Spirooxindoles: Promising scaffolds for anticancer agents

European Journal of Medicinal Chemistry 97 (2015) 673e698

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Review article

Spirooxindoles: Promising scaffolds for anticancer agents Bin Yu, De-Quan Yu*, Hong-Min Liu* School of Pharmaceutical Sciences and New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450001, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 May 2014 Received in revised form 20 June 2014 Accepted 25 June 2014 Available online 27 June 2014

The search for novel anticancer agents with more selectivity and lower toxicity continues to be an area of intensive investigation. The unique structural features of spirooxindoles together with diverse biological activities have made them privileged structures in new drug discovery. Among them, spiro-pyrrolidinyl oxindoles have been extensively studied as potent inhibitors of p53eMDM2 interaction, finally leading to the identification of MI-888, which could achieve rapid, complete and durable tumor regression in xenograft models of human cancer with oral administration and is in advanced preclinical research for cancer therapy. This review highlights recent progress of biologically active spirooxindoles for their anticancer potentials, mainly focusing on the discussions of SARs and modes of action. This article also aims to discuss potential further directions on the development of more potent analogues for cancer therapy. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Spirooxindoles Oxindole Synthesis Cancer therapy

1. Introduction Carcinogenesis is a highly complex multi-step process induced by a number of carcinogens which leads to the development of cancer [1]. In cancer, cells divide and grow uncontrollably, forming malignant tumors, which may invade nearby normal cells, even spread to more distant parts of the body through the lymphatic system or bloodstream. Data from the international agency for research on Cancer GLOBOCAN database and also the World Health Organization Global Health Observatory and the United Nations World Population Prospects report showed that an estimated 14.1 million new cancer cases occurred with 8.2 million deaths in 2012 [2]. Many natural and also synthetic anticancer agents such as paclitaxel and cisplatin are available in the market and some are in clinical trials. However, these drugs are always associated with serious side effects such as bone marrow depression, alopecia and nephrotoxicity owing to their non-selective action. Although some antiproliferative drugs like tamoxifen are highly selective to cancer cells. These agents are not very effective to kill the existing tumor cells. More importantly, their prolong use may develop uterine and endometrial cancers [3]. Hence, developing the new therapeutic drugs for cancer treatment is much more important to improve the efficiency and efficacy of the drugs on the cancer cells.

* Corresponding authors. E-mail addresses: [email protected] (B. Yu), [email protected] (D.-Q. Yu), [email protected] (H.-M. Liu). http://dx.doi.org/10.1016/j.ejmech.2014.06.056 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

Spiro compounds have always been prevalent in organic synthesis due to the pronounced biological activities [4]. In particular, the spirocyclic oxindoles have emerged as attractive synthetic targets because of their prevalence in numerous natural products and biologically active molecules [5e7]. The key structural characteristic of these compounds is the spiro ring fused at the C3 position of the oxindole core with varied heterocyclic motifs (Fig. 1). These spirooxindoles seem to be promising candidates for drug discovery, since they incorporate both oxindoles and other heterocyclic moieties simultaneously. Two representative examples are NITD609 (1) and MI-888 (2) (Fig. 2), which are currently in preclinical evaluation for the treatment of malaria and human cancer, respectively. NITD609 rapidly inhibits protein synthesis in Plasmodium falciparum, an effect that is ablated in parasites bearing nonsynonymous mutations in the gene encoding the P-type cation-transporter ATPase4 [8]. MI-888, as a potent inhibitor of p53eMDM2 interaction (Ki ¼ 0.44 nM), is capable of achieving rapid, complete, and durable tumor regression in two types of xenograft models of human cancer with oral administration [9]. Spirooxindoles have continued to gain attention in recent years, to better show this trend, we made a statistic analysis about the number of publications regarding spirooxindoles (Fig. 3). The number of publications has continued to increase over the last decade, with the most significant increase occurring in 2011e12 (531 reports published). Besides, these publications were also classified based on the spiro ring fused at C3 position of oxindole core (Fig. 4). It is evident that most of these publications were about those with 5 or 6-membered ring fused at C3 position (1112 and 464 publications, respectively; ‘A’ denotes any atom in the ring).

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Fig. 1. Scaffold of spirocyclic oxindoles. Fig. 3. A decade of spirooxindoles.

