r XXXX American Chemical Society A dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
B dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
were designed to a...
C dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
oxidizing reagent ...
D dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
and 8h were toxic ...
E dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
ethylenedioxy deri...
F dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
Similarly, R-enant...
G dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
main apoptotic pat...
H dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
A549 cells, and 8e...
I dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
of the targeted mo...
J dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
for C20H15NO7: C, ...
K dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
(2) Gupta, S.; Das...
L dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
(36) Hitotsuyanagi...
of 12

Polyalkoxybenzenes JMC2011

Published on: Mar 4, 2016

Transcripts - Polyalkoxybenzenes JMC2011

  • 1. r XXXX American Chemical Society A dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 ARTICLE pubs.acs.org/jmc Polyalkoxybenzenes from Plants. 5. Parsley Seed Extract in Synthesis of Azapodophyllotoxins Featuring Strong Tubulin Destabilizing Activity in the Sea Urchin Embryo and Cell Culture Assays Marina N. Semenova,†,‡ Alex S. Kiselyov,§ Dmitry V. Tsyganov,|| Leonid D. Konyushkin,|| Sergei I. Firgang,|| Roman V. Semenov,|| Oleg R. Malyshev,|| Mikhail M. Raihstat,|| Fabian Fuchs,^ Anne Stielow,^ Margareta Lantow,^,# Alex A. Philchenkov,3 Michael P. Zavelevich,3 Nikolay S. Zefirov,O Sergei A. Kuznetsov,^ and Victor V. Semenov*,‡,|| † Institute of Developmental Biology, RAS, 26 Vavilov Street, 119334 Moscow, Russian Federation ‡ Chemical Block Ltd., 3 Kyriacou Matsi, 3723 Limassol, Cyprus § CHDI Foundation, 6080 Center Drive, Suite 100, Los Angeles California 90045, United States ) N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation ^ Institute of Biological Sciences, University of Rostock, 3 Albert-Einstein-Strasse, D-18059 Rostock, Germany # Department of Pathology, Immunology and Laboratory Medicine, University of Florida, 1600 Archer Road, Gainesville Florida 32610, United States 3 R. E. Kavetsky Institute of Experimental Oncology, Pathology, and Radiobiology, National Academy of Sciences of Ukraine, 45 Vasyl0 kivska Street, 03022 Kyiv, Ukraine O Lomonosov Moscow State University, GSP-1, Leninskie Gory, 119991 Moscow, Russian Federation bS Supporting Information ’ INTRODUCTION Podophyllotoxin (PT) (Figure 1) is a lignan of aryltetralin family found in plants of Podophyllum genus. It is a strong microtubule destabilizing agent that binds to the colchicine site of tubulin.1,2 PT and its derivatives exhibit anticancer, antiviral, and insecticide activities based on their ability to dissociate microtubules.3À9 However, clinical trials of PT as an anticancer agent were unsuccessful due to its considerable toxicity.3 Nu- merous attempts have been made to improve PT safety profile while maintaining its potency. For example, semisynthetic PT derivatives, including etoposide (Figure 1), teniposide, and etopophos, are currently in clinical use for the treatment of a variety of malignancies. A reported mechanism of action for these compounds is distinct from that of the parent PT and involves strong DNA-topoisomerase II inhibition leading to late S-G2 cell cycle arrest and subsequent apopotosis.10À13 An alternative nontubulin and nonantitopoisomerase mode of action for some PT derivatives has been proposed recently, associated with cell death and sub-G1 apoptotic cell accumulation without any significant cell cycle arrest.11,14 PT structure (Figure 1) features four chiral centers, making it a challenge for the structureÀactivity relationship studies. Abso- lute stereochemistry of substituents at the C1 atom (the ring C) was reported to have major influence on compound’s antitumor activity.3 Several synthetically feasible structural analogues of PT Received: June 8, 2011 ABSTRACT: A series of 4-azapodophyllotoxin derivatives with modified rings B and E have been synthesized using allylpo- lyalkoxybenzenes from parsley seed oil. The targeted molecules were evaluated in vivo in a phenotypic sea urchin embryo assay for antimitotic and tubulin destabilizing activity. The most active compounds identified by the in vivo sea urchin embryo assay featured myristicin-derived ring E (4e, 6e, and 8e). These molecules were determined to be more potent than podophyl- lotoxin. Cytotoxic effects of selected molecules were further confirmed and evaluated by conventional assays with A549 and Jurkat human leukemic T-cell lines including cell growth inhibition, cell cycle arrest, cellular microtubule disruption, and induction of apoptosis. The ring B modification yielded 6-OMe substituted molecule 8e as the most active compound. Finally, in Jurkat cells, compound 8e induced caspase-dependent apoptosis mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic pathway.
  • 2. B dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE were designed to address epimerization at positions 1À4.3 For example, 4-aza-PT derivatives with a chiral center C1 (Figure 1) showed antiproliferative and pro-apoptotic activity in HeLa and Jurkat cells (4a and 18a)15,16 and inhibited growth of human liver cancer cells HepG2 (4a and 4i).17 Two racemic analogues of 4-aza-PTs (4a and 6a) were more cytotoxic against P388 murine leukemia cells than the parent PT.18,19 Several 4-aza-PT deriva- tives were reported to be potent tumor inhibitors (ex., 4a)20 and vascular-disrupting agents.21 In addition, insecticidal (larvicidal) activity possibly mediated by microtubule alterations was re- ported for this class of molecules.6 However, experimental evidence for the mechanism of action and cellular targets of 4-aza-PTs has been obtained only recently. Namely, it was shown that some of 4-aza-PTs with hetero aryl, benzo hetero aryl, or tetrahydronaphthalene rings A and B exhibited cytotoxicity involving microtubule depolymerization, G2/M cell cycle arrest, and apoptotic cell death.22,23 Considering synthetic feasibility, antimitotic, antiproliferative, and tubulin destabilizing activity of 4-aza-PT derivatives, these compounds continue to attract a considerable interest. In the present study, we prepared a series of 4-aza-PT derivatives using plant polyalkoxybenzenes easily available from parsley and dill seed extracts.24,25 It has been suggested that rotation of the axial E-ring in the parent PT molecule is restricted as it is almost orthogonal to the other rings.26 Therefore a modification of the rings B and E with additional methoxy functionality could affect the rotation of the ring E yielding PT derivatives with promising antimitotic potencies. Moreover, tetraalkoxybenzene pharmaco- phore is featured in several natural products with reported antiproliferative activity including Cactaceae tetrahydroiso- quinolines27 and flavonoids and isoflavonoids.28À30 In designing the evaluation sequence for test molecules, we aimed at identifying potent novel aza-analogues of PT and confirming their specific antitubulin activity via a variety of cell-based assays. The initial in vivo sea urchin embryo assay allowed for the identification of hits featuring desirable antimi- totic potency and phenotypic effect (vide infra) in comparison with the parent molecule (PT). This primary screen based on the simple organism model has been carried out to yield active molecules that were likely to affect tubulin dynamics as shown by us earlier.31À34 Tubulin targeting activity of these compounds would be further confirmed in A549 human lung epithelial carcinoma cell line. A detailed insight into the effects of selected active compounds on cellular proliferation, cell cycle progression, and microtubule distribution in cultured A549 cells was intended to confirm their specific tubulin destabilizing mode of action. Moreover, the link between compound-induced microtubule disruption and respective downstream intracellular mechanisms responsible for initiation of caspase-mediated apoptotic events was studied. All molecules were evaluated in a phenotypic sea urchin embryo assay in order to assess their antiproliferative and tubulin-destabilizing activities.31 The assay includes (i) fertilized egg test for antimitotic activity displayed by cleavage alteration/ arrest and (ii) behavioral monitoring of a free-swimming blas- tulae treated immediately after hatching. In our earlier validation efforts, we concluded that lack of forward movement, settlement to the bottom of the culture vessel and rapid spinning of an embryo around the animalÀvegetal axis suggests a tubulin- destabilizing activity caused by a molecule (video illustrations are available at http://www.chemblock.com). An assay setup provides the possibility to identify a molecule with antimitotic or nonspecific cytotoxic activity and a novel mode of action.32 Data generated by the sea urchin embryo assay have been further confirmed using conventional cell-based and in vitro tubulin polymerization assays.33,34 As a result, we identified several promising candidates for the advanced in vivo evaluation. ’ CHEMISTRY 4-Aza-PTs 4À13 and intermediate quinolines 40 À150 were synthesized as described earlier6,15À20,35À37 via the cyclization of 4-chloro-acetoacetic acid with the corresponding amines, fol- lowed by the addition of aldehydes 2aÀk in the presence of an Figure 1. Structures of podophyllotoxin, etoposide, and general struc- ture of synthesized 4-aza-analogues. Scheme 1a a Reagents and conditions: (a) (i) powdered KOH, (n-Bu)4N+ BrÀ , heat, 100 °C, 40 min; (ii) O3, CHCl3ÀMeOHÀpyridine (80:20:3 v/v), À15 °C, 1À2 h;25 (b) C6H6ÀAcOH, reflux, 8 h; (c) CF3COOH, rt, 24 h; (d) AcOHÀNaBH3CN, rt, 15 h; (e) ref 43; (f) EtOHÀEt3N, reflux, 3 h. Compounds 4À13 (fÀk), 17i, 18e, 19e were synthesized from the corresponding commercial aldehydes.
