. K. Patel, Asi. Pac. J. Trop. Biomed., 2 (2012) 660 (b) A. Abe, H. Yamada, Anticancer Drugs,20 (2009) 373. 13 (a) F. A. Khan, A. Maalik, Z. Iqbal, I. Malik, Eur. J. Pharmacol.,721 (2013) 391; (b) S. P.

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B. Ovenden, J. L. Nielson, C. H. Liptrot, R. H. Willis, D. M. Tapiolas, A. D. Wright, C. A. Motti, Phytochem. Lett.,4 (2011) 69.

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Chem. Lett., 22 (2012) 3506.

16 G. D. Cuny, N. P. Ulyanova, D. Patnaik, J. Liu, X. Lin, K. Auerbach, S. S. Ray, J. Xian, M.

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A. Glicksman, R. L. Stein, J. M.G. Higgins, Bioorg. Med. Chem. Lett., 22 (2012) 2015. 17 (a) J. Bain, L. Plater,M. Elliott, N. Shpiro, C. J.Hastie, H.McLauchlan, I.Klevernic, J. S. C.Arthur, D. R.Alessi, P.Cohen, Bio. chem. J., 408 (2007) 297; (b) A.Seifert, L. A.Allan, P. R. Clarke., FEBS J.275( 2008) 6268; (c) N.Göckler, G.Jofre, C.Papadopoulos, U.Soppa, F. J.Tejedor, W. Becker, FEBS J. 276 (2009) 6324; (d) Y.Ogawa, Y.Nonaka, T.Goto, E.Ohnishi, T.Hiramatsu, I.Kii, M.Yoshida, T.Ikura, H.Onogi, H.Shibuya, T.Hosoya, N.Ito, M.Hagiwara, Nat. Commun. 1 (2010) 86. 18 L. Liu, Y. Y. Xu, Z. Q. Yang, J. N. Xiang, G. Y. Xu, Chin. Chem. Lett.,23 (2012) 1230.

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19 B. Xin, W. Tang, Y. Wang, G. Lin, H. Liu, Y. Jiao, Y. Zhu, H. Yuan, Y. Chen, T. Lu, Bioorg. Med. Chem. Lett., 22 (2012) 4783. 20 R. Cao, W. Fan, L. Guo, Q. Ma, G. Zhang, J. Li, X. Chen, Z. Ren, L. Qiu, Eur. J. Med.

RI PT

Chem., 60 (2013) 135. 21B. Bai, X. Y. Li, L. Liu, Y. Li, H. J. Zhu, Bioorg. Med. Chem. Lett.,24 (2014) 96.

22 C. M. Roggero, J. M. Giulietti, S. P. Mulcahy, Bioorg. Med. Chem. Lett., 24 (2014) 3549.

SC

23 H. Chen, P. Gao, M. Zhang, W. Liao, J. Zhang, New J. Chem., 38 (2014) 4155.

24 Y. Ling, C. Xu, L. Luo, J. Cao, J. Feng, Y. Xue, Q. Zhu, C. Ju, F. Li, Y. Zhang, Y. Zhang, X. Ling, J. Med. Chem.,58 (2014) 9214.

M AN U

25 Y. F. Chen, Y. C. Lin, J. P. Chen, H. C. Chan, M. H. Hsu, H. Y. Lin, S. C. Kuo, L. J. Huang, Bioorg. Med. Chem. Lett.,25 (2015) 3873.

26 A. Kamal, M. Sathish, V. L. Nayak, V. Srinivasulu, B. Kavitha, Y. Tangella, D. Thummuri, C. Bagul, N. Shankaraiah, N. Nagesh, Bioorg. Med. Chem., 23 (2015) 5511. 27 N. A. Lunagariya, V. M. Gohil, V. Kushwah, S. Neelagiri, S. Jain, S. Singh, K. K.

TE D

Bhutani,Bioorg. Med. Chem. Lett.,26 (2016) 789.

28 L. P. P. Liew, J. M. Fleming, A. Longeon, E. Mouray,I. Florent, M. L. B. Kondracki, B. R. Copp, Tetrahedron.,70 (2014) 4910.