This review is organized based on the type of ring fused at C3 position of oxindole core and mainly focuses on the discussions of structureeactivity relationships (SARs) and modes of action. The chemical synthesis of spirooxindoles is beyond the scope of this review although the general synthetic strategies are briefly given below. 2. Chemistry The synthesis, especially asymmetric synthesis of spirooxindoles has always been a research field of great interest due to their biological activity and applications for pharmaceutical lead discovery. A number of methods have been developed for the synthesis of such compounds and the chemistry was comprehensively reviewed by several groups [4-7,10-15]. The general synthetic strategies are summarized in Fig. 5. (1) Reactions based on 1, 3-dipolar cycloaddition (also known as [3 þ 2] cycloaddition). The azomethine ylides (1, 3dipoles), which are always generated in situ from isatinderived compounds and a-amino acids through the thermal decarboxylation, react with activated alkenes (dipolarophiles), giving pyrrolidine-containing spirooxindoles with high regioselectivity and stereoselectivity (Method A). Alternatively, cycloaddition can also be fulfilled between azomethine ylides and electron-withdrawing Knoevenagel alkenes originated from isatin (Method B). (2) Reactions based on domino KnoevenageleMichaelecyclization. Due to the high reactivity of the C3 carbonyl group in isatin, the Knoevenagel condensations always occur in the presence of nucleophilic methylene substrates, forming corresponding Knoevenagel alkenes. These electrondeficient alkenes (Michael acceptors) are capable of initiating further domino Michaelecyclization (Method C). (3) Reactions based on Michaelecyclization of isatins. Due to the high reactivity of the C3 carbonyl group, nucleophilic attack to the C3 carbonyl group of isatin, followed by further cyclization gives the oxo-spirooxindoles (Method D). (4) Reactions based on C3 aminalization. Aminalization of isatins at C3 position leads to corresponding aminals, which

Fig. 2. Two representative examples of spirooxindoles NITD609 and MI-888.

are subjected to further transformation to give nitrogencontaining spirooxindoles (Method E).

3. Spirooxindoles with anticancer activities A number of spirooxindoles have shown different degrees of anticancer activities mainly based on the spiro rings fused at the C3 position of oxindole scaffold and substituents on the oxindole nucleus. Among these, many natural spirooxindoles like Spirotryprostatins A and B also show excellent anticancer activities [16]. More importantly, some synthesized spirooxindoles such as MI888 (Fig. 2) have been in preclinical research for the treatment of human cancers [9]. All these have inspired more medicinal chemists to design analogues to study SARs and modes of action. To help researchers gain a deep insight into SARs and mechanisms of such compounds, this review gives the detailed discussions of SARs and mechanisms reported. 3.1. Spiro-pyrrolidinyl oxindoles Two novel diketopiperazine alkaloids (Spirotryprostatins A and B, Fig. 6) were first isolated from the secondary metabolites of Aspergillus fumigatus [16] and synthesized in 1999 [17]. Biological

Fig. 4. Number of reports for each type of spirooxindoles.

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Fig. 5. The general synthetic strategies of spirooxindoles.

evaluation showed that Spirotryprostatins A (3) and B (4) inhibited the cell cycle progression of tsFT210 cells at the G2/M phase with IC50 values of 197.5 mM and 14.0 mM, respectively. Further evaluation indicated that Spirotryprostatin A and its analogues 5 and 9 showed little effect towards MCF-7 cells (IC50 > 300 mM) but inhibited the growth of MDA MB-468 cells (IC50 ¼ 110 mM and 85 mM, respectively). Compounds 6e8 were active against MCF-7

(IC50 < 30 nM) and MDA MB-468 cells (IC50 < 100 nM) and more potent than natural Spirotryprostatins A. These findings indicated that the isopropylidene side chain of spirotryprostatin A was not necessary for biological activity. Compound 7 lacking the diketopiperazine system was also quite active as cell cycle inhibitors. Five oxindole alkaloids, namely isopteropodine (10), pteropodine (11), isomitraphylline (12), mitraphylline (14), uncarine F

Fig. 6. Several natural oxindole alkaloids.

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Fig. 7. Oxindole alkaloids isolated from uncaria tomentosa.

(15) (Fig. 7), were obtained from Uncaria tomentosa [18]. Except for mitraphylline, isopteropodine, pteropodine, isomitraphylline and uncarine F inhibited proliferation of acute lymphoblastic leukemia cells (CEM-C7H2 cells). Among them, pteropodine and uncarine F induced apoptosis of CEM-C7H2 cells, which was independent of CD95/Fas signaling and Bcl-2 overexpression. However, pteropodine and uncarine F could not induce the death of G0/G1arrested C7H2-6E2 cells. Although mitraphylline failed to inhibit the growth of CEM-C7H2 cells, mitraphylline could inhibit the growth of neuroblastoma SKN-BE, glioma GAMG [19], human Ewing's sarcoma MHH-ES-1 and breast cancer MT-3 cell lines [20] in a dose-dependent manner (12.3 mM for SKN-BE, 20 mM for GAMG, 17.15 ± 0.82 mM for MHH-ES-1 and 11.80 ± 1.03 mM for MT-3, respectively). From the evidence of different cytotoxicity of mitraphylline and isomitraphylline against CEM-C7H2 cells, it is evident that the stereochemistry has a certain effect on the cytotoxicity. Samuel Kaiser and co-authors reported that cell incubation (buffered medium pH 7.4 at 37  C) could induce the isomerization of oxindole alkaloids, which helped to explain the similar cytotoxic activities of non-isomerized and isomerized oxindole alkaloids against T24 and RT4 human bladder cancer cell lines [21]. Macrophyllionium (15) and Macrophyllines A (16) and B (17), together with four other oxindole alkaloids (18e21) (Fig. 8) were isolated from the aerial parts of Uncaria macrophylla and evaluated for their cytotoxic activities [22]. However, all these oxindole