  • 3. C dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE oxidizing reagent p-chloranil (Scheme 1). Aldehydes 2aÀe were synthesized using natural polyalkoxybenzenes extracted from parsley seed oil.25 For the SAR study, a panel of 4-aza-PTs with the ring E derived from commercially available aldehydes 2fÀk has been obtained. Quinolines 40 À130 were reduced with NaBH3CN in a glacial acetic acid to yield compounds 4À13 in 12À86% yields. 4-Aza-PTs 4À13 were stable as solids, however, they were readily oxidized to respective quinolines 40 À130 when dissolved in DMSO or ethanol. Dihydropyridopyrazole deriva- tives 17À19 were obtained by a three-component condensation of respective aminopyrazole, aldehyde 2, and tetronic acid 16 as reported in the literature.16 Notably, compounds 4g, 40 g, 4f, and 40 f are aza-analogues of the natural lignans taiwanin C and chinensin.36,38À42 ’ RESULTS AND DISCUSSION All synthesized aza-PTs 4À19 and 40 À150 were evaluated for their antiproliferative, antimitotic, and microtubule destabilizing activities using in vivo sea urchin embryo assay. Cytotoxicity, cellular microtubule depolymerization, and apoptosis-inducing effects of selected compounds have been further assessed using A549 human lung epithelial carcinoma and Jurkat human leukemic T-cell lines. Evaluation of 4-aza-PTs in the Phenotypic Sea Urchin Embryo Assay. Effects of 4-aza-PTs on the sea urchin embryo are summarized in Table 1 and Table S1 of the Supporting Information. PT and etoposide were used as references. As evidenced from Table 1, a number of 4-aza-PTs (4a,c,eÀg, iÀk, 5e, 6a,e, 7e, 8e,h, 9e, 11e, 110 e, 12a, and 13e) displayed significant cleavage alteration, cleavage arrest, and embryo spinning, suggesting their antimitotic microtubule destabilizing activity. Fig- ure 2 illustrates typical developmental changes of an embryo when treated with a representative compound 4e. The observed pheno- typic changes suggest direct tubulin dynamic effects of 4-aza-PT derivatives. Interestingly, molecules 4a, 4e, and 8h were reported as larvicidal agents against Phaedon cochleariae beetle, whereas 4j, 4k, Table 1. Effects of 4-Aza-podophyllotoxins on Sea Urchin Embryo and Cancer Cells sea urchin embryo effects EC (nM)a cytotoxicity IC50 (nM) compd cleavage alteration cleavage arrest embryo spinning A549b P388c A549 cell total microtubule disruption (μM)d PT 20 50 500 7.47 ( 0.41 10.4 0.1 etoposide 2000 >68000 >68000 7140e 1180e NDf 4a 50 500 2000 2.71 ( 0.92 4.5 0.1 40 a >4000 >4000 >4000 11928.33 ( 5368.30 >252900 >5 4b 1000 2000 >5000 NDf NDf NDf 4c 100 500 4000 NDf NDf NDf 4d 1000 4000 >10000 299.32 ( 58.35 NDf 1 4e 5 50 50 55.18 ( 7.54 NDf 1 4f 10 50 200 NDf 85.4 NDf 4g 5 100 2000 595.58 ( 46.18 130.7 1 4i 50 200 2000 >3200 385.4 NDf 4j 5 20 100 NDf 15.7 NDf 4k 5 20 100 63.00 ( 5.21 17.2 NDf 5a 2000 >2000 >2000 291.43 ( 143.53 NDf 5 5e 50 500 2000 297.63 ( 164.54 NDf >5 5i 2000 >2000 >2000 NDf NDf NDf 6a 200 1000 1000 9.20 ( 1.22 4.1 1 6e 5 50 100 64.67 ( 11.34 NDf 0.5 7e 10 50 500 282.27 ( 96.99 NDf 5 8e 0.5 5 50 15.14 ( 2.98 NDf 1 8h 2 10 200 NDf NDf NDf 9e 20 200 2000 533.83 ( 111.47 NDf >5 10f >4000 >4000 >4000 NDf NDf NDf 11e 5 50 100 159.05 ( 8.24 NDf NDf 110 e 200 2000 5000 NDf NDf NDf 12a 50 200 100 56.54 ( 6.83 NDf 0.5 13e 2 20 50 264.09 ( 52.32 NDf 5 130 e 100 1000 >5000 NDf NDf NDf 140 e 100 >4000 >5000 NDf NDf NDf a The sea urchin embryo assay was conducted as described previously;31 fertilized eggs and hatched blastulae were exposed to 2-fold decreasing concentrations of compounds; duplicate measurements showed no differences in effective threshold concentration (EC) values. b A549: human lung epithelial carcinoma cells; data are expressed as the mean ( SD from the doseÀresponse curves of three independent experiments. c P388: murine leukemia cells; data from ref 19. d A549 cells were incubated with compounds at concentrations of 0.1, 0.5, 1, and 5 μM during 8 h. Data are expressed as compound concentration that caused total microtubule disassembly from three independent experiments (see Figure 4B,DÀF as an example). e Data from ref 44. f ND: not determined.
  • 4. D dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE and 8h were toxic against Spodoptera frugiperda larvae.6 We believe that less potent compounds 4b,d, 18e, 60 b, and 130 e were tubulin destabilizers as well, although they did not cause embryo spinning. As evidenced from the detailed microscopic analysis, these com- pounds induced formation of tuberculate eggs typical of tubulin destabilizers (Figure 2C).31 In our hands, topoisomerase inhibitor etoposide featured cleavage alteration but failed to induce cleavage arrest and embryo spinning. When applied to hatched blastulae, etoposide at 4À5 μM caused larval (pluteus) malformations. At higher concentrations (10À20 μM), marked abnormalities of gastrulation and morphogenesis were detected, whereas at 40À68 μM, developmental abnormalities and eventual embryo death were observed after 2.5 h of treatment. Because of their marginal solubility in DMSO, ethanol, and seawater, we were unable to test 80 e, 110 a, and 120 a or reach higher concentrations for 4b,d, 18e, 60 b, and 130 e. 140 e showed nontubulin antiproliferative activity, as evidenced by the lack of embryo spinning and arrested tuberculate eggs. StructureÀActivity Studies in the Sea Urchin Embryo Assay. 1. The Ring C: Saturated vs Unsaturated Compounds. A number of 1,4-aza-PT analogues (4aÀg,iÀk, 5e, 6a,e, 7e, 9e, and 13e) featuring 1,4-dihydropyridine system for the ring C exhibited potent antimitotic activity with EC50 values of 0.5 nMÀ2 μM in the sea urchin assay. These results are in agreement with the published data.19 Conversely, the respective aromatic pyridine derivatives 40 aÀg,iÀk, 50 e, 60 a,e, 70 e, 90 e, and 130 e) were inactive up to 4 μM concentration or exhibited moderate cleavage alteration effect (Table 1; Table S1, Support- ing Information), with a notable exception of 110 e, featuring myristicin substituent for the ring E. This molecule did exhibit a tubulin destabilizing activity although it was significantly lower than that for the corresponding unsaturated analogue 11e. In addition, aromatization of the ring C yielded decrease in cyto- toxicity and microtubule effects, presumably due to the inactive conformation of the ring E.3 Several saturated compounds were found to lose their activity in DMSO solution at room temperature. For example, the antimitotic effect of 4k on sea urchin embryos decreased significantly (by ca. 5-fold) within 2 days after storage in DMSO. According to the literature data, aromatization of 4a to yield 40 a required harsh experimental conditions such as refluxing acetic acid.37 We observed this conversion to take place in DMSO at room temperature, as confirmed by the time course NMR studies of 4k. Specifically, in 2 and 6 days, the rate of its conversion to the respective aromatic analogue 40 k were 25% and 45%, respec- tively. A similar activity reduction was observed for 4i and 6a, whereas DMSO stocks of compounds 4f, 8e, 9e, and 11e retained their activities for 2À4 days. 2. Other Substitutions: Rings A, B, and E. Introduction of a methoxy-group to C8 position of the ring B dramatically de- creased antiproliferative activity in sea urchin embryo assay (Table 1, compounds 4a . 5a, 4e . 5e, and 4i . 5i). Replacement of methylenedioxy group (the ring A) with ethy- lenedioxy substituent did not result in a definitive outcome. For example, the potency of respective ethylenedioxy derivative (Table 1, 4a vs 6a) in the sea urchin embryo cleavage stages was reduced significantly, similar to the reported insecticidal activity.6 However, for the pair 4e vs 6e, the activity was not affected.19 Replacement of the ring A with two methoxy substit- uents was reported to either result in a marked reduction of cytotoxicity19,45,46 or was negligible.16 Similar derivatives featur- ing a single methoxy group at C7 displayed reduced potency compared to the parent 4-aza-PT molecule (Table 1, 4e vs 7e). On the contrary, 6-methoxy derivative 8e exhibited the strongest antiproliferative effect of all tested 4-aza-PTs being ca. 40 times more potent than the parent PT. Another 6-methoxy analogue 8h displayed high activity in the sea urchin assay as well as pronounced larvicidal effect.6 Compound 9e lacking the ring A was less active, however it did retain the antimitotic activity. Substitution of the ring A with cyclopentane moiety was reported to result in a moderate decrease in cytotoxicity.19 In our assay system, this modification slightly enhanced the in vivo effect (Table 1, 4e and 13e), however a nontubulin antiproliferative activity of 5,6-cyclopentyl derivative 140 e was also detected. In contrast, replacement of the ring A with a cyclohexyl group did not influence the antimitotic potency at the sea urchin embryo cleavage stages (Table 1, 4e and 11e; 4a and 12a). At later developmental stages of the embryo (ex., after hatching), 12a displayed formidable toxicity. Similarly, cyclohexyl derivative 12a exhibited cytotoxicity against human cervical (HeLa) and breast (MCF-7) carcinomas cells at nanomolar concentration range.23 A replacement of the AB ring system of 4-aza-PTs with pyrazole was reported to furnish derivatives that retained both cytotoxic and proapoptotic activities at 5 μM concentration.15,16 However, cellular target(s) or mode of action for these molecules have not been determined. In our hands, all tested pyrazole- containing compounds 17a,i, 18aÀc, and 19aÀc,e failed to affect sea urchin embryo development at any stage up to 4 μM concentration, the only exception being myristicin-derived 18e that exhibited weak tubulin-destabilizing properties (Table S1, Supporting Information). As described above, 1,4-dihydropyridine derivatives (the ring C) showed the best activities in the sea urchin embryo assay. We further evaluated the effect of C1 substituent (the ring E) on compounds activity. Notably, molecules featuring m-methoxybenzene- or myr- isticin-derived substituent (Table 1, 4e,j, 5e, 6e, 7e, 8e, 9e, 11e, and 13e) consistently exhibited the highest potency. 2,3-Dimethoxy and Figure 2. Effect of 4e on sea urchin embryo development. Time after fertilization (21 °C): 6 h. (A) Intact embryo at early blastula stage. Fertilized eggs were exposed continuously to 4e at 10 nM (B) and 100 nM (C). Note abnormal cleavage of cells with different size and shape that are irregularly positioned and formation of large multinucleated blastomeres (B). Tuberculate shape of an arrested egg seen on (C) is typical for tubulin destabilizing agents. Figure 3. Structures of 4e enantiomers.