29 S. Fortin, G. Bérubé, Expert Opin. Drug Discov., 8 (2013) 1029.

EP

30 (a) F. C.Savariz, M. A. Foglio, J. E. deCarvalho, A. L. T. G. Ruiz, M. C. T. Duarte, M. F. da Rosa, E. Meyer, M. H. Sarragiotto, Molecules, 17 (2012) 6100; (b) V.A. Barbosa, P. Baréa,

AC C

R.S. Mazia, T. Ueda-Nakamura, W.F. da Costa, M.A. Foglio, A.L.T.G. Ruiz, J.E. de Carvalho, D. B.V. Costa, C.V. Nakamura, M.H. Sarragiotto, Eur. J. Med. Chem. 124 (2016) 1093. 31 B. Shi, R. Cao, W. Fan, L. Guo, Q. Ma, X. Chen, G. Zhang, L. Qiu, H. Song, Eur. J. Med. Chem., 60 (2013) 10.

32 (a) Q. Wu, Z. Bai, Q. Ma, W. Fan, L. Guo, G. Zhang, L. Qiu, H. Yu, G. Shao, R. Cao, Med. Chem. Comm., 5 (2014) 953; (b) W. Chen, G. Zhang, L. Guo, W. Fan, Q. Ma, X. Zhang, R. Du, R. Cao, Eur. J. Med. Chem. 124 (2016) 249.

ACCEPTED MANUSCRIPT

33 J. Chatwichien, S. Basu, M. E. Murphy, M. T. Hamann, J. D. Winkler, Tet. Lett., 56 (2015) 3515. 34 R. Sun, R. Liu, C. Zhou, Z. Ren, L. Guo, Q. Ma, W. Fan, L. Qiu, H. Yu, G. Shao, R. Cao,

RI PT

Med. Chem. Comm., 6 (2015) 2170.

SC

35 P. Singh, A. Anand, V. Kumar, Eur. J. Med. Chem., 85 (2014) 758.

36 S. S. Chauhan, A. K. Singh, S. Meena, M. Lohani, A. Singh, R. K. Arya, S. H. Cheruvu, J. Sarkar, J. R. Gayen, D. Datta, P. M. S. Chauhan, Bioorg. Med. Chem. Lett., 24 (2014) 2820.

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37 A. Kamal,V. Srinivasulu,V. L. Nayak,M. Sathish,N. Shankaraiah,C. Bagul,N. V. S. Reddy,N. Rangaraj,N. Nagesh, Chem. Med. Chem., 9 (2014) 2084. 38 A. Kamal,M. P. N. Rao,P. Swapna,V. Srinivasulu,C. Bagul,A. B. Shaik,K. Mullagiri,J. Kovvuri,V. S. Reddy,K. Vidyasagar, N. Nagesh, Org. Biomol. Chem., 12 (2014) 2370. 39 N. Shankaraiah, K. P. Siraj, S. Nekkanti, V. Srinivasulu, P. Sharma, K. R. Senwar, M.

TE D

Sathish, M. V. P. S. Vishnuvardhan, S. Ramakrishna, C. Jadala, N. Nagesh, A. Kamal, Bioorg. Chem., 59 (2015) 130.

40 P. Salehi, K. B. Harikandei, M. Bararjanian, A. A. Harrasi,M. A. Esmaeili, A. Aliahmadi,Med. Chem. Res.,25 (2016) 1895.

EP

41 N. Shankaraiah, C. Jadala, S. Nekkanti, K. R. Senwar, N. Nagesh, S. Shrivastava, V.G.M. Naidu, M. Sathish, A. Kamal, Bioorg. Chem., 64 (2016) 42.

AC C

42 (a) C. Biot, W. Castro,C. Y. Botte, M. Navarro, Dalton Trans.,41 (2012) 6335. (b) C. G. Hartinger, P. Dyson,J. Chem. Soc. Rev., 38 (2009) 391 (c) G. Gasser,N.Metzler-Nolte,Curr. Opin. Chem. Biol., 16 (2012) 84 (d) Bioorganometallics: Biomolecules,Labeling, Medicine; Jaouen, G. Ed.; Wiley-VCH: Weinheim, Germany, 2006. 43 S. Y. Chen, M. Y. Qin, J. H. Wu, L. Wang, H. Chao, L. N. Ji, A. L. Xu, Eur. J. Med. Chem., 70 (2013) 120. 44 (a) K. K. W. Lo and K. Y. Zhang, RSC Adv., 2012, 2, 12069; (b) C.- H. Leung, H.-J. Zhong, D. S.-H. Chan and D.-L. Ma, Coord. Chem. Rev., 257 (2013) 1764

ACCEPTED MANUSCRIPT

45 L. He, S. Liao, C. P. Tan, Y. Y. Lu, C. X. Xu, L. N. Jia, Z. W. Mao, Chem. Comm., 50 (2014) 5611. 46 Q. M. Jin, Y. Lua, J. L. Jin, H. Guo, G. W. Lin, Y. Wanga, T. Lu, Inorg. Chimm. Acta., 421

RI PT

(2014) 91.