alkaloids were inactive against HL-60, A549, SMMC-7721, MCF-7 and SW480 cell lines (IC50 > 40 mM). Apart from natural oxindole alkaloids, synthesized spiropyrrolidinyl oxindoles have drawn wide attention as non-peptide small molecule inhibitors of p53eMDM2 interaction for cancer therapy since the structural basis of the p53eMDM2 interaction has been established by X-ray crystallography [23]. X-ray crystal structure analysis showed that the interaction between p53 and MDM2 was primarily mediated by three hydrophobic residues (Phe19, Trp23 and Leu26) of p53 peptide and a small but deep hydrophobic cleft in MDM2. Since the indole ring of the Trp23 residue of p53 is buried deeply inside the hydrophobic cavity in MDM2 and its NH group forms a hydrogen bond with the backbone carbonyl in MDM2, Trp23 appears to be the most critical for binding of p53 to MDM2 [24]. Based on this fact, a library of spirooxindoles as p53eMDM2 inhibitors were initially discovered using a de novo structure based design strategy, two alkaloids (Spirotryprostatin A and Alstonisine) contained the spiro-pyrrolidinyl oxindole core that was used as a scaffold to design possible inhibitors by docking into the MDM2 structure using the GOLD program (Fig. 9) [25]. These spirooxindoles can closely mimic the Trp23 of p53 in both hydrogen-bonding formation and hydrophobic interactions with MDM2, and the spiro-pyrrolidinyl oxindole ring provides a rigid scaffold from which two hydrophobic groups can be projected to mimic the side chain of Phe19 and Leu26.

Fig. 8. Oxindole alkaloids isolated from uncaria macrophylla.

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Fig. 9. Spirotryprostatin and alstonisine-based design of potent inhibitors of p53eMDM2 interaction.

Spirooxindoles 22e28 (Fig. 10) displayed different levels of activity in an FT-based assay with Ki values ranging from 0.086 mM to 8.46 mM (1.52 mM for natural p53 peptide). Compound 22 bond to MDM2 with a Ki value of 8.46 mM due to the fact that it fitted poorly into the binding pocket occupied by the side chain of Phe19 and Leu26, respectively. However, compound 22 provided a template for further optimization. Further modifications focusing on the variations of R2 group and the position of the chlorine atom at the phenyl ring yielded compounds 23e28. Among them, compound 25 showed the best affinity to MDM2 (Ki ¼ 0.086 mM). Docking studies suggested that the chlorooxindole ring in compound 25 occupied the Trp23 pocket, the 3-chlorophenyl group projected into the Phe19 pocket, and the t-butyl group filled the Leu26 pocket. Besides, compound 25 represented excellent inhibition against LNCaP cells with wild-type p53 (IC50 ¼ 0.086 mM) but relatively weak inhibition against human prostate cancer PC-3 cells with a deleted p53 (IC50 ¼ 22.5 mM) and had good selectivity between cancer and normal cells with wild-type p53. Compound 25 had an IC50 value of 10.5 mM in inhibition of normal human prostate epithelial cell growth, 13 times less toxic than to LNCaP cancer cells. Compared to compound 25, compound 28 (known as MI-61, Fig. 10) with a bromo atom at the meta-position of the phenyl ring showed a significantly decreased activity (Ki > 10 mM), indicating that substituent on the oxindole core played an important role in its binding to MDM2 [26]. Although these spirooxindoles achieved high binding affinities to MDM2, they were still significantly less potent than the most potent peptide-based inhibitors probably due to the fact that the additional interaction between MDM2 and peptide-based inhibitors was still not captured by these spirooxindoles. Analysis of the X-ray structure of the MDM2ep53 complex showed that Leu22

Fig. 10. Several spirooxindoles that inhibit p53eMDM2 interaction.