  • 5. E dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE ethylenedioxy derivatives (ex., 4f,g) were less potent, displaying activities slightly higher than the parent PT. (see Table 1). Respec- tive p-methoxybenzene and 3,4,5-trimethoxybenzene analogues showed further reduced antitubulin effects in the assay (Table 1, 4a,i, 5a,i, and 6a), whereas 4-aza-PT molecules substituted with dillapiol-, apiol-derived or tetramethoxybenzene moieties 4bÀd were the least active. Compound 4k with unsubstituted benzene ring E exhibited strong antimitotic tubulin destabilizing activity, suggesting that methoxy groups in the ring E may not directly affect antitubulin activity. Similarly, 30 ,40 ,50 -demethoxy PT retained sig- nificant cytotoxicity.47 For the molecules featuring 8-methoxy moiety, the myristicin-derived analogue 5e displayed the highest activity(5evs5a,i).Asimilar patternwasobservedfortherespective ethylenedioxy derivatives 6a and 6e. In the next round of structureÀactivity studies, the effect of stereochemistry on compound’s activity has been studied. It was reported previously that (+)-configuration of antitubulin isoqui- noline derivatives was essential for their cytotoxicity and inhibi- tion of tubulin polymerization.48 For 4-aza-PTs, only R-enantiomer of 8h was found to be cytotoxic against cultured insect cells and larvae, showing cellular behavior consistent with its microtubule effect.6 Figure 4. Effects of selected compounds on the interphase microtubule networks in A549 lung carcinoma cells as analyzed by indirect immunofluorescence microscopy. Cells were treated with 0.1% DMSO (A) and with test compounds (1 μM) for 8 h: 4a (B), 40 a (C), 4e (D), 6a (E), and 8e (F). Bar: 25 μm. Note microtubule disappearance in B, DÀF.
  • 6. F dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE Similarly, R-enantiomer of 4-aza-PT mimetic with naphtha- lene A,B-ring system was about 1000 times more potent in HeLa and MCF-7 cells than S-enantiomer.23 In the present study, a racemic 4e was separated by chiral HPLC to yield pure enantio- mers (Figure 3 and Figure S1, Supporting Information). Both R-isomer and a respective racemate of 4e were found to cause cleavage alteration and arrest of the sea urchin embryo at 5 and 50 nM, respectively. The respective S-isomer was less active, ex- hibiting cleavage abnormalities at 50 nM and cleavage arrest at 200 nM. Both enantiomers triggered embryo spinning, suggest- ing their tubulin destabilizing properties. In summary, the sea urchin embryo profiling of the new aza- PT derivatives suggested that the most potent representative in each structural group featured myristicin-derived ring E (ex., 4e, 5e, 6e, 8e, 11e, and 13e, Table 1). Notably, methylenedioxy ring A was not required for the activity. For example, introduction of 6-methoxy or 7-methoxy groups into the ring B yielded potent molecules (ex., 7e, 8e, and 8h). Cell Growth Inhibitory Activity. Following outcome of the sea urchin embryo assay, we evaluated the inhibitory effect of selected active compounds on proliferation of A549 human lung carcinoma cells and Jurkat leukemia T-cells using MTT49 and trypan blue assays, respectively. Relevant A549 cellular IC50 data for 4a,d,e,g,i,k, 5a,e, 6a,e, 7e, 8e, 9e, 11e, 12a, 13e, 40 a, and reference compounds (PT, etoposide) are summarized in Table 1. Reported cytotoxicity data for selected molecules against P388 murine leukemia cells are included as well (Table 1).19 Notably, in Jurkat cells, 8e and PT exhibited IC50 values of 17.5 and 25 nM, respectively. In general, sensitivity of sea urchin embryos to 4-aza-PTs at the cleavage stage, when cells divide synchronously every 35À40 min, was markedly higher than that of cancer cells, probably due to their higher frequency of mitosis. Key features of the most active molecules identified in both assays included: (i) 1,4- dihydropyridine template for the ring C (4a vs 40 a), (ii) a myristicin-derived substitution in the ring E (8e), (iii) 6-methoxy substituent replacing the ring A (4e vs 8e), and (iv) lack of 8-methoxy substitution in the ring B (4a vs 5a, 4e vs 5e). Results of structureÀactivity relationship studies for the ring E correlated well between both cellular (A549 and P388) and sea urchin embryo assays. Compounds 4f and 4j exhibited high cytotoxicity against P388 cells (Table 1).19 Molecules featuring 3,4,5-tri- methoxybenzene substituent (4a and 6a) as well as the parent PT were the most cytotoxic agents against both human A549 and murine P388 cancer cell lines, 4a being more potent than PT. Notably, derivatives endowed with the myristicin moiety showed the greatest antimitotic activities in sea urchin embryo assay. Similarly, in Jurkat cells a myristicin derivative 8e was more potent than PT. Replacement of the ring A with cyclopentyl or cyclohexyl moieties markedly decreased cytotoxicity against A549 cells (4e vs 13e; 4a vs 12a), but this effect was almost negligible in the sea urchin embryo test. The observed discre- pancy could be attributed to the differences between mitotic spindle microtubules in frequently dividing sea urchin blasto- meres vs predominantly interphase microtubules in cultured cancer cells targeted by tubulin destabilizing agents. The Effect on Microtubule Distribution in Cells. Next, we evaluated the effect of selected active compounds on microtubule stability and distribution in cultured A549 cells. Specifically, cells were incubated with test articles (0.1À5 μM) or PT (0.1 and 5 μM) as a positive control and 0.5% DMSO as a negative control. Microtubules were subsequently labeled by the indirect immu- nofluorescence method and analyzed by fluorescence micro- scopy. Figure 4 exemplifies the microtubule destabilization effect of selected 4-aza-PTs in A549 cells. As shown in Table 1, compounds 4a,d,e,g, 6a, 6e, 7e, 8e, 12a, and 13e identified as tubulin destabilizers in the sea urchin embryo assay also affected cellular microtubule structure (Figure 4B,DÀF). It should be noted that 5a and 5e precipitated from 10 mM stock solutions in DMSO, therefore the data pertinent to these molecules could not be accurately interpreted. Compound 40 a (an aromatic analogue of 4a) featuring low cyto- toxicity failed to induce sea urchin embryo development alterations and to depolymerize cellular microtubules (Figure 4C). Surprisingly, compound 9e that exhibited modest cytotoxicity without the disruption of interphase microtubules in A549 cells did show a considerable antimitotic tubulin destabilizing activity in the sea urchin assay. The effect could be attributed to a somewhat selective interaction of this molecule with spindle microtubules in the sea urchin blastomeres vs interphase microtubules in cultured cancer cells. Treatment with PT at 0.1 and 5 μM resulted in complete disassembly of cellular microtubules (data not shown). Cell Cycle Analysis. The ability of selected 4-aza-PTs to interfere with A549 and Jurkat cell cycle progression was estimated by propidium iodide staining followed by flow cyto- metry analysis. In A549 cells, PT as well as its aza-analogues 4a,g and 6e caused G2/M cell cycle arrest, exhibiting EC50 values of 16.5 ( 0.35, 16.2 ( 0.71, 230 ( 7.07, and 119 ( 1.41 nM, respectively. Compound 40 a failed to affect the cell cycle progression even at 50 μM concentration. Notably, all three cell-based assays ranked the tested molecules similarly. Cyto- toxicity and microtubule dynamics effects of 4-aza-PTs decreased in the following order: 4a > 6e > 4g . 40 a. In Jurkat cells, both 8e and PT were potent G2/M blockers as well. In contrast, etopo- side caused a pronounced cell cycle arrest in G0/G1 phase (Figure 5). Apoptosis-Inducing Activity. It has been well established that tubulinbindingcompounds interferewiththe dynamics andstructure of both mitotic spindle and interphase microtubules, eventually leading to cell death by apoptosis. However the link between microtubule alteration and respective intracellular mechanisms responsible for initiation of apoptotic events remains unclear.50À52 Caspases (intracellular proteases) play a key role in the apoptotic response. Their activation by specific signals triggers proteolysis of cellularsubstratestherebyexecutingapoptoticevents.53 Therearetwo Figure 5. Cell cycle distribution of Jurkat cells treated with 8e (20 nM), PT (25 nM), and etoposide (2 μM) for 24 h. The cells were stained with propidium iodide to analyze DNA content by flow cytometry.
