Synthesis and in vitro cytotoxicity of haloderivatives of noscapine

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Synthesis and in vitro cytotoxicity of haloderivatives of noscapine
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  Synthesis and in vitro cytotoxicity of haloderivatives of noscapine Akhilesh Kumar Verma, a,* Sandhya Bansal, b Jaspal Singh, a Rakesh Kumar Tiwari, a V. Kasi Sankar, a Vibha Tandon b and Ramesh Chandra a,b,* a Synthetic Organic Chemistry Research Laboratory, Dr. B. R. Ambedkar Center for Biomedical Research,University of Delhi, Delhi 110007, India b Medicinal Chemistry Research Laboratory, Dr. B. R. Ambedkar Center for Biomedical Research,University of Delhi, Delhi 110007, India Received 5 May 2006; revised 26 May 2006; accepted 26 May 2006Available online 19 June 2006 Abstract—  Three haloderivatives of noscapine  2  –  4  were synthesized chemoselectively and their in vitro cytotoxicity was assessed byMTT assay on U-87 human glioblastoma cell lines. At 50  l M concentration after 72 h, 9-chloronoscapine  2 , 9-bromonoscapine  3 (EM011), and 9-iodonoscapine  4  killed 87.8%, 51.2%, and 56.8% cells, respectively, however noscapine kills only 40% of the cells;revealing 9-chloronoscapine as a potential cytotoxic agent than noscapine and 9-bromonoscapine (EM011). At low concentration(1  l M) 9-bromonoscapine (46.7%) and 9-chloronoscapine (45.7%) did not show any significant difference.   2006 Elsevier Ltd. All rights reserved. 1. Introduction Microtubule-targeting agents such as the vinca alkaloids(vinblastine, vincristine, vindesine, etc.) and taxanes(paclitaxel and docetaxel) are important chemothera-peutic drugs for the treatment of cancer. 1,2 Antitumoragents that affect microtubule dynamics are of greatmedical interest and are now commonly used in currentchemotherapy regimens. 3,4 The clinical use of thesedrugs has been hampered, however, by the side effectsand limited effectiveness, increased drug resistance in tu-mors, 5 poor bioavailability, and poor solubility. 6 Thus,there is still a need for effective microtubule-directeddrugs with improved solubility and therapeutic index.Noscapine  1  is a naturally occurring phthalideisoquin-oline alkaloid obtained from opium. It has been usedorally in humans as an antitussive agent and displaysa favorable toxicity profile. 7 Additionally, it has beenknown for some time that noscapine can act as a weakanticancer agent in certain in vivo models. 8 Recently,Joshi et al. have performed several studies to evaluatethe mechanism of action of this anticancer effect andfound that noscapine can disrupt tubulin dynamics. 9 Noscapine inhibits the progression of murine lympho-ma, melanoma, and human breast tumors implantedin nude mice with little or no toxicity to the kidney,heart, liver, bone-marrow, spleen, or small intestineand does not inhibit primary humoral immuneresponses in mice. The lead compound noscapine iscurrently undergoing phase I/II clinical trials for cancertreatment at University of Southern California, USA.EM011, a brominated derivative of noscapine, possess-es 5- to 10-fold higher anticancer activity in compari-son to noscapine in preclinical models and it ispotently effective against vinblastine-sensitive lineCEM. 10–12 Although noscapine appears to be a weakinhibitor of microtubule polymerization, its low costand ready availability allow for further exploration of this natural product.In the present work, we are reporting the chemoselectivesynthesis of 9-halonoscapines  2  –  4  without effecting lac-tone ring and their in vitro cytotoxicity on U-87 humanglioblastoma cell lines. 