was also crucial in the overall interaction between MDM2 and p53 [23]. Structure-based optimization of this class of compounds as inhibitors of MDM2ep53 interaction was further carried out, generating new effective inhibitors (Fig. 11), which captured the additional interaction between Leu22 in p53 and MDM2 [27]. Ding et al. first designed compound 29, in which the dimethylamine in compound 25 was replaced by a morpholin-4-yl-ethylamine group, a chemical group which has been extensively used in drug design. Compound 29 was determined to bind to MDM2 with a Ki value of 13 nM in a FT-based binding assay. Analysis of the X-ray structure of compound 29eMDM2 complex showed that compound 29 not only mimicked the four hydrophobic residues (Phe19, Leu22, Leu26 and Trp23) in p53 for their interaction with MDM2 but also captured the interaction between Glu17 in p53 and Lys90 in MDM2. Considering that fluorine substitution on a phenyl ring has been frequently used as an effective strategy to increase the metabolic stability of drug molecules, compounds 30e32 with a fluoro substituent at the 2, 4, 5 position of the 40 -(m-chlorophenyl) ring were also designed to test their affinities to MDM2. A fluorine substitution at C4 (compound 31) resulted in a 2-fold reduction in binding affinity to MDM2, but substitution at C2 (compound 30) or C5 (compound 32) improved the binding affinity. With compound 29 as the template, compound 33 (also known as MI-63) was designed and synthesized with a Ki value of 3 nM (12- and 2000-times more potent than Nutlin-3 and the natural p53 peptide, respectively). Besides, compound 35, an enantiomer of 33, had a weak binding to MDM2 (Ki ¼ 4.0 mM), 1000-times less potent than MI-63. This finding suggested the stereospecific binding of MI-63 (33) to MDM2. Compound 34, in which the oxygen atom was replaced by a carbon atom, had a Ki value of 39 nM, 13-times less potent than compound 33, confirming the significant contribution of this oxygen atom for binding to MDM2. Interestingly, MI-63 (33) displayed a more than 10,000-fold specificity for MDM2 protein over Bcl-2/ Bcl-xL proteins. Further investigation showed that compound 33 was 3- and 5-times more potent than compound 29 and Nutlin-3 in the growth assay of LNCaP prostate cancer cells with wild-type p53 with the IC50 values of 280, 800 and 1500 nM, respectively [28]. Synergy was observed when MI-63 was used in combination with doxorubicin [29]. However, for PC-3 cells with deleted p53, MI-63 had an excellent cellular specificity (IC50 ¼ 18 mM) and the minimal toxicity to normal prostate epithelial cells as high as 5 mM. Activation of p53 always resulted in an increase of the levels of p21cip1/waf and MDM2 proteins, two p53 targeted genes [30]. After

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Fig. 11. New inhibitors that capture the interaction between Leu22 in p53 and MDM2.

treatment LNCaP cells with wild-type p53 with compound 33, the levels of p53, MDM2 and p21cip1/waf proteins were increased in a dose-dependent manner. However, compound 35 with a weak binding affinity had a minimal effect. Since MDM2 protein is an E3 ubiquitin ligase and is known to effectively degrade p53 protein upon binding [31], the very high levels of p53 and MDM2 protein in LNCaP cells treated with 33 suggested that MDM2 protein was unable to degrade p53 protein when blocked by 33. Compounds 33 and 35 did not increase the levels of p53 and MDM2 protein in PC-3 cells with deleted p53. As an analog of compound 29, compound 36 (MI-43) was found to induce the accumulation of p53 and downstream target genes (MDM2, p21, Noxa and Puma) in wt p53-containing A549 and H460 cells but was much less effective to p53-null H522 and H1299 cells [26]. Mechanistically, MI-43 induced G0 or G1 arrest and apoptosis in a dose-dependent manner due to p21 and Puma/Noxa induction, respectively. Interestingly, MI-43 was much less toxic to normal fetal lung fibroblast MRC5 cells at low concentration and sensitized A549 cells to etoposide-induced apoptosis. Follow-up research showed that reactivation of p53 by MI-43 led to p21-mediated cell cycle arrest and selective cell death in colon cancer [32]. The fluorescence polarization-based binding assay determined that MI-43 bond to recombinant MDM2 protein with a Ki value of 18 nM (2 and >300-times more potent than Nutlin-3 and a natural p53

Fig. 12. The structure of MI-319.