  • 7. G dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE main apoptotic pathways, namely the extrinsic pathway that involves membrane-bound death receptors and leads to activa- tion of caspase-8 and the mitochondria-related caspase-9-depen- dent intrinsic pathway. Both pathways converge onto the effector caspase-3 activated by the apical (initiator) caspases-8 and -9.53,54 Caspase-2 combines both initiator and effector features.53 Its processing requires caspase-9 and caspase-3.55 Generally, microtubule interfering agents induce caspase- dependent apoptotic cell death through intrinsic caspase-9- dependent pathway.50À52,56 For instance, tubulin destabilizers combretastatin A-4 and its analogues, as well as nocodazole, trigger apoptosis through the intrinsic pathway accompanied by an activation of caspase-9 and caspase-3.57À59 However, other tubulin-targeting compounds were found to induce apoptosis in human cancer cells via the activation of caspase-8 and -2 rather than caspase-9, suggesting the extrinsic pathway as a primary apoptotic mechanism.60À63 Whether this difference is cell-spe- cific or dependent on the compound remains unclear. Because numerous malignancies are characterized by molecular defects in the apoptotic pathways,54 the investigation of apoptotic mechan- isms is of particular importance. There are a few data concerning the participation of effector caspases-3 and -7 in apoptosis caused by PT derivatives.64À66 For 4-aza-PTs, caspase-3 activation in Jurkat and A549 cells has been described.16,22,23 Up to now there is no evidence on the involve- ment of apical caspases-9 and -2 in cell death caused by PT derivatives. In the present study, the details of apoptosis induc- tion by compound 4a found to be the most cytotoxic against Figure 6. Jurkat cells treated with PT or 8e: flow cytometry assessment of hypodyploid cells (A), cells with active form of caspase-3 (B), and with cleaved PARP (C). (a) Control (untreated cells); (b) PT, 50 nM, 48 h; (c) 8e, 50 nM, 48 d. In (d), cells were pretreated with caspase inhibitor z-VAD- FMK (50 μM) for 2 h, followed by the incubation with 8e as in (c). M1 comprises the hypodiploid cells (A), cells with active form of caspase-3 (B), and cleaved PARP-positive cells (C) with corresponding percentages given in each plot.
  • 8. H dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE A549 cells, and 8e, the most potent in the sea urchin embryo assay, were studied in A549 and Jurkat cells, respectively. The proapoptotic activity of compound 8e was examined in Jurkat cells. Apoptosis induction, cell cycle distribution, expres- sion of active effector caspase-3 in cells, and cleavage of PARP were assessed quantitatively by flow cytometry. Procaspase-8 and the active form of apical caspase-2 and -9 were analyzed by Western blot. As evidenced by the flow cytometric assay, after 48 h, both 8e and PT increased the hypodiploid subpopulation peak (sub-G1) indicative of apoptosis from 12% (control) to 55% of the total cell population (Figure 6A). To confirm the involve- ment of caspases in the 8e-induced Jurkat cell death, an effect of pan-caspase inhibitor Z-VAD-fmk67 has been examined. Jurkat cells were preincubated with Z-VAD-fmk at 50 μM for 2 h, followed by incubation with 50 nM of 8e for additional 48 h. Pretreatment with Z-VAD-fmk decreased the content of apop- totic cells from 55% to 28% (Figure 6A), suggesting that cell death induced by 8e was related to caspase activation. Approxi- mately 30% of cells treated with 8e or PT possessed active form of caspase-3, as compared to 7% in untreated cells (Figure 6B). Cleaved PARP, the product resulting from caspase-3 activation, has been identified in ca. 40% of the cells treated by 8e or PT (Figure 6C). Activation of caspases-9, -2, and -8 in cells treated with 8e was further analyzed by Western blot. The exposure of Jurkat cells to 8e resulted in the processing of procaspase-9 and -2, as evidenced by the appearance of their active forms (Figure 7A,B). The intensities of bands corresponding to active forms of caspase-9 and -2 upon 24 h of treatment with 8e or PT were higher than those at 48 h, while the intensity of procaspase-8 55/57 kDa traces was unaltered (Figure 7C), suggesting lack of active caspase-8. As expected, etoposide caused the reduction of procaspase-8 level in Jurkat cells (Figure 7D).68 Finally, the ability of compound 4a to trigger apoptotic and necrotic events in A549 cells was analyzed using double staining with Annexin VÀFITC and 7-AAD (7-amino actinomycin D) dyes. The procedure allows for the differentiation between living (intact) (Annexin VÀFITCÀ /7-ADDÀ ), early apoptotic (Annexin VÀ FITC+ /7-ADDÀ ), late apoptotic (Annexin VÀFITC+ /7-ADD+ ), and necrotic cells (Annexin VÀFITCÀ /7-ADD+ ).69,70 Compound 4a as well as nocodazole (positive control) induced a significant increase of early apoptosis and to a lesser degree necrosis without affecting late apoptosis (Figure S2, Supporting Information). The effects of 4a were concentration-independent, suggesting that this compound might trigger apoptosis and necrosis even at lower concentrations. 4a was more potent apoptotis- and necrosis-indu- cing agent than nocodazole. On the basis of these data, we concluded that compounds 4a and 8e caused G2/M cell cycle arrest and apoptotic response in A549 and Jurkat human cancer cells, notably, that in Jurkat cells 8e as well as PT induced caspase-dependent apoptosis mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic pathway. PT-Related Compounds: Mode of Action. In the sea urchin embryoassay, deathofarrested eggs exposedtotubulindestabilizing agents was observed after 6À8 h of treatment with a test article. It has been suggested that apoptosis never occurs during sea urchin embryo cleavage, first appearing only at blastula stage just before hatching.71 This explains the absence ofquick cell divisionarrest and death at cleavage after treatment by various DNA-targeting agents known to trigger apoptosis such as etoposide, aloe-emodin, apigen- in, 5-fluorouracil, and 4-phenylbutyric acid.31 Correspondingly, the formation of multinucleated cells (Figure 1B) typical of tubulin destabilizers is an inherent morphological characteristic of mitotic catastrophe, a cell death different from apoptosis.72 Tubulin destabilizers including colchicine, nocodazole, combre- tastatins, phenstatin, indibulin, dolastatin 15, vinblastin, and others interacting with different binding sites on tubulin caused embryo spinning when applied at the swimming blastula stage. Spinning continued for several hours, followed by slow movements near the bottom of the vessel suggesting embryo viability.31,33,34,73 In con- trast, PT andsomeof4-aza-PTs (4a,e,g,j, 6e, 7e, 8e,h, 12a, and13e) induced sea urchin embryo disintegration and death after spinning independently of compound concentration, probably owing to yet another microtubule-unrelated effect. In addition, compound 140 e caused cleavage alteration at 100 nM, whereas it failed to produce cleavage arrest and embryo spinning, suggesting nontubulin antu- proliferative activity. Similarly, 9e exerted cytotoxic effect against A549 cells without microtubule disruption (Table 1). These results are correlated well with the reported ability of PT derivatives to induce cell death and sub-G1 apoptotic cell accumulation without cell cycle arrest and interphase microtubule damage.10,14 Moreover, in a series of 4-aza-PTs with hetero aryl or benzo hetero aryl system instead of the A and B rings, chemical modifications of the ring E resulted in a significant decrease of antitubulin activity and retention of cytotoxicity.22 A combination of our results and literature data suggests an alternative tubulin-independent mechanism of 4-aza- PTs embryotoxicity that could be evident at postcleavage stages of the sea urchin embryo development. ’ CONCLUSIONS In conclusion, we synthesized a series of 4-aza-PT analogues from the easily accessible plant-derived polyalkoxybenzenes. A developed reaction sequence allows for an expedited synthesis Figure 7. Western blot analysis of procaspase-8, cleaved caspase-2, and caspase-9 in total lysates of Jurkat cells. The lysates treated with PT or 8e were prepared in SDSÀLaemmli sample buffer. Immunoblotting was performed using monoclonal anticaspase-9 (A), anticaspase-2 (B), and antiprocaspase-8 (C, D) antibodies. β-Actin served as the loading control. Lanes: (1, 6) control (untreated cells); (2) PT, 50 nM, 24 h; (3) PT, 50 nM, 48 h; (4) 8e, 50 nM, 24 h; (5) 8e, 50 nM, 48 h; (7) etoposide, 2 μM, 24 h.