2. Results and discussion2.1. Synthesis 3-(9-Chloro-4-methoxy-6-methyl-5,6,7,8,-tetrahydro-[1,3]-dioxolo [4,5-  g  ] isoquinoline-5-yl)-6,7-dimethoxy-3 H  -iso-benzofurane-1-one  2  was prepared by the chlorination 0968-0896/$ - see front matter    2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.bmc.2006.05.069 Keywords : Synthesis; Noscapine; Halo-noscapine; U-87 glioblastomacell line; Cell proliferation.*Corresponding authors. Tel.: +91 11 55782976/27666272; fax: +91 1127666248; e-mail: akhilesh682000@yahoo.comBioorganic & Medicinal Chemistry 14 (2006) 6733–6736  of noscapine (RS)  1  by dropwise addition of sulfurylchloride at 0–5   C with vigorous stirring followed bystirring at 25   C for 8–10 h in 92% yield (Scheme 1). 13–15 The chlorination takes place regioselectively at 9position. Iododerivative of noscapine 3-(9-iodo-4-meth-oxy-6-methyl-5,6,7,8,-tetrahydro-[1,3]dioxolo[4,5-  g  ]iso-quinoline-5-yl)-6,7-dimethoxy-3 H  -isobenzofurane-1-one 4  was prepared by the iodination of noscapine  1  usingiodinemonochloride in chloroform and HCl at 0–5   Cin 74% yield (Scheme 1). Completion of the reactionwas monitored by thin-layer chromatography. 9-Bro-monoscapine 3-(9-bromo-4-methoxy-6-methyl-5,6,7,8,-tetrahydro-[1,3]dioxolo [4,5-  g  ] isoquinoline-5-yl)-6,7-dimethoxy-3 H  -isobenzofurane-1-one  3  was preparedas described previously. 16 Disappearance of one aromat-ic singlet proton of C-9 at   6.30 ppm in the  1 H NMR of the starting compound confirms the chlorination andiodination at C-9 position. Structures of compounds 2  –  4  were fully characterized by the  1 H NMR,  13 CNMR, and mass spectroscopic data. 2.2. Cell cytotoxicity The cytotoxicity of synthesized derivatives of noscapinewas investigated using human glioma cell line U-87 byusing MTT assay 17,18 studying their effect on cell surviv-al and cell proliferation. For survival studies cells wereincubated with noscapine and its analogues  2  –  4  contin-uously and then washed to remove the drug and cell sur-vival was determined following the addition of 1, 10,and 50  l M drug. At concentrations of 1, 10, and50  l M noscapine killed 18%, 27%, and 40% of the cells,respectively. On the other hand, chloronoscapine  2 , bro-monoscapine  3 , and iodonoscapine  4  at 1, 10, and50  l M concentrations killed 45.7%, 61.5%, 87.8% (chlo-roderivative  2 ); 46.7%, 47.1%, 51.2% (bromoderivative 3 ); and 34.4%, 46.0%, 56.8% (iododerivative  4 ) cells,respectively (Figs. 1 and 2). At low concentration(1  l M) bromonoscapine (46.7%) and chloronoscapine(45.7%) showed more toxicity than iodonoscapine(34.4%).log P   of all the haloderivatives were calculated usingChem 3D software and was found to be 3.18, 3.45,and 3.98 for compound  2  (9-chloronoscapine),  3  (9-bro-monoscapine), and  4  (9-iodonoscapine), respectively. NOOOOMeO OMeOMe MeBrNOOOOMeO OMeONOOMeO OMeOMeClOONOOOOMeO OMeOMeI 1234 i iiiii Scheme 1.  Reagents and conditions: (i) SO 2 Cl 2 , CHCl 3 , 25   C, 10 h;(ii) Br 2 /H 2 O, 25   C, 2 h; (iii) ICl, CHCl 3 , 25   C, 10 h. 1 0204060801000 24 48 72 Time (hrs)      %    s    u    r    v     i    v    a     l     %    s    u    r    v     i    v    a     l     %    s    u    r    v     i    v    a     l     %    s    u    r    v     i    v    a     l 1(1)1(10)1(50) 3 020406080100 0 24 48 72 Time(hrs) 3(1)3(10)3(50) 2 0204060801000 24 48 72 Time(hrs) 2(1)2(10)2(50) 4 020406080100 0 24 48 72 Time(hrs) 4(1)4(10)4(50) Figure 1.  