Acknowledgements

SK acknowledges Science and Engineering Research Board (SERB), New Delhi for providing financial assistance under grant no. EMR/2015/001687. Financial assistance

SC

from University Grants Commission (UGC), New Delhi, India, under UGC-JRF Fellowship with Ref. No. 23/12/2012(ii)EU-V is gratefully acknowledged (AS).

M AN U

Captions: Figure 1: β-Carbolines and their derivatives.

Figure 2: Most potent benzylamino-β-carboline derivative 14a. Figure 3: Most potent β-carboline derivative 17a. Figure 4: Compounds with apoptotic potential.

TE D

Figure 5: Structure of β-carbolines as haspine-kinase inhibitors. Figure 6: Most potent β-carbolines against B-Raf kinase. Figure 7: Most potent N2-benzylated β-carbolines Figure 8: Most active C-1 aryl substituted β-carbolines. Figure 9: Structures of biologically active eudistomins.

EP

Figure 10: Most potent of synthesized β−carboline tripeptide derivaives.

acids.

AC C

Figure 11: Highly potent derivatives of synthesized series of β-carbolines hydroxamic Figure 12: Most potent of 3,9-substitutedβ-carbolines. Figure 13: Most potent β-carboline-dithiacarbamates. Figure 14: Highly potent compound of the synthesized series. Figure 15: Most active 1-indolyl-substituted β-carboline. Figure 16: Most potentβ-carbolines-4-benzylidene-4H-oxazol-5-one hybrid. Figure 17: Most potent bivalent β-carbolines. Figure 18: Most potent of synthesized series of bivalent β-carbolines.

ACCEPTED MANUSCRIPT

Figure 19: Neokauluamine. Figure 20: Most potent piperazine tethered β-carboline hybrids. Figure 21: Most potent β-carboline-chalcone hybrids.

RI PT

Figure 22: Most potent hybrids156a and 157a that cleave PBR322 plasmid DNA upon UV irradiation.

Figure 23: Highly potent hybrids exhibiting strong cytotoxicity against different cancer cell lines. Figure 24: Most potent compound of the synthesized series.

SC

Figure 25: Highly potentβ-carboline hybrids prepared via click chemistry. Figure 27: β-carboline-Ru(II) complex.

M AN U

Figure 26: Most potent β-carboline-phthalimide and β-carboline napthalimide Figure 28: Most potent anticancer Ru(II)-β-carbolinecomplexes against A325 cell line. Figure 29: β-carboline-Ir (III) complexes, 175a and 175b. N N H

CH3

Harmane

N

H3CO

N H

N

HO

N H

CH3

Harmine

CH3

Harmol

TE D

COOH N

N

H3CO

N H

Harmaline

CH3

OH NH Callophycin A

AC C

EP

Figure 1: β-Carbolines and their derivatives

Figure 2: Most potent benzylamino-β-carboline derivative 14a.

RI PT

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

Figure 3: Most potent β-carboline derivative 17a.

AC C

EP

Figure 4: Compounds with apoptotic potential.

Figure 5: Structure of β-carbolines as haspine-kinase inhibitors.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Figure 6: Most potent β-carbolines against B-Raf kinase.

Figure 7: Most potent N2-benzylated β-carbolines.

RI PT

ACCEPTED MANUSCRIPT

M AN U

SC

Figure 8: Most active C-1 aryl substituted β-carbolines.

AC C

EP

TE D

Figure 9: Structures of biologically active eudistomins.

Figure 10: Most potent of synthesized β−carboline tripeptide derivaives.

ACCEPTED MANUSCRIPT

NHOH O NHOH N H N

O

N H

O

N

N H

N H 103a

103c

O

O N H

O

NHOH

N N H

M AN U

103d

O

SC

N

103b

NHOH

N H

N H

RI PT

O

Figure 11: Highly potent derivatives of synthesized series of β-carbolines hydroxamic

EP

TE D

acids.