peptide). Besides, MI-43 failed to show any appreciable binding to Bcl-2/Bcl-xL proteins, indicating >1000-fold specificity for MDM2 over Bcl-2/Bcl-xL proteins. MI-43 bond to MDM2 protein with a Ki value of 18 nM and is 300 times more potent than a native p53 peptide. MI-43 blocked the intracellular MDM2ep53 interaction and induced p53 accumulation in both normal and cancer cells with wild-type p53 without causing p53 phosphorylation. Induction of p53 led to modulation of the expression of p53 target genes, including up-regulation of p21 and MDM2 in normal primary human cells and in colon cancer cells with wild-type p53. The cell growth inhibition and cell death induction by MI-43 was p53dependent. Furthermore, induction of cell cycle arrest by MI-43 was dependent on p53 and p21. In normal cells, MI-43 induced cell cycle arrest but not apoptosis. Although MI-63 (compound 33) bond to MDM2 with high affinity [27] and even effectively induced apoptosis ex vivo in chronic lymphocyte leukemia patient samples with functional p53 [33], MI63 had a poor PK profile and was not suitable for in vivo evaluation, extensive modifications of MI-63 yielded MI-219 (37) with a desirable PK profile (Fig. 11) [34]. Computational modeling predicted that MI-219 mimicked the Phe19, Leu22, Leu26 and Trp23 residues in p53 and achieved optimal interactions with MDM2 (Ki ¼ 5.0 nM, >1000-fold more potent than p53 peptide). MI-219 bond to MDM2 with a high affinity and selectivity (>10, 000-fold more potent for MDM2 over MDMX, a homolog of MDM2). The binding affinity of MI-219 to MDM2 can be reduced by MDMX. MI219 dose-dependently induced p53 accumulation at the posttranscriptional level and up-regulation of MDM2 and p21 (two p53-target genes) at the transcriptional level in the SJSA-1, LNCaP and 22Rv1 cancer cells and normal cells with wild-type p53 but had no effect in cell lines with mutated/deleted p53. However, MI-219 failed to up-regulate PUM in normal PrEC cells. MI-219 inhibited cell growth in cancer cell lines with wild-type p53 with IC50 values ranging between 0.4 and 0.8 mM and induced apoptosis in cancer cells but not in normal cells. Considering the excellent oral bioavailability, the in vivo activation of p53 by MI-219 was investigated in Xenograft tumors, showing that MI-219 activated p53, inhibited cell proliferation and induced apoptosis in a time-

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dependent manner in Xenograft tumors. MI-219 represented robust in vivo antitumor activity but was not toxic to normal tissues. The lack of toxicity of MI-219 to normal tissues was probably attributed to the fact that activation of p53 did not require posttranscriptional modifications and took place in the presence of MDM2. The preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphocytes were further examined by means of an in vitro and ex vivo approach [35], showing that MI-219 altered the functional activity of HDM2 through enhanced autoubiquitination and degradation and triggered an earlier response predominantly in the form of apoptotic cell death. MI-319 (38, Fig. 12), which was a closely related to MDM2 antagonists MI-219 and Nutlin-3 in terms of the mode of action, bond to MDM2 with an affinity slightly higher than that of MI-219 and Nutlin-3 (Ki ¼ 9.6 nM) [36]. MI-319 had an anti-lymphoma potency equal to that of MI-219 and Nutlin-3 against WSU-FSCCL (a B-cell follicular lymphoma line) and could increase the life span of orally treated follicular lymphoma bearing animals. Analysis of the predicted binding model of MI-63 (Fig. 13) showed that the morpholinyl group partially exposed to solvent [27], this finding suggested that this region may not play a detrimental effect on binding to MDM2 and cellular activity. Modifications of this region (Fig. 13) were further carried out to investigate the SARs on binding, cellular activity and PK parameters [37]. MI126 and MI-122 with a methylpiperazinyl group and a methylpiperidinyl group bond to MDM2 with Ki values of 1.5 and 2.0 nM, respectively and had an improved PK profile over MI-63

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(Ki ¼ 1.7 nM). However, their Cmax and AUC values were still relatively low with oral dosing, probably due to the fact that the morpholinyl, methylpiperazinyl and methylpiperidinyl groups in MI63, MI-126 and MI-122 were protonated and charged at physiological condition. So MI-142 and MI-147 with neutral groups were designed, these two compounds showed similar binding ability to MDM2 and cellular activity but had very different PK profiles (MI142 with 1-ethoxy-2-methoxyethanyl tail had a very poor PK profile). MI-147 with a butyl-1, 2-diol tail showed significant PK profile over MI-63, MI-126 and MI-122. With MI-147 as the template, MI188 without the 2-F substituent was designed, but it was 7 and 2e3 times less potent than MI-147 in binding affinity and cell growth inhibition, indicating that the 2-F substituent in the phenyl ring was essential. Based on the chemical structure of MI-188, MI-219 was designed to examine the 5-F substituent in the oxindole ring on binding, cellular ability and PK parameters. MI-219 was less potent than MI-188 in binding and cellular ability, but its PK profile was significantly improved over MI-188. Furthermore, MI-220 with an opposite configuration in the butyl-1, 2-diol tail had similar binding affinity to MDM2, cellular ability and PK profiles in terms of their Cmax and AUC values, showing that the chirality did not have a significant impact on these aspects. MI-221 with a shorter tail was slightly inferior to MI-219 and MI-220 in terms of PK profiles, although MI-221 was as potent as MI-219 and MI-220 in binding to MDM2 and in cell growth inhibition in SJSA-1 and HCT-116 cells. As the most potent MDM2 inhibitor, MI-147 was chosen to test its ability to activate p53 in cancer cells. MI-147 effectively and dose-dependently activated p53 and overexpressed MDM2 in SJSA-

Fig. 13. MI-63-based further modifications.