Cytotoxicity of noscapine and its analogues  2  –  4  using the MTT assay on human malignant glioma cells U87. Cells were grown in thepresence of drugs at various concentrations up to 72 h and metabolic activity of these cells was measured using MTT. Results are shown as the %survival. 111222333444 0204060801001201 Conc.microMolarCytotoxicity Chart    %    C  y   t  o   t  o  x   i  c   i   t  y 10 50 Figure 2.  Showing the cytotoxicity of haloderivatives 1–4 at differentconcentrations.6734  A. K. Verma et al. / Bioorg. Med. Chem. 14 (2006) 6733–6736   log P   values of compounds  2  –  4  suggested the more sol-ubility of compound  2  in comparison to compounds  3 and  4 . 2.3. Cell proliferation Effects on proliferation of exponentially growing cellswere also studied. In these experiments, cells were incu-bated with noscapine  1  and 9-chloronoscapine  2  for 24,48, and 72 h to observe growth. Kinetics of cell growthtreated with noscapine  1  and chloronoscapine  2  differsignificantly from the kinetics of nontreated cells anddoubling time was not comparable to that of nontreatedcells (Fig. 3). 3. Conclusion In conclusion, we have synthesized haloderivatives of isoquinoline alkaloid noscapine chemoselectively ingood yields, and their in vitro cytotoxicity was assessedby MTT assay on U-87 human glioblastoma cell line.9-Chloronoscapine  2  was found to be the most cytotoxicagent killing 87.8% cells at 50  l M concentration after72 h. The enhanced cytotoxicity of 9-chloronoscapine 2  in comparison to 9-bromonoscapine (EM011) and itsready synthesis lend hope that it can be taken up forthe development of novel anticancer agent. Furtherstudies are ongoing and the results will be reported indue course. 4. Experimental4.1. General All reagents used were of AR grade. Melting pointswere determined using a Thomas Hoover meltingpoint apparatus and are uncorrected.  1 H (300 MHz)and  13 C NMR (75 MHz) spectra were recorded on aBruker 300 NMR spectrometer in CDCl 3  (with TMSfor  1 H and chloroform- d   for  13 C as internal referenc-es) unless otherwise stated. Mass spectrum was record-ed on Hybrid Quadrupole-TOF LC n MS n MS massspectrometer (Q. Star XL). Infrared spectra ( t max )were recorded on Perkin-Elmer FTIR spectrophotom-eter as thin films on KBr plates (for oils) or KBr discs(for solids). Column chromatography was performedon silica gel (230–400 mesh). The reactions were mon-itored by thin-layer chromatography (TLC) using alu-minum sheets with silica gel 60 F 254  (Merck). All of the reactions were carried out under nitrogenatmosphere. 4.2. Synthesis of 3-(9-chloro-4-methoxy-6-methyl-5,6,7,8,-tetrahydro-[1,3]dioxolo[4,5-  g  ]isoquinoline-5-yl)-6,7-dimethoxy-3 H  -isobenzofurane-1-one (2) To a stirred solution of noscapine (1 g, 2.4187 mm) inchloroform (50 ml), sulfuryl chloride (3 equiv) in 30 mlchloroform was added dropwise at 0–5   C. Reactionmixture was allowed to come at room temperatureand stirring was continued for 8–10 h. Completionof the reaction was monitored by thin-layer chroma-tography. The reaction mixture was poured into200 ml of water and extracted twice with chloroform.The organic extract was washed with brine and driedover anhydrous sodium sulfate. After removal of thesolvent in vacuo, the residue was purified with columnchromatography using chloroform/methanol (5%) asan eluent to give the desired product  3  as white nee-dles, Yield 92%, mp 168.2–168.1   C.  1 H NMR(300 MHz; CDCl 3 ; Me 4 Si): 7.03 (d,  J   = 8.0 Hz, 1H)6.31 (d,  J   = 8.1 Hz, 1H), 6.02 (s, 2H), 5.51 (d, J   = 4.32 Hz, 1H), 4.34 (d,  J   = 4.3 Hz, 1H), 4.09 (s,3H), 3.98 (s, 3H), 3.88 (s, 3H), 2.75–2.61 (m, 2H),2.51–2.44 (m, 4H), 2.00–1.94 (m, 1H);  13 C (75 MHz;CDCl 3 ; Me 4 Si): 167.5, 152.3, 147.5, 139.3, 134.6,126.1, 120.3, 118.4, 108.6, 102.4, 93.5, 81.7, 64.3,61.8, 59.4, 57.8, 54.9, 46.1, 45.2, 39.8, 20.6, 18.3.LC–MS  m / z : 448.4 (M+1). 