AC C

Figure 12: Most potent of 3,9-substitutedβ-carbolines.

Figure 13: Most potent β-carboline-dithiacarbamates.

RI PT

ACCEPTED MANUSCRIPT

MeO

M AN U

N

SC

Figure 14: Highly potent compound of the synthesized series.

N H

N H

TE D

127

OMe

AC C

EP

Figure 15: Most active 1-indolyl-substituted β-carboline.

Figure 16: Most potentβ-carbolines-4-benzylidene-4H-oxazol-5-one hybrid.

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Figure 17: Most potent bivalent β-carbolines.

ACCEPTED MANUSCRIPT

SC

RI PT

Figure 18: Most potent of synthesized series of bivalent β-carbolines.

TE D

M AN U

Figure 19: Neokauluamine.

AC C

EP

Figure 20: Most potent piperazine tethered β-carboline hybrids.

Figure 21: Most potent β-carboline-chalcone hybrids.

RI PT

ACCEPTED MANUSCRIPT

Figure 22: Most potent hybrids 156a and 157a that cleave PBR322 plasmid DNA upon

M AN U

SC

UV irradiation.

AC C

EP

cell lines.

TE D

Figure 23: Highly potent hybrids exhibiting strong cytotoxicity against different cancer

Figure 24: Most potent compound of the synthesized series.

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

M AN U

Figure 25: Highly potentβ-carboline hybrids prepared via click chemistry.

AC C

EP

Figure 26: Most potent β-carboline-phthalimide and β-carboline napthalimide

Figure 27: β-carboline-Ru(II) complex.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

Figure 28: Most potent anticancer Ru(II)-β-carbolinecomplexes against A325 cell line.

AC C

EP

Figure 29: β-carboline-Ir (III) complexes, 175a and 175b.

ACCEPTED MANUSCRIPT

COOH

O

O COOH

NH2

a

b

NH

1

c

NH

N H

N H

O

O N N H 4

N H 3

2

Cl

Cl

N3

N

Cl

H N

Cl

NH2

h

N

N

Cl

N

N H

HN R

H2N

14

N N H

R

o

O

HN

EP

l

N H

12

N H

R

15

Ph Cl

O2N

TE D

NH2

HN

N H 13

n

O

NH

m

N O

Ph

16

NH N

N

i

O

O

N

O2N

N H 10

9

8

O

H N

Cl

M AN U

N H

N H

NH

SC

6

g

e

N H 5

N H

N H 7

O

Cl

NHNH2

f

N

Cl

RI PT

d

O

O

j NH N

Ph Cl

NH2

k

N O2N

N H 11

Ph R = NHCOn-Bu, NHCOPh, NHMe, Methyyl, Ethyl n-propyl, isoppropyl, n-butyl, n-pentyl, phenyl, O-pyridyl m-pyridyl, p-pyridyl, CF3, CF2CF3, CF2CF2CF3

AC C

Scheme 1: (a) HCHO, 2.5M NaOH, (b) SOCl2, MeOH (c) TCAA, TEA, DMF, 00C (d) NCS, AcOEt, reflux, 18h (e) NH2NH2.H2O, 1-pentanol, reflux, 7h (f) NaNO2, 2N HCl (g) 50% AcOH, reflux, 1h (h) RCHO, AcOH, NaBH3CN, MeOH (i) NaNO2, TFA(excess) (j) 1N HCl,H2O reflux, 12h (k) PhCHO, AcOH, NaBH3CN, MeOH (l) SnCl2.2H2O, 12N HCl, MeOH (m) (RCO)2O/DMAP or RCOOH, DCC, DMAP or RCOCl, CHCl3, rt (n) (1) NH2NH2:H2O (20 eq.), (2) 10% Pd/C (0.5eq) MeOH, reflux, 5h. (o) (1) Aldehyde (1 eq.) CH3COOH (pH=4), (2) NaBH3CN (1.5 eq.), MeOH, rt, 4h and then 1h.