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Fig. 14. Isomerization of MI-219 in polar solvent.

1 and HCT-116 cells with wild-type p53 but not in isogenic HCT-116 cells with deleted p53. Besides, MI-147 dose-dependently induced cell death in the SJSA-1 cell lines with wild-type p53, which was specific and p53-dependent. This cell death was consistent with its ability to up-regulate Puma and Nova (two potent proapoptotic proteins). The therapeutic potential of MI-147 was further evaluated in SJSA-1 xenograft model, showing that MI-147 was highly effective in inhibition of tumor growth as an oral agent and no significant weight loss or toxicity was observed in animals treated with MI-147 and in combination with Irinotecan. It is well known that the stereochemistry is an essential dimension in pharmacology and has a great impact on ADME of therapeutic molecules. Drug chirality is now a major theme in the design, discovery, development, launching and marketing of new drugs [38]. An excellent work regarding the relationship between the stereochemistry and binding affinities to MDM2 of spirooxindoles was carried out by Wang and co-authors [39]. Three other isomers (46e48) were obtained when MI-219 (37) was treated with MeOH, MeCN or H2O (Fig. 14). In aqueous solution, MI-219 underwent a reversible retro-Mannich reaction affording the respective ring-opened transition state (a-TS) in which the pyrrolidine ring was opened. Recyclization of a-TS afforded four stereoisomers. Binding kinetics of isomers 37, 46e48 to MDM2 were investigated using the fluorescence-polarization assay, showing that isomer 46 had the highest binding affinity, followed by isomer 47, 37 and 48. Isomer 46 was >300-times more potent than isomer 48 and was 18-times more potent than isomer 37. Interestingly, there was no significant difference in the activity against cellular growth of the

SJSA-1 cell line due to the rapid isomerization of both isomer 37 and 46 in the cell culture medium. Isomer 46 was more potent than isomer 37 in vivo in activation of p53 and induction of apoptosis and had much better in vivo anticancer activity in the SJSA-1 xenograft model than isomer 46. Besides, no significant weight loss or other signs of toxicity for either isomer was observed. Although isomer 46 had a strong antitumor activity and shrank tumors by 91% in the SJSA-1 xenograft model, it failed to achieve complete tumor regression probably due to its less ideal pharmacokinetic properties. Extensive modifications of such compounds ultimately led to the identification of MI-888 (Fig. 15), which was capable of achieving rapid and complete tumor regression in animal models of human cancer upon oral administration and bond to MDM2 with a Ki value of 0.44 nM, being 10-times more potent that its cis-trans isomer 49.

Fig. 15. MI-888 and its cis-trans isomer.

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Fig. 16. MI-888 analogues with different cyclic side chains.

MI-888 came from extensive modifications on the side chain by introducing the conformationally constrained groups [9]. Compounds 50e53 (Fig. 16) with a cyclic side chain bond to MDM2 with high affinities (Ki ¼ 0.61e1.1 nM) and were as potent as isomer 46 (Fig. 14) but 10 times more potent than MI-219. However, compound 54 with a tert-butanol side chain was 3 times less potent than isomer 46. Compounds 50e53 were evaluated for their metabolic stability in mouse, rat and human microsomes. Compound 53 was quite stable, suggesting that trisubstituted alcohol containing side chain may block or retard the potential oxidation by cytochrome P450 enzymes. To test this hypothesis, compounds 55e56 and MI-888 were designed. Microsomal stability evaluation confirmed that these three compounds were very stable in mouse, rat and human microsomes. More importantly, MI-888 was the most potent MDM2 inhibitor based on their potencies in inhibition of cell growth in SJSA-1 and RS4; 11 acute leukemia cell lines (IC50 ¼ 0.08 and 0.06 mM, respectively). MI-888 represents the most potent and efficacious MDM2 inhibitor discovered to date and warrants extensive evaluation as a potential clinical development candidate for the treatment of human cancer. The aryl “A” and “B” rings in Nutlin and MI-219 (Fig. 17) adapt a ‘Cis’ configuration, much less is known about the ‘Trans’ configuration as shown in MI-888. Bradford Graves et al. designed a series of compounds with a novel core scaffold (Scaffold 1, Fig. 18). Extensive modifications and further biological evaluation finally led to the identification of RG7388 (58) [40]. RG7388 was 2- and 8-

times more potent than Nutlin in binding to MDM2 and in vitro antiproliferative activity, respectively. Besides, RG7388 dosedependently induced p53 stabilization, cell cycle arrest and apoptosis in cancer cells with wild-type p53 and achieved impressive in vivo efficacy against SJSA-1 osterosarcoma xenograft in nude mice. To identify more potent and selective MDM2 antagonists with desirable in vitro ADMET and in vivo PK properties, further modifications were carried out to discover MDM2 inhibitor RO8994 (59, Fig. 18), which was found to be highly efficacious against human tumor xenografts in nude mouse models. A slight modification was made by converting terminal carboxylic acid to a carboxamide moiety. This aromatic group was critical in stabilizing stability

Fig. 17. Structures of Nutlin and MI-219.