  • 9. I dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE of the targeted molecules in 12À86% overall yields from naturally occurring building blocks. The resulting compounds were ex- tensively evaluated in both phenotypic sea urchin embryo and cellular assays linking their observed cytotoxicity to a potent antimitotic tubulin destabilizing effect. Notably, the phenotypic sea urchin embryo assay allowed for rapid and reproducible identification of antitubulin molecules. These compounds were further reconfirmed as potent antimitotic agents by a panel of conventional cell-based assays. The most potent representative in each structural group featured myristicin-derived ring E (4e, 6e, 8e, 11e, and 13e), as evidenced by the in vivo sea urchin embryo assay. The activity of these new compounds compares favorably to the reported antimitotic agents containing myristi- cin-derived pharmacophore including corresponding phenstatin analogue and combretastatin A-2.25,73 The ring B modification yielded 6-methoxy substituted molecule 8e as the most active compound. Compound 4a featuring 3,4,5- trimethoxybenzene substituent was the most cytotoxic against A549 human lung epithelial carcinoma cells, being more potent than the parent PT. The activity of 4-aza-PT derivatives correlated well between the sea urchin phenotypic and conventional cell-based assays. In Jurkat cells, both 8e and PT induced caspase-dependent apop- tosis mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic pathway. The structureÀactivity data reported in this paper warrant further synthetic studies and biological evaluation of novel heterocyclic myristicine derivatives. Four specific mol- ecules (4e, 7e, 8e, and 8h) have been selected for advanced NCI60 human tumor cell line anticancer drug screen. Their antiproliferative activity will be reported separately. ’ EXPERIMENTAL SECTION Chemistry. Materials and Methods. NMR spectra were col- lected on a Bruker DR-500 instrument [working frequencies of 500.13 MHz (1 H) and 75.47 (13 C)]. Mass spectra were obtained on a Finnigan MAT/INCOS 50 instrument (70 eV) using direct probe injection. Elemental analysis was accomplished with the automated Perkin-Elmer 2400 CHN microanalyzer. HPLC was performed using a Laboratorni Pristroje (Praha, Czech Republic) chromatograph. Ozonolysis was conducted using a custom-designed apparatus (Science and Technology Park, St. Petersburg State Polytechnic University, Russia) equipped with an IR detector of O3 concentration (Japan) and an automated shut- down circuit. The device allowed for the controlled generation of ozone, with a maximal capacity of 10 g of O3 per h from O2. The compound purity has been determined by NMR, HPLC, and elemental analyses. Purity of compounds 40 À150 , 4À13, and 17À19 was determined to be g95%. Isolation of Plant Allylpolyalkoxybenzenes24 . Liquid CO2 extraction of parsley and dill seeds was carried out earlier by Company Karavan Ltd. (Krasnodar, Russia). Allylpolymethoxybenzenes 1bÀe with 98À99% purity were obtained by high-efficiency distillation using a pilot plant device at N. D. Zelinsky Institute of Organic Chemistry, RAS (Moscow, Russia). The seed essential oils of parsley varieties cultivated in Russia contained 70À75% of 1b (var. Sakharnaya), 21% of 1d (var. Slavyanovskaya), and 40À46% of 1e (var. Astra). Indian dill seeds were purchased from Vremya & Co. (St. Petersburg, Russia). The dill seed essential oil contained 30À33% of 1c. General Procedure A for the Synthesis of Quinolines 40 À15036 . A solution of aromatic amine (50 mmol), ethyl 4-chloroa- cetoacetate (50 mmol) and acetic acid (3 mL) in 100 mL of benzene was refluxed for 8 h. The reaction mixture was cooled to room temperature, and the precipitated product was collected by vacuum filtration and washed with EtOH (30 mL). In most cases, the resulting products 3 were obtained with >85% purity as judged by NMR analysis. A solution of corresponding anilinolactone 3 (2 mmol), appropriate aromatic aldehyde (2aÀk) (2.2 mmol), and p-chloranil (2 mmol) in TFA (3 mL) was stirred at room temperature for 24 h. The reaction mixture was quenched with water (50 mL) and extracted with CH2Cl2 (3 Â 20 mL). The organic layers were combined, washed with 5% NaHCO3 (2 Â 20 mL), water (2 Â 20 mL), dried over anhydrous sodium sulfate, and concentrated in vacuum to obtain the crude product. Flash chromatography purification (petroleum ether/EtOAc, 4:1À1:1) af- forded targeted quinolines 40 À150 as a white solid. General Procedure B for the Synthesis of 4-Aza-podo- phyllotoxin Derivatives 4À13. A suspension of quinoline 40 À130 (0.26 mmol) in a glacial AcOH (3.96 mmol, 3 mL) was treated with NaBH3CN (0.78 mmol), and the resulting mixture was stirred for 3 h at room temperature and treated with the ice water (15 mL). The precipitate was filtered, washed with ethanol (2 Â 1 mL), and the product was purified by column chromatography (petroleum ether/ EtOAc, 2:1) to afford the targeted 4-aza-podophyllotoxins 4À13 as a white solid. NMR analysis confirmed the absence of intermediates 40 À130 . General Procedure C for the Synthesis of Dihydropyrido- pyrazole Derivatives 17À19. A mixture of of corresponding pyrazole (1 mmol), tetronic acid 16 (1 mmol), triethylamine (0.05 mL), and appropriate aldehyde (2aÀk) (1 mmol) in EtOH (5 mL) was refluxed for 3 h. The reaction mixture was cooled to +4 °C, and the precipitated product was collected by vacuum filtration and washed with EtOH (2 mL) at 0 °C. Purification by flash chroma- tography (petroleum ether/EtOAc, 4:1À2:1) afforded the targeted 4-aza-dihydropodophylltoxins 17À19 as a white solid. Synthetic and Analytical Data for Selected 4-Aza-podo- phyllotoxins. Data for the other compounds are presented in Supporting Information. 9-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)[1,3]dioxolo[4,5- g]furo[3,4-b]quinolin-8(6H)-one (40 c). The title compound was obtained according to the general procedure A. Yield 27%, mp 241À244 °C. 1 H NMR (DMSO-d6): δ 3.35 (s, 3H, OCH3-60 ), 4.02 (s, 3H, OCH3-70 ), 5.42 (d, J = 15.4 Hz, 1H, OCH2-6), 5.49 (d, J = 15.4 Hz, 1H, OCH2-6), 6.11 and 6.13 (s, 2H, OCH2O-20 ), 6.28 (s, 2H, OCH2O-2), 6.51 (s, 1H, H-40 ), 6.89 (s, 1H, H-10), 7.52 (s, 1H, H-4). EIMS m/z 409 [M]+ (25), 394 (1), 351 (15), 350 (68), 224 (23), 167 (100). Anal. Calcd for C21H15NO8: C, 61.62; H, 3.69; N, 3.42. Found: C, 61.69; H, 3.72; N, 3.36. 9-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)-6,9-dihydro- [1,3]dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one (4c). The ti- tle compound was obtained according to the general procedure B. Yield 26%, mp 295À298 °C. 1 H NMR (DMSO-d6): δ 3.73 (s, 3H, OCH3-60 ), 3.94 (s, 3H, OCH3-70 ), 4.83 (d, J = 15.6 Hz, 1H, OCH2-6), 4.95 (d, J = 15.7 Hz, 1H, OCH2-6), 5.13 (s, 1H, H-9), 5.88 and 5.94 (s, 2H, OCH2O-2), 5.89 and 5.91 (s, 2H, OCH2O-20 ), 6.24 (s, 2H, H-40 ), 6.42 (s, 1H, H-10), 6.49 (s, 1H, H-4), 9.81 (s, 1H, NH-5). EIMS m/z 411 [M]+ (20), 380 (100), 366 (17), 230 (39), 172 (7). Anal. Calcd for C21H17NO8: C, 61.32; H, 4.17; N, 3.40. Found: C, 61.29; H, 4.14; N, 3.44. 9-(7-Methoxy-1,3-benzodioxol-5-yl)-6,9-dihydro[1,3] dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one (4e). The title compound was obtained according to the general procedure B. Yield 55%, mp 308À310 °C. 1 H NMR (DMSO-d6): δ 3.80 (s, 3H, OCH3-70 ), 4.83 (s, 1H, H-9), 4.84 (d, J = 15.7 Hz, 1H, OCH2-6), 4.97 (d, J = 15.7 Hz, 1H, OCH2-6), 5.90 and 5.91 (s, 2H, OCH2O-20 ), 5.90 and 5.96 (s, 2H, OCH2O-2), 6.33 (d, J = 1.4 Hz, 1H, H-40 ), 6.52 (s, 1H, H-10), 6.56 (d, J = 1.4 Hz, 1H, H-60 ), 6.64 (s, 1H, H-4), 9.87 (s, 1H, NH-5). EIMS m/z 381 [M]+ (5), 231 (14), 230 (100), 152 (24), 151 (7). Anal. Calcd