ACCEPTED MANUSCRIPT

NH N

Ph

Cl

Ph

N

N H 13

H2N

NH

a, b N HN H R 17

Entry 17a 17b 17c 17d 17e 17f

R Methyl Ethyl n-propyl iso-propyl n-butyl benxyl

Emtry 17g 17h 17i 17j 17k

R pyridin-2-ylmethyl pyridin-3-ylmethyl pyridin-4-ylmethyl furan-2-ylmethyl thiophen-2-ylmethyl

RI PT

Cl

M AN U

SC

Scheme 2: (a) (CH2O)n 5eq, NaOMe, MeOH reflux (b) NaBH4, MeOH reflux, 1h.

Scheme 3: (a) (1) Aldehyde (1 eq.) CH3COOH (pH=4), (2) NaBH3CN (1.5 eq.), MeOH, rt, 4h

EP

TE D

and then 1h

AC C

Scheme 4: (a) Br(CH2)nNHBoc, Cs2CO3, DMF, rt (b) HCl, MeOH, rt; (c) Br(CH2)nNPhth, NaH, DMF, rt (d) NH2NH2.H2O, EtOH, 65 oC.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

Scheme 5: (a) Me2NCHCHNO2, TFA, rt, 30 min (b) LiAlH4, THF, rt (c) R1CHO or CF3CHOH(OEt), MeOH, cat. HCl, rt (d) MnO2, 5% Pd/C, DMF, microwave, 150 oC; (f)

AC C

EP

Br(CH2)nNPhth, NaH, DMF, rt (g) NH2NH2.H2O, MeOH, DCM.

ACCEPTED MANUSCRIPT

Me

N

HO

a

O

O

O

n O N O

O

O

O

35

34

M AN U

Me

N

O

n

e

EP AC C

n

d

d

n H2N 38

37

N

Me

Me

N

t-BuO

O

33

N

n

N

N

O

N

N Me

N

MeO

f or g

Me

N

MeO

n

n

O

N

O

24

H2N 39

Me

N

O

N

MeO

N

O S Me N O H

H2N 36

Me

N

t-BuO

TE D n

d

N

N HO

SC

33

24

Me

N

N

N

N

c

n

n

n

Me

N

b TfO

N

O S Me N O H

RI PT

Me

N

MeO

N

N

N

R 40 R= NMe2 41 R=NHCHO 42 R=NHMe

Scheme 6: (a) HBr, AcOH, MW, 130oC; (b) Tf2O, i-Pr2EtN,DCM, 0oC to rt (c) MeSO2NH2, Pd2(dba)3, xPhOS, K3PO4, toluene, 110oC, 1h (d) NH2NH2.H2O, MeOH, DCM (e)

h

ACCEPTED MANUSCRIPT

Me2NCH(O-t-Bu)2, DMF, 120 oC, 1h (f) CH2O,HCO2H, MW, 150 oC (g) EtOCHO, MW, 160 o

I

I

a

b

N H

c

N H

44

46

45

RI PT

C (h) LiAlH4, THF, rt.

N N PhO2S SO2Ph

47

f PhO2S

N SO2Ph 50

e

N

PhO2S

g R

N

Br

N3 49

M AN U

N

SC

d

I

48

R

h N

N

N SO2Ph 51

52

TE D

N H

R = Phenyl, 4-methylphenyl, 4-methoxyphenyl, 2-methoxyphenyl Cyclopropyl, 3-nitrophenyl, 3-formylphenyl, 4-fluorophenyl 4-acteylphenyl, 2-bromophenyl

Scheme 7: (a) I2, KOH, DMF, rt, 4h (b) PhSO2Cl, benzene, aq. NaOH, Bu4N+HSO4-, rt, 12h

EP

(c) Phenyl acetylene, Pd(PPh3)2Cl2, CuI, Et3N, N2 (d) NBS, AIBN, CCl4, reflux (e) NaN3, petroleum/water, Bu4N+HSO4-, reflux, 12h (f) I2, CH3NO2, 105oC, 20h (g) R-B(OH)2,

AC C

Pd(PPH3)2Cl2, K3PO4, toluene, 45-60oC, 20h (h) 40% NaOH, dioxane, 80oC, 3h.