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Fig. 18. Scaffold inspired discovery of RO2468 and RO5353 using bioisosteric replacements.

metabolically and improving cellular potency/selectivity, PK profiles and in vivo efficacy for both RG7388 and RO8994 [41]. Since the interaction in the Trp23 pocket played an important role in binding to MDM2, further exploration of bioisosteric replacements [42,43] on the phenyl moiety of 6-chlorooxindole, while preserving other important architectural features in RO8994 for optimal binding and pharmacological properties, was prioritized, leading to the identification of two highly potent, selective, and orally active p53eMDM2 inhibitors, RO2468 (60) and RO5353 (61), which achieved impressive in vivo efficacy against SJSA-1 osterosarcoma xenograft in nude mice and displayed tumor regression at 10 mg/ kg. No significant weight loss was observed for both compounds. Compounds collections based on the natural-product-inspired scaffolds continue to be rich sources for new drug candidates, especially in the area of cancer therapeutics [44]. As mentioned above, spiro-pyrrolidinyl oxindole scaffold defines the characteristic structural core of a large family of oxindole alkaloids with pronounced and diverse bioactivities. Based on this rationale, a library of spirooxindoles with multiple stereocentres were enantioselectively synthesized by means of an asymmetric Lewis-catalyzed 1, 3-dipolar cycloaddition of an azomethine ylide to a substituted 3-

methylene-2-oxindole using a chiral catalyst formed from a N, Pferrocenyl ligant and CuPF6(MeCN)4 [45] (Fig. 19). Among the 39 synthesized compounds, only compound 62 with a different relative configuration induced phenotypic changes, such as accumulation of round-shaped cells with condensed DNA. Both enantiomers of 62 were subjected to fluorescence activated cell sorting analysis, revealing that ()-62 but not (þ)-62 caused accumulation of cells in G2/M phase in BSC-1, HCT116 p53þ/þ and p53 / and Hela cells. Interestingly, ()-62 did not act by inhibition of p53eMDM2 interaction, but rather via an interference with microtubule polymerization. This difference may be related to the different spatial arrangement of functional groups for ()-62 as compared with the diastereomeric spirooxindoles-based p53eMDM2 inhibitors. The mode of action of ()-62 also differed from the biological function of natural oxindole alkaloid spirotryprostatin, which directly inhibited tubulin polymerization [16]. Richard Jove et al. designed 12 analogues of natural oxindole alkaloid spirotryprostatin B (6 pairs of diastereoisomers) and evaluated their antitumor activity against melanoma cells (Fig. 20) [46]. The SARs study showed that the diastereoisomer relationship was not a major factor in deciding the antitumor activity.

Fig. 19. Ferrocene catalyzed synthesis of spiro-pyrrolidinyl oxindoles.

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Fig. 20. Analogues of natural spirotryprostatin B (SOID-1-12).

Substituents at the 6-position but not 7-position of the phenyl group were important to improve their antitumor properties. Compounds with bromo or methoxy group had better antitumor property. Among them, SOID-8 (72) displayed the best antitumor activity in both melanoma cells (A2058 and A373) and had little effect on the growth of normal cells. SOID-8 induced the apoptosis of A2058, A375, SK-MEL-5 and SK-MEL-28 human melanoma cells and an increase of poly (ADP-ribose) polymerase cleavage and downregulated the expression of Mcl-1. In addition, SOID-8 timeand dose-dependently reduced tyrosine phosphorylation of STAT3. This inhibition was associated with decreased levels of phosphorylation of JAK2, an upstream kinase that mediates STAT3 phosphorylation at Tyr705. SOID-8 inhibited IL-6-induced activation of STAT3 and JAK2 in melanoma cells and suppressed melanoma tumor growth in a mouse xenograft model, accompanied with a decrease of phosphorylation of JAK2 and STAT3. Dispirooxindoles 75e80 were efficiently synthesized in a highly regioselective manner via a 1, 3-cycloaddition reaction between 2(arylmethylene)-2, 3-dihydro-1H-inden-1-ones and azomethine ylides, which were generated in situ via decarboxylative condensation of sarcosine and isatins (Table 1) [47]. These compounds were further evaluated for their anticancer activity against 56 human cancer cell lines (leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate and kidney). The tested