  • 10. J dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE for C20H15NO7: C, 62.99; H, 3.96; N, 3.67. Found: C, 63.06; H, 4.03; N, 3.60. 9-Phenyl-6,9-dihydro[1,3]dioxolo[4,5-g]furo[3,4-b]quin- olin-8(5H)-one (4k). The title compound was obtained according to the general procedure B. Yield 73%, mp 290À293 °C (lit.6 mp 295À298 °C). 1 H NMR (DMSO-d6): δ 4.85 (d, J = 15.7 Hz, 1H, OCH2-6), 4.91 (s, 1H, H-9), 4.94 (d, J = 15.7 Hz, 1H, OCH2-6), 5.89 and 5.95 (s, 2H, OCH2O-2), 6.53 (s, 1H, H-10), 6.56 (s, 1H, H-4), 7.15 (t, J = 7.2 Hz, 1H, H-40 ), 7.19 (d, J = 7.2 Hz, 2H, H-20 ,60 ), 7.26 (d, J = 7.2 Hz, 2H, H-30 ,50 ), 9.87 (s, 1H, NH-5). EIMS m/z 307 [M]+ (20), 230 (21), 78 (12), 77 (100). Anal. Calcd for C18H13NO4: C, 70.35; H, 4.26; N, 4.56. Found: C, 70.29; H, 4.22; N, 4.63. 10-Methoxy-9-(7-methoxy-1,3-benzodioxol-5-yl)-6,9- dihydro[1,3]dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one (5e). The title compound was obtained according to the general procedure B. Yield 26%, mp 321À326 °C. 1 H NMR (DMSO-d6): δ 3.66 (s, 3H, OCH3-10), 3.77 (s, 3H, OCH3-70 ), 4.78 (d, J = 15.7 Hz, 1H, OCH2-6), 4.89 (d, J = 15.7 Hz, 1H, OCH2-6), 4.89 (s, 1H, H-9), 5.89 and 5.96 (s, 2H, OCH2O-2), 5.89 and 5.90 (s, 2H, OCH2O-20 ), 6.22 (d, J = 1.4 Hz, 1H, H-40 ), 6.33 (s, 1H, H-4), 6.42 (d, J = 1.4 Hz, 1H, H-60 ), 9.93 (s, 1H, NH-5). EIMS m/z 411 [M]+ (3), 384 (0.5), 260 (100), 245 (25), 152 (31), 151 (9). Anal. Calcd for C21H17NO8: C, 61.32; H, 4.17; N, 3.40. Found: C, 61.36; H, 4.18; N, 3.38. 6-Methoxy-9-(7-methoxy-1,3-benzodioxol-5-yl)-4,9-dih- ydrofuro[3,4-b]quinolin-1(3H)-one (8e). The title compound was obtained according to the general procedure B. Yield 60%, mp 278À280 °C. 1 H NMR (DMSO-d6): δ 3.70 (s, 3H, OCH3-6), 3.80 (s, 3H, OCH3-70 ), 4.84 (d, J = 15.5 Hz, 1H, OCH2-3), 4.86 (s, 1H, H-9), 4.97 (d, J = 15.5 Hz, 1H, OCH2-3), 5.89 and 5.90 (s, 2H, OCH2O-20 ), 6.31 (d, J = 1.4 Hz, 1H, H-40 ), 6.44 (d, J = 2.5 Hz, H-5), 6.52 (dd, J = 8.6, 2.5 Hz, 1H, H-7), 6.53 (d, J = 1.4 Hz, 1H, H-60 ), 6.99 (d, J = 8.6 Hz, H-8), 9.94 (s, 1H, NH-4). EIMS m/z 367 [M]+ (14), 217 (15), 216 (100), 152 (16), 151 (7). Anal. Calcd for C20H17NO6: C, 65.39; H, 4.66; N, 3.81. Found: C, 65.42; H, 4.69; N, 3.74. 9-(3,5-Dimethoxyphenyl)-6-methoxy-4,9-dihydrofuro- [3,4-b]quinolin-1(3H)-one (8h). The title compound was obtained according to the general procedure B. Yield 43%, mp 229À233 °C (lit.6 mp 254 °C). 1 H NMR (DMSO-d6): δ 3.68 (s, 6H, 2 Â OCH3-30 , 50 ), 3.70 (s, 3H, OCH3-6), 4.85 (d, J = 15.8 Hz, 1H, OCH2-3), 4.85 (s, 1H, H-9), 4.98 (d, J = 15.8 Hz, 1H, OCH2-3), 6.30 (t, J = 2.2 Hz, 1H, H-40 ), 6.32 (d, J = 2.2 Hz, 2H, H-20 ,60 ), 6.45 (d, J = 2.6 Hz, H-5), 6.52 (dd, J = 8.5, 2.6 Hz, 1H, H-7), 6.99 (d, J = 8.5 Hz, H-8), 9.94 (s, 1H, NH-4). EIMS m/z 353 [M]+ (12), 217 (15), 216 (100), 138 (16). Anal. Calcd for C20H19NO5: C, 67.98; H, 5.42; N, 3.96. Found: C, 67.91; H, 5.38; N, 4.04. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-3-methyl-1,4,7, 8-tetrahydro-5H-furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (18b). The title compound was obtained according to the general procedure C. Yield 47%, mp 295À297 °C. 1 H NMR (DMSO-d6): δ 1.83 (s, 3H, CH3-3), 3.69 (s, 3H, OCH3-40 ), 3.71 (s, 3H, OCH3-70 ), 4.78 (d, J = 15.7 Hz, 1H, OCH2-7), 4.85 (d, J = 15.7 Hz, 1H, OCH2-7), 4.99 (s, 1H, H-4), 5.98 and 5.99 (s, 2H, OCH2O-20 ), 6.23 (s, 1H, H-60 ), 10.03 (s, 1H, NH-8), 11.79 (s, 1H, NH-1). EIMS m/z 371 [M]+ (31), 341 (14), 340 (74), 327 (5), 326 (21), 312 (16), 296 (14), 191 (11), 190 (100), 182 (10). Anal. Calcd for C18H17N3O6: C, 58.22; H, 4.61; N, 11.32. Found: C, 58.30; H, 4.68; N, 11.23. Biology. Materials and Methods. Sea Urchin Embryo Assay. Adult sea urchins Paracentrotus lividus were collected from the Mediterranean Sea at the Cyprus coast and kept in an aerated seawater tank. Gametes were obtained by intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered seawater and fertilized by adding drops of a diluted sperm. Embryos were cultured at room temperature under gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered seawater. The embryos were observed with a light microscope Biolam (LOMO, S.-Petersburg, Russia). For treatment with the test com- pounds, 5 mL aliquots of embryo suspension were transferred to 6-well plates and incubated as a monolayer at a concentration up to 2000 embryos/mL. Stock solutions of compounds were prepared in DMSO at 5À10 mM concentrations, followed by a 10-fold dilution with 95% EtOH. This procedure enhanced solubility of the test compounds in the salt-containing medium (seawater), as evidenced by microscopic exam- ination of the samples. The maximal tolerated concentrations of DMSO and EtOH in the in vivo assay were determined to be 0.05% and 1% respectively. Higher concentrations of either DMSO (g0.1%) or EtOH (>1%) caused nonspecific alteration and retardation of the sea urchin embryo development independent of the treatment stage. Podophyllo- toxin (Aldrich) and topoiomerase II inhibitor etoposide (LANS, Ver- opharm, Russia, 20 mg/mL in 96% EtOH) served as reference com- pounds. The antiproliferative activity was assessed by exposing fertilized eggs (8À20 min after fertilization, 43À55 min before the first mitotic cycle completion) to 2-fold decreasing concentrations of the compound. Cleavage alteration and arrest were clearly detected at 2.5À5.5 h after fertilization. The effects were quantitatively estimated as a threshold concentration resulting in cleavage alteration and embryo death before hatching or full mitotic arrest. At these concentrations, all tested tubulin destabilizers caused 100% cleavage alteration and embryo death before hatching, whereas at 2-fold lower concentrations, the compounds failed to produce any effect. For tubulin destabilizing activity, the compounds were tested on free-swimming blastulae just after hatching (9À10 h after fertilization), originated from the same embryo culture. Embryo spin- ning was observed after 15 min to 20 h of treatment, depending on the nature and concentration of the compound. Both spinning and lack of forward movement were interpreted to be the result of the tubulin destabilizing activity of a molecule according to previous studies.31 Video illustrations are available at http://www.chemblock.com). Com- pound substructure search in sea urchin embryo screening database is available free online at http://www.zelinsky.ru. ’ ASSOCIATED CONTENT bS Supporting Information. Effects of compounds 40 À150 and 17À19 on the sea urchin embryo development, experimen- tal details regarding syntheses and analytical data, biological assay methods. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *Phone: +7 (916) 620-9584. Fax: +7 (499) 137-2966. E-mail: vs@zelinsky.ru. ’ ACKNOWLEDGMENT This work was supported by Alexander von Humboldt Foundation (no. 3.4-Fokoop-Rus/1015567, Germany) and a grant from Chemical Block Ltd. ’ ABBREVIATIONS USED PT, podophyllotoxin;SAR, structureÀactivity relationship;7-AAD, 7-amino actinomycin D;PARP, poly ADP-ribose polymerase ’ REFERENCES (1) Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.; Knossow, M. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 2004, 428, 198–202.