4.3. Synthesis of 3-(9-iodo-4-methoxy-6-methyl-5,6,7,8,-tetrahydro-[1,3]dioxolo[4,5-  g  ]isoquinoline-5-yl)-6,7-dime-thoxy-3 H  -isobenzofurane-1-one (4) To a stirred solution of noscapine (1 g, 2.4187 mm) inchloroform (50 ml), iodinemonochloride (3 equiv) in30 ml chloroform was added dropwise at 0   C. Reac-tion mixture was allowed to come at room tempera-ture and stirring was continued for 10 h. TLCmonitored completion of the reaction. The reactionmixture was poured in 200 ml of water and extractedtwice with chloroform. The organic extract waswashed with brine and dried over anhydrous sodiumsulfate. After removal of the solvent in vacuo, the res-idue was purified with column chromatography usingCHCl 3 /MeOH (5%) as an eluent to give the desired 1 00.511.50 24 48 72 Time(hrs) Control1(1)1(10)1(50)Control3(1)3(10)3(50) 3 00.511.50 24 48 72 Time(hrs)    C  e   l   l  n  u  m   b  e  r   (  m   i   l   l   i  o  n  s   /  m   l   )   C  e   l   l  n  u  m   b  e  r   (  m   i   l   l   i  o  n  s   /  m   l   ) Figure 3.  Effects of 1, 10, and 50  l M of noscapine ( 1 ) and chloronoscapine ( 3 ) on growth kinetics of U87 cells. Cells were grown in presence of thesedrugs and number of proliferating cells was counted using hemocytometer at different time intervals. Results are shown as number of cells (in millionsper milliliter). A. K. Verma et al. / Bioorg. Med. Chem. 14 (2006) 6733–6736   6735  product ( 4 ): white powder, Yield 74%, mp 186–190   C. d H  (300 MHz; CDCl 3 ; Me 4 Si): 7.15 (d,  J   = 8.1 Hz,1H), 6.93 (d,  J   = 8.1 Hz, 1H), 6.11 (s, 2H), 5.42 (d, J   = 4.8 Hz, 1H), 4.26 (d,  J   = 4.8 Hz, 1H), 3.85 (s,3H), 3.76 (s, 3H), 3.72 (s, 3H), 2.78–2.72 (m, 2H),2.55–2.50 (m, 2H), 2.32 (s, 3H),  13 C (75 MHz; CDCl 3 ;Me 4 Si): 168.2, 155.3, 151.8, 148.4, 146.6, 143.1, 140.3,120.4, 119.6, 113.3, 101.5, 85.9, 82.3, 61.9, 56.7, 55.7,54.5, 54.2, 51.2, 39.8, 30.1, 18.7. LC–MS  m / z : 539.9(M+1). 4.4. Cell culture The human glioma cell line U 87 was maintained asmonolayers at 37   C in 25 cm 2 tissue culture flasks (Tar-sons, India) using Dulbecco’s modified Eagle’s medium(Sigma) supplemented with 5% fetal calf serum (Biolog-icals, Israel). Cells were passaged routinely in exponen-tial growth phase twice a week using 0.05% trypsin– EDTA solution in phosphate-buffered saline (PBS) fortrypsinization. All experiments were performed withasynchronously growing cells in the exponential growthphase (24 h after plating). 4.5. Cytotoxicity assay Cytotoxicity was determined using the MTT [3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-2 H  -tetrazolium bro-mide] assay 17,18 using a 96-well microtiter plate.Three thousand cells per well were plated in 200  l lof the complete medium and treatment with these li-gands was performed 24 h after plating. For % surviv-al determination, cells were exposed continuously withvarying concentrations of drug and MTT assays wereperformed at the end of the fourth day. At the end of the treatment, control and treated cells were incubatedwith MTT at a final concentration of 0.5 mg/ml for2 h at 37   C and then the medium was removed.The cells were lysed and the formazan crystals weredissolved using 150  l l DMSO. The absorbance wasread at 570 nm using 630 nm as reference wavelengthusing ELISA reader. 4.6. 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