ACCEPTED MANUSCRIPT

NH2 a

NH N H 54

NH N H

COOH

Cl O O S f

N H

COOMe

N

N

R1

58

M AN U

Compound

SC

N H

CONH2

59a 59b 59c 59d 59e 59f 59g 59h

N COOMe

55

R1 HN O O S

N H 59

c

56

RI PT

N H 53

b

Propyl 3-Diethylaminopropyl 3-Hydroxypropyl 3-Hydroxypropyl 3-Dimethylaminopropyl 2,2-Dimethoxyethyl 2,3-Dihydroxypropyl 2-Hydroxyethyl

e

59i 59j 59k 59l 59m 59n 59o 59p

N N H

CONH2

Compound

d

R1 3-Methoxypropyl Cyclopropyl Phenyl p-Tolyl 4-Fluorophenyl Pyridin-2-yl 2-Thiazolyl Hydrogen

TE D

Scheme 8: (a) conc.HCl, glyoxylic acid, 30% KOH, rt (b) SOCl2, CH3OH, reflux, 6 h (c)

AC C

EP

KMnO4, DMF, rt 24 h (d) MeOH, NH3.H2O, rt (e) ClSO3H, 0οC (f) R1NH2, NH3. H2O, rt.

Scheme 9: (a) HCHO, NaOH, rt, 3 h and then reflux 3 h (b) SeO2, AcOH, reflux 12 h (c) ClSO3H, 0 oC (d)Amines, rt

CONH2 57

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 10: (a) DMF/NaH, R1Br (b) HOAc/HBr (c) DMF/NaH, R2Br (d) benzyl bromide,

EP

TE D

ethyl acetate, reflux.

Scheme 11: (a) aldehyde, CH2Cl2, molecular sieve, rt, then TFA, toluene, 60 oC (b) DDQ,

AC C

CH2Cl2, rt, 0.5 h (c) CH3I orCH3CH2Br, K2CO3, DMF, rt (d) For R3 = H, LiAlH4, THF, rt, 1 h, For R3 = CH3, CH3MgBr, THF, rt, 6–10 h.

ACCEPTED MANUSCRIPT

NH2 NH

a N H

N H

NH

b

O

N H 75

74

53

N

c

O

N H

OTf 76

HN

N e

N H 78

RI PT

d

N H

O S N O

N

77

SC

Scheme 12: (a) Cl3COOOCCl3,Et3N, toluene, HBr (b) DDQ, dioxane (c) Tf2O, pyridine (d) 1benzenesulfonyl-1H-indole-3-boronic acid,5% Pd2(dba)3, Et3N, PPh3, Ethanol:toluene (e)

TE D

M AN U

KOH, MeOH : THF (1:1).

EP

Scheme 13: (a) NMM, DCC, HOBt (b) 2M NaOH (c) NH2-AA-OBzl, NMM, DCC, HOBt (d)

AC C

HCl in Ethyl acetate.

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

Scheme 14: (a) CH3COOH (b) SOCl2and CH3OH (c) SeO2 and CH3COOH (d) 6 M NaOH (e)

TE D

NH2-Trp-Trp-AA-OBzl, NMM, DCC, HOBt.

EP

Scheme 15: (a) H+(acidic) or OH−(basic), RCHO, reflux, 2−4 h (b)KMnO4, DMF, reflux, 6 h (c)Ethyl chloroformate 93, N-methylmorpholine, NH2(CH2)nCOOMe 94, rt, 5−8 h

AC C

(d)NH4O-K+, MeOH, rt.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 16: (a)SOCl2, MeOH, 0 °C, 1h, and then reflux (b)CH3I, NaH, CH2Cl2, rt, 2−4 h (c)NH2-NH2·H2O, CH3OH, 0 °C, 1h (d)NaNO2, CH3OH, 0 °C, 6 h. (e)NH2(CH2)nCOOMe 99,

AC C

EP

TE D

toluene reflux, 1h (f)NH4O-K+, MeOH.

Scheme 17: (a) substituted arylmethyl halide, KOH, THF (b) 50% ethanol, NaOH (c) LiAlH4, THF.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 18: (a) LiBH4, THF, 0 0C to rt (b) DPPA, DBU, dry THF (c) TPP, acetonitrile/water

AC C

EP

TE D

(1:1), rt (d) alkyl halide, CS2, TEA, pyridine, 0 0C (e) MeI, NaH, dry DMF.

Scheme 19: (a) conc. HNO3, conc. H2SO4, 0-2 0C, 30 min. (b) Pd/C, NaBH4, MeOH, rt, 3h (c) RCOCl, anhydrous dichloromethane, rt, 6-10 h.