  • 11. K dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE (2) Gupta, S.; Das, L.; Datta, A. B.; Poddar, A.; Janik, M. E.; Bhattacharyya, B. Oxalone and lactone moieties of podophyllotoxin exhibit properties of both the B and C rings of colchicine in its binding with tubulin. Biochemistry 2006, 45, 6467–6475. (3) Desbene, S.; Giorgi-Renault, S. Drugs that inhibit tubulin polymerization: The particular case of podophyllotoxin and analogues. Curr. Med. Chem.: Anti-Cancer Agents 2002, 2, 71–90. (4) Chen, S.-W.; Wang, Y.-H.; Jin, Y.; Tian, X.; Zheng, Y.-T.; Luo, D.-Q.; Tu, Y.-Q. Synthesis and anti-HIV-1 activities of novel podophyl- lotoxin derivatives. Bioorg. Med. Chem. Lett. 2007, 17, 2091–2095. (5) Saitoh, T.; Kuramochi, K.; Imai, T.; Takata, K.; Takehara, M.; Kobayashi, S.; Sakaguchia, K.; Sugawara, F. Podophyllotoxin directly binds a hinge domain in E2 of HPV and inhibits an E2/E7 interaction in vitro. Bioorg. Med. Chem. 2008, 16, 5815–5825. (6) Frackenpohl, J.; Adelt, I.; Antonicek, H.; Arnold, C.; Behrmann, P.; Blaha, N.; B€ohmer, J.; Gutbrod, O.; Hanke, R.; Hohmann, S.; van Houtdreve, M.; Losel, P.; Malsam, O.; Melchers, M.; Neufert, V.; Peschel, E.; Reckmann, U.; Schenke, T.; Thiesen, H.-P.; Velten, R.; Vogelsang, K.; Weiss, H.-C. Insecticidal heterolignans—tubuline po- lymerization inhibitors with activity against chewing pests. Bioorg. Med. Chem. 2009, 17, 4160–4184. (7) Xu, H.; Lv, M.; Tian, X. A review on hemisynthesis, biosynthesis, biological activities, mode of action, and structureÀactivity relationship of podophyllotoxins: 2003À2007. Curr. Med. Chem. 2009, 16, 327–349. (8) Xu, H.; Xiao, X. Natural products-based insecticidal agents 4. Semisynthesis and insecticidal activity of novel esters of 2-chloropodo- phyllotoxin against Mythimna separata Walker in vivo. Bioorg. Med. Chem. Lett. 2009, 19, 5415–5418. (9) Xu, H.; He, X.-Q. Natural products-based insecticidal agents 6. Design, semisynthesis, and insecticidal activity of novel monomethyl phthalate derivatives of podophyllotoxin against Mythimna separata Walker in vivo. Bioorg. Med. Chem. Lett. 2010, 20, 4503–4506. (10) Damayanthi, Y.; Lown, J. W. Podophyllotoxins: current status and recent developments. Curr. Med. Chem. 1998, 5, 205–252. (11) Canel, C.; Moraes, R. M.; Dayan, F. E.; Ferreira, D. Podophyl- lotoxin. Phytochemistry 2000, 54, 115–120. (12) Meresse,P.;Dechaux,E.;Monneret,C.;Bertounesque,E.Etoposide: discovery and medicinal chemistry. Curr. Med. Chem. 2004, 11, 2443–2466. (13) Duca, M.; Guianvarc’h, D.; Meresse, P.; Bertounesque, E.; Dauzonne, D.; Kraus-Berthier, L.; Thirot, S.; Leonce, S.; Pierre, A.; Pfeiffer, B.; Renard, P.; Arimondo, P. B.; Monneret, C. Synthesis and biological study of a new series of 40 -demethylepipodophyllotoxin derivatives. J. Med. Chem. 2005, 48, 593–603. (14) Castro, M. A.; del Corral, J. M. M.; Garcia, P. A.; Rojo, M. V.; de la Iglesia-Vicente, J.; Mollinedo, F.; Cuevas, C.; San Feliciano, A. Synthesis and biological evaluation of new podophyllic aldehyde derivatives with cytotoxic and apoptosis-inducing activities. J. Med. Chem. 2010, 53, 983–993. (15) Magedov, I. V.; Manpadi, M.; Rozhkova, E.; Przheval’skii, N. M.; Rogelj, S.; Shors, S. T.; Steelant, W. F.; Van Slambrouck, S.; Kornienko, A. Structural simplification of bioactive natural products with multicomponent synthesis: dihydropyridopyrazole analogues of podophyllotoxin. Bioorg. Med. Chem. Lett. 2007, 17, 1381–1385. (16) Magedov, I. V.; Manpadi, M.; Slambrouck, S. V.; Steelant, W. F.; Rozhkova, E.; Przheval’skii, N. M.; Rogelj, S.; Kornienko, A. Discovery and investigation of antiproliferative and apoptosis-inducing properties of new heterocyclic podophyllotoxin analogues accessible by a one-step multi- component synthesis. J. Med. Chem. 2007, 50, 5183–5192. (17) Shi, C.; Wang, J.; Chen, H.; Shi, D. Regioselective synthesis and in vitro anticancer activity of 4-aza-podophyllotoxin derivatives cata- lyzed by L-proline. J. Comb. Chem. 2010, 12, 430–434. (18) Hitotsuyanagi, Y.; Kobayashi, M.; Morita, H.; Itokawa, H.; Takeya, K. Synthesis of (À)-4-aza-4-deoxypodophyllotoxin from (À)- podophyllotoxin. Tetrahedron Lett. 1999, 40, 9107–9110. (19) Hitotsuyanagi, Y.; Fukuyo, M.; Tsuda, K.; Kobayashi, M.; Ozeki, A.; Itokawa, H.; Takeya, K. 4-Aza-2,3-dehydro-4-deoxypodophyllotoxins: simple aza-podophyllotoxin analogues possessing potent cytotoxicity. Bioorg. Med. Chem. Lett. 2000, 10, 315–317. (20) Husson, H.-P.; Giorgi-Renault, S.; Tratrat, C.; Atassi, G.; Pierre, A.; Renard, P.; Pfeiffer, B. Dihydrofuro[3,4-b]quinolin-1-one com- pounds. U.S. Patent 6,548,515, 2003. (21) Labruere, R.; Gautier, B.; Testud, M.; Seguin, J.; Lenoir, C.; Desbene-Finck, S.; Helissey, P.; Garbay, C.; Chabot, G. G.; Vidal, M.; Giorgi-Renault, S. Design, synthesis, and biological evaluation of the first podophyllotoxin analogues as potential vascular-disrupting agents. ChemMedChem. 2010, 5, 2016–2025. (22) Kamal, A.; Suresh, P.; Mallareddy, A.; Kumar, B. A.; Reddy, P. V.; Raju, P.; Tamboli, J. R.; Shaik, T. B.; Jain, N.; Kalivendi, S. V. Synthesis of a new 4-aza-2,3-didehydropodophyllotoxin analogues as potent cytotoxicand antimitotic agents. Bioorg. Med. Chem. 2011, 19, 2349–2358. (23) Magedov, I. V.; Frolova, L.; Manpadi, M.; Bhoga, U. D.; Tang, H.; Evdokimov, N. M.; George, O.; Georgiou, K. H.; Renner, S.; Getlik, M.; Kinnibrugh, T. L.; Fernandes, M. A.; van Slambrouck, S.; Steelant, W. F. A.; Shuster, C. B.; Rogelj, S.; van Otterlo, W. A. L.; Kornienko, A. Anticancer properties of an important drug lead podophyllotoxin can be efficiently mimicked by diverse heterocyclic scaffolds accessible via one- step synthesis. J. Med. Chem. 2011, 54, 4234–4246. (24) Semenov, V. V.; Rusak, V. A.; Chartov, E. M.; Zaretsky, M. I.; Konyushkin, L. D.; Firgang, S. I.; Chizhov, A. O.; Elkin, V. V.; Latin, N. N.; Bonashek, V. M.; Stas’eva, O. N. Polyalkoxybenzenes from plant raw materials 1. Isolation of polyalkoxybenzenes from CO2 extracts of Umbelliferae plant seeds. Russ. Chem. Bull. 2007, 56, 2448–2455. (25) Semenov, V. V.; Kiselyov, A. S.; Titov, I. Y.; Sagamanova, I. K.; Ikizalp, N. N.; Chernysheva, N. B.; Tsyganov, D. V.; Konyushkin, L. D.; Firgang, S. I.; Semenov, R. V.; Karmanova, I. B.; Raihstat, M. M.; Semenova, M. N. Synthesis of antimitotic polyalkoxyphenyl derivatives of combretas- tatinusing plant allylpolyalkoxybenzenes.J.Nat.Prod.2010, 73, 1796–1802. (26) (a) Petcher, T. J.; Weber, H. P.; Kuhn, M.; Von Wartburg, A. Crystal structure and absolute configuration of 20 -bromopodophyllo- toxin-0.5 ethyl acetate. J. Chem. Soc., Perkin Trans. 2 1973, 2, 288–292. (b) Rithner, C. D.; Bushweller, C. H.; Gender, W. J.; Hoogasian, S. Dynamic nuclear magnetic resonance and empirical force field studies of podophyllotoxin. J. Org. Chem. 1983, 48, 1491–1495. (27) Shulgin, A. T. Tinkal: The Continuation; Transform Press: Berkeley, CA, 1977; p 646. (28) Lichius, J. J.; Thoison, O.; Montagnac, A.; Pais, M.; Gueritte- Voegelein, F.; Sevenet, T.; Cosson, J. P.; Hadi, A. H. Antimitotic and cytotoxic flavonols from Zieridium pseudobtusifolium and Acronychia porteri. J. Nat. Prod. 1994, 57, 1012–1016. (29) Walle, T. Methoxylated flavones, a superior cancer chemopre- ventive flavonoid subclass? Semin Cancer Biol. 2007, 17, 354–362. (30) Quintin, J.; Buisson, D.; Thoret, S.; Cresteil, T.; Lewin, G. Semisynthesis and antiproliferative evaluation of a series of 30 -amino- flavones. Bioorg. Med. Chem. Lett. 2009, 19, 3502–3506. (31) Semenova, M. N; Kiselyov, A. S.; Semenov, V. V. Sea urchin embryo as a model organism for the rapid functional screening of tubulin modulators. BioTechniques 2006, 40, 765–774. (32) Semenova, M. N.; Tsyganov, D. V.; Yakubov, A. P.; Kiselyov, A. S.; Semenov, V. V. A synthetic derivative of plant allylpolyalkox- ybenzenes induces selective loss of motile cilia in sea urchin embryos. ACS Chem. Biol. 2008, 3, 95À100. (33) Semenova, M. N.; Kiselyov, A. S.; Titov, I. Y.; Raihstat, M. M.; Molodtsov, M.; Grishchuk, E.; Spiridonov, I.; Semenov, V. V. In vivo evaluation of indolyl glyoxamides in the sea urchin embryo model: correlation with in vitro tubulin dynamics effects. Chem. Biol. Drug Des. 2007, 70, 485–490. (34) Kiselyov, A. S.; Semenova, M. N.; Chernyshova, N. B.; Leitao, A.; Samet, A. V.; Kislyi, K. A.; Raihstat, M. M.; Oprea, T.; Lemcke, T.; Lantowe, M.; Weiss, D. G.; Ikizalp, N. N.; Kuznetsov, S. A.; Semenov, V. V. Novel derivatives of 1,3,4-oxadiazoles are potent mitostatic agents featuring strong microtubule depolymerizing activity in the sea urchin embryo and cell culture assays. Eur. J. Med. Chem. 2010, 45, 1683–1697. (35) Schmidt, D. G.; Seemuth, P. D.; Zimmer, H. Substituted .gamma.-butyrolactones.Part31.2,4(3H,5H)-Furandione:heteroannulations with aromatic o-amino carbonyl compounds and condensations with some vic-polyones. J. Org. Chem. 1983, 48, 1914–1916.