ACCEPTED MANUSCRIPT

O

R

1

NH2

R1

H

R

N H

R2

a N H

N Boc

121 121a R1= R2= H 121b R1=H, R2= COOMe 121c R1=OMe, R2= H

R

NH

NH 123 123a R= R1= R2= H 123b R= R1=H, R2= COOMe 123c R= R1=OMe, R2= H

122 122a R= H 122b R= OMe

b R2 R1

N N H

O

RI PT

R2

NH

c d

SC

124 R = R1 = R2 = H

124b R= R1 = H, R2 = COOMe 125 R= R1 = H, R2 = COOH 124c R= R1 = OMe, R2 = H 126

M AN U

Scheme 20: (a) 5 or 10% HOAc/H2O, reflux, 1.5-5 h, 37-77% (b) DDQ,CH2Cl2/H2O, rt, 3090 min, 11-37% (c) LiOH, THF/H2O (3:1), reflux, 12 h, 83% (d)BBr3, CH2Cl2, N2, -78 0C, 40 min, rt, 3 h, 87%.

OCH3

O

COOH a

N

R1 128

N

N H

O b

R1

TE D

N H

N H

129

N H

EP

O

N

N H

R1

130 c

R1= H, p-OCOCH3, p-OCH3 R2= m-NO2, H

AC C

OH

O

O O N

R2

N N H

R1

131

Scheme 21: (a) NH2CH2COOCH2CH3, DMAP, DCC, pyridine (b) Na2CO3, H2O: MeOH(2:1), reflux (c) Aldehyde (ArCHO, AcONa, (CH3CO)2O.

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ACCEPTED MANUSCRIPT

Scheme 22: (a) CH3COOH, appropriate aldehyde, reflux, 3 h (b) CH3COOH, K2CO3, NaHSO3, NaOH, stirred at 100

0

C, 20 min. (c) C2H5OH/SOCl2, reflux, 4h (d)

AC C

EP

TE D

Xylene/Sulphur, reflux, 8h (e) DMF, NaH, dibromoalkane.

Scheme 23: (a) DMF, K2CO3, dibromoalkane.

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ACCEPTED MANUSCRIPT

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SC

Scheme 24: (a) 1. MeOH/CH2Cl2 2. NaBH3CN,MeOH.

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TE D

Scheme 25: (a) Piperazine, NaBH3CN.

Scheme 26: (a) OHCCH(OMe)2, 5% TFA, CH2Cl2 (b) KMnO4, THF, rt, 17 h (c)RI, NaH, DMF, rt, 4 h. (d)AcOH/H2O (2:3 ratio), 120 oC, 1 h (e) 10% NaOH,MeOH, rt.

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ACCEPTED MANUSCRIPT

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Scheme 27: (a) Ba(OH)2/MeOH, rt, 8h (b) NH2NH2.H2O, acetic acid, reflux.

Scheme 28: (a) substituted o-phenylenediamine, EtOH, (La(NO3)3·6H2O) (b) pyridine-2,3-

AC C

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TE D

diamine, EtOH, (La(NO3)3·6H2O), 60 °C.

Scheme 29: (a) Substituted acetophenones, Ba(OH)2/MeOH, rt, 8h.

RI PT

ACCEPTED MANUSCRIPT

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Scheme 30: (a) 20 % CuSO4.5H2O, 40 % sodium ascorbate, t-BuOH:H2O (4:1).

AC C

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TE D

Scheme 31: (a) CuSO4. 5H2O, Na. Ascorbate, tert-BuOH:H2O (1:1), rt, 3h.

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ACCEPTED MANUSCRIPT

Scheme 32: Reagents and conditions: (a) PPA, o-phenylenediamine, 180 0C, 6 h (b) NiCl2. 6H2O, THF:H2O (4:1), r.t, 4 h (c) CuCl2. 2H2O, DMF:H2O (1:1), r.t, 4 h (d) CoCl2. 6 H2O,

AC C

EP

TE D

THF:H2O (4:1), r.t, 2 h (e) CisPt(DMSO)2Cl2, MeOH, r.t, 20 min.

ACCEPTED MANUSCRIPT

Research Highlights:

β-carbolines as excellent scaffolds for synthetic manipulations with enormous anticancer potential. Recent developments (2011–2016) on anticancer activities of β-carboline are discussed.

AC C

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RI PT

Particular emphasis has been given on SAR and mechanism of action