  • 12. L dx.doi.org/10.1021/jm200737s |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE (36) Hitotsuyanagi, Y.; Kobayashi, M.; Fukuyo, M.; Takeya, K.; Itokawa, H. A facile synthesis of the 4-aza-analogs of 1-arylnaphthalene lignans chinensin, justicidin B, and taiwanin C. Tetrahedron Lett. 1997, 38, 8295–8296. (37) Tratrat, C.; Giorgi-Renault, S.; Husson, H.-P. A multicompo- nent reaction for the one-pot synthesis of 4-aza-2,3-didehydropodo- phyllotoxin and derivatives. Org. Lett. 2002, 4, 3187–3189. (38) Ogiku, T.; Yoshida, S.; Ohmizu, H.; Iwasaki, T. Efficient syntheses of 1-arylnaphthalene lignan lactones and related compounds from cyanohydrins. J. Org. Chem. 1995, 60, 4585–4590. (39) Cow, C.; Leung, C.; Charlton, J. L. Antiviral activity of aryln- aphthalene and aryldihydronaphthalene lignans. Can. J. Chem. 2000, 78, 553–561. (40) Ban, H. S.; Lee, S.; Kim, Y. P.; Yamaki, K.; Shin, K. H.; Ohuchi, K. Inhibition of prostaglandin E(2) production by taiwanin C isolated from the root of Acanthopanax chiisanensis and the mechanism of action. Biochem. Pharmacol. 2002, 64, 1345–1354. (41) Chang, S.-T.; Sheng-Yang Wang, S.-Y.; Yueh-Hsiung Kuo, Y.-H. Resources and bioactive substances from Taiwania (Taiwania cryptomerioides). J. Wood Sci. 2003, 49, 1–4. (42) Foley, P.; Eghbali, N.; Anastas, P. T. Silver-catalyzed one-pot synthesis of arylnaphthalene lactone natural products. J. Nat. Prod. 2010, 73, 811–813. (43) Momose, T.; Toyooka, N.; Takeuchi, Y. A laboratory synthesis of 4-hydroxy-2(5H)-furanone (β-tetronic acid). Heterocycles 1986, 24, 1429–1431. (44) Jin, Y.; Chen, S. W.; Tian, X. Synthesis and biological evaluation of new spin-labeled derivatives of podophyllotoxin. Bioorg. Med. Chem. 2006, 14, 3062–3068. (45) Cisney, M. E.; Shilling, W. L.; Hearon, W. M.; Goheen, D. W. Conidendrin. II. The stereochemistry and reactions of the lactone ring. J. Am. Chem. Soc. 1954, 76, 5083–5087. (46) Dantzig, A.; LaLonde, R. T.; Ramdayal, F.; Shepard, R. L.; Yanai, K.; Zhang, M. Cytotoxic responses to aromatic ring and config- urational variations in alpha-conidendrin, podophyllotoxin, and sikki- motoxin derivatives. J. Med. Chem. 2001, 44, 180–185. (47) Berkowitz, D. B.; Maeng, J.-H.; Dantzig, A. H.; Shepard, R. L.; Norman, B. H. Chemoenzymatic and ring E-modular approach to the (À)-podophyllotoxin skeleton. Synthesis of 30 ,40 ,50 -tridemethoxy-(À)- podophyllotoxin. J. Am. Chem. Soc. 1996, 118, 9426–9427. (48) Goldbrunner, M.; Loidl, G.; Polossek, T.; Mannschreck, A.; von Angerer, E. Inhibition of tubulin polymerization by 5,6-dihydroindolo- [2,1-alpha]isoquinoline derivatives. J. Med. Chem. 1997, 40, 3524–3533. (49) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. (50) Bhalla, K. N. Microtubule-targeted anticancer agents and apoptosis. Oncogene 2003, 22, 9075–9086. (51) Mollinedo, F.; Gajate, C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis 2003, 8, 413–450. (52) Gascoigne, K. E.; Taylor, S. S. How do anti-mitotic drugs kill cancer cells? J Cell Sci. 2009, 122, 2579–2585. (53) Pop, C.; Salvesen, G. S. Human caspases: activation, specificity, and regulation. J. Biol. Chem. 2009, 284, 21777–21781. (54) Ghobrial, I. M.; Witzig, T. E.; Adjei, A. A. Targeting apoptosis pathways in cancer therapy. Cancer J. Clin. 2005, 55, 178–194. (55) Inoue, S.; Browne, G.; Melino, G.; Cohen, G. M. Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway. Cell Death Differ. 2009, 16, 1053–1061. (56) Viola, G.; Cecconet, L.; Leszl, A.; Basso, G.; Brun, P.; Salvador, A.; Dall’Acqua, F.; Diana, P.; Barraja, P.; Cirrincione, G. Pyrrolotetra- zinones deazaanalogues of Temozolomide induce apoptosis in Jurkat cell line: involvement of tubulin polymerization inhibition. Cancer Chemother. Pharm. 2009, 64, 1235–1251. (57) Beswick, R. W.; Ambrose, H. E.; Wagner, S. D. Nocodazole, a microtubule depolymerising agent, induces apoptosis of chronic lym- phocytic leukaemia cells associated with changes in Bcl-2 phosphoryla- tion and expression. Leukemia Res. 2006, 30, 427–436. (58) Vitale, I.; Antoccia, A.; Cenciarelli, C.; Crateri, P.; Meschini, S.; Arancia, G.; Pisano, C.; Tanzarella, C. Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells. Apoptosis 2007, 12, 155–166. (59) Romagnoli, R.; Baraldi, P. G.; Cruz-Lopez, O.; Cara, C. L.; Carrion, M. D.; Brancale, A.; Hamel, E.; Chen, L.; Bortolozzi, R.; Basso, G.; Viola, G. Synthesis and antitumor ativity of 1,5-disubstituted 1,2,4- triazoles as cis-restricted combretastatin analogues. J. Med. Chem. 2010, 53, 4248–4258. (60) Chen, Y.-C.; Lu, P.-H.; Pan, S. L.; Teng, C.-M.; Kuo, S.-C.; Lin, T.-P.; Ho, Y.-F.; Huang, Y.-C.; Guh, J.-H. Quinolone analogue inhibits tubulin polymerization and induces apoptosis via Cdk1-involved signal- ing pathways. Biochem. Pharmacol. 2007, 74, 10–19. (61) McElligott, A. M.; Maginn, E. N.; Greene, L. M.; Siobhan McGuckin, S.; Hayat, A.; Browne, P. V.; Butini, S.; Campiani, G.; Catherwood, M. A.; Vandenberghe, E.; Williams, D. C.; Zisterer, D. M.; Mark Lawler, M. The novel tubulin-targeting agent pyrrolo- 1,5-benzoxazepine-15 induces apoptosis in poor prognostic subgroups of chronic lymphocytic leukemia. Cancer Res. 2009, 69, 8366–8375. (62) Hsieh, C.-C.; Kuo, Y.-H.; Kuo, C.-C.; Chen, L.-T.; Cheung, C.-H. A.; Chao, T.-Y.; Lin, C.-H.; Pan, W.-Y.; Chang, C.-Y.; Chien, S.-C.; Chen, T.-W.; Lung, C.-C.; Chang, J.-Y. Chamaecypanone C, a novel skeleton microtubule inhibitor, with anticancer activity by trigger caspase 8-Fas/FasL dependent apoptotic pathway in human cancer cells. Biochem. Pharmacol. 2010, 79, 1261–1271. (63) Maginn, E. N.; Browne, P. V.; Hayden, P.; Vandenberghe, E.; MacDonagh, B.; Evans, P.; Goodyer, M.; Tewari, P.; Campiani, G.; Butini, S.; Williams, D. C.; Zisterer, D. M.; Lawler, M. P.; McElligott, A. M. PBOX-15, a novel microtubule targeting agent, induces apoptosis, upregulates death receptors, and potentiates TRAIL-mediated apoptosis in multiple myeloma cells. Br. J. Cancer 2011, 104, 281–289. (64) Qi, Y.; Liao, F.; Zhao, C.; Lin, Y.; Zuo, M. Cytotoxicity, apoptosis induction, and mitotic arrest by a novel podophyllotoxin glucoside, 4DPG, in tumor cells. Acta Pharmacol. Sin. 2005, 26, 1000–1008. (65) Yong, Y.; Shin, S. Y.; Lee, Y. H.; Lim, Y. Antitumor activity of deoxypodophyllotoxin isolated from Anthriscus sylvestris: Induction of G2/M cell cycle arrest and caspase-dependent apoptosis. Bioorg. Med. Chem. Lett. 2009, 19, 4367–4371. (66) Shin, S. Y.; Yong, Y.; Kim, C. G.; Lee, Y. H.; Lim, Y. Deoxypodophyllotoxin induces G2/M cell cycle arrest and apoptosis in HeLa cells. Cancer Lett. 2010, 287, 231–239. (67) Ko, S. C.; Johnson, V. L.; Chow, S. C. Functional characterization of Jurkat T cells rescued from CD95/Fas-induced apoptosis through the inhibitionofcaspases.Biochem.Biophys.Res.Commun.2000,270,1009–1015. (68) Sohn, D.; Schulze-Setoff, K.; J€anicke, R. U. Caspase-8 can be activated by interchain proteolysis without receptor-triggered dimerization during drug-induced apoptosis. J. Biol. Chem. 2005, 280, 5267–5273. (69) Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phospha- tidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 1995, 184, 39–51. (70) Koopman, G.; Reutelingsperger, C. P.; Kuijten, G. A.; Keehnen, R. M.; Pals, S. T.; van Oers, M. H. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994, 84, 1415–1420. (71) Thurber, R. V.; Epel, D. Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus. Dev. Biol. 2007, 303, 336–346. (72) Castedo, M.; Perfettini, J. L.; Roumier, T.; Andreau, K.; Medema, R.; Kroemer, G. Cell death by mitotic catastrophe: a molecular definition. Oncogene 2004, 23, 2825–2837. (73) Titov, I. Y.; Sagamanova, I. K.; Gritsenko, R. T.; Karmanova, I. B.; Atamanenko, O. P.; Semenova, M. N.; Semenov, V. V. Bioorg. Med. Chem. Lett. 2011, 21, 1578–1581.

Related Documents