compounds showed moderate or no activity at all against most of the tested cancer cell lines. Interestingly, these compounds exhibited selective inhibition against SK-MEL-2 cell lines. SARs studies showed that the methyl group attached to the nitrogen of the amide was essential to the observed anticancer activity. The 4chlorophenyl group attached to the pyrrolidine ring was much more beneficial to the anticancer activity than the 4methoxyphenyl and thienyl groups. As a follow-up work, two series of spirooxindoles 82 and 83 were synthesized following previously reported method and evaluated for their anticancer activities against different human cancer cell lines utilizing the in vitro sulfo-rhodamine B (SRB) standard method (Table 2) [48,49]. Most of the compounds 82 showed moderate anticancer activities against HCT116, MCF-7 and HEPG2 cell lines (data not listed in Table 2). Compound 82a displayed the best inhibitory effect against HEPG2 cell line with an IC50 value of 12.16 mM, slightly less potent than Doxorubicin (IC50 ¼ 7.36 mM). 3D-QSAR pharmacophore modeling showed that the major structural factor affecting the potency of these compounds was related to their basic skeleton. Compounds 83 exhibited mild antitumor properties against HEPG2, HELA and PC-3 cell lines. Among them, compound 83b demonstrated rather higher anticancer activity against HELA cells (IC50 ¼ 6.96 mM), slightly more potent than Doxorubicin (IC50 ¼ 7.71 mM). Compounds 83aec

Table 1 Antitumor property of the tested compounds against selected melanoma cancer cell line (SK-MEL-2) at a dose of 10 mM.

Compounds

R1

R2

Percentage growth of SK-MEL-2

75 76 77 78 79 80

H CH3 H CH3 H CH3

4-ClC6H4 4-ClC6H4 4-MeOC6H4 4-MeOC6H4 2-thienyl 2-thienyl

6.14 11.05 8.70 6.92 6.58 5.11

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Table 2 Antitumor activity of compounds 82a and 83aec against selected human cancer cell lines. Structures

82a

IC50 (mM) HCT116 46.24

MCF-7 46.83

HEPG2 12.16

e

6.86

5.46

7.36

Compounds 83a 83b 83c

HELA 11.86 6.96 10.90

PC-3 17.47 19.50 32.03

HEPG2 7.42 8.09 6.99

e

7.71

8.83

7.36

Compounds

Doxorubicin

Doxorubicin

had similar anticancer activity against HEPG2 with the reference drug Doxorubicin. Perumal et al. reported the synthesis of three series of novel spirooxindoles-pyrrolidine derivatives via the 1, 3-dipolar cycloaddition using different amino acids and dipolarophiles and further evaluated their anticancer properties against A549 human lung adenocarcinoma cancer cell line (Fig. 21) [50]. Compounds 85 showed prominent cytotoxic activity in vitro at different concentrations (200, 100, 50 mg/mL). Among N-alkyl substituted derivatives, the activity depended on the length of carbon chain

probably due to the increased lipophilicity (7.22% for N-methyl derivative 84b and 56.74% for N-hexyl derivative 84c, Table 3). 5-Halo substituted compounds showed decreased inhibitory effect while 5nitro substituted compound 84d had better anticancer activity compared to unsubstituted compounds. Further modifications of substitutions attached to the nitrogen atom of the amide (R1) led to the identification of N-propargyl substituted compound 84e with the highest anticancer activity against A549 cells (IC50 ¼ 50 mg/mL). Further modifications focused on the variations of the substitutions attached to the indole ring and the replacement of

Fig. 21. Three series of novel spirooxindoleepyrrolidine derivatives.

B. Yu et al. / European Journal of Medicinal Chemistry 97 (2015) 673e698 Table 3 Cell inhibition of selected spirooxindoles again A549 cells at the concentration of 100 mg/mL. Compounds

R1

R2

R3

Cell inhibition (%)

84a 84b 84c 84d 84e 85a 85b 85c 86a 86b

H Me Hexyl H Propargyl H H Me H H

H H H NO2 H H H H H H

e e e e e H Methoxyl Methoxyl Methoxyl H

20.91 7.22 56.74 36.65 59.31 23.2 83.0 85.0 83.3 87.8

oxindole ring by the imidazole group. The cell inhibition increased slightly at the concentration of 100 mg/mL when the oxindole ring was replaced by the imidazole group (85a). Compounds with a methoxyl group (85b) attached to the indole ring (R3) showed better activity than the unsubstituted compounds, especially when a methyl group was attached to the oxindole ring (85c). Interestingly, the derivatives containing thiazolidine ring (86a) had better activity. Besides, these compounds could cause apoptotic DNA fragmentation and induce apoptosis of A549 cells. Molecular docking studies showed that the most potent compound 86b bond very well with ALK receptor (tyrosine kinase receptor) with the binding energy of 8.47 kcal/mol. Another series of novel spiropyrrolidine oxindoles containing thiazolidine ring were diastereoselectively synthesized from isatin, chalcone and 2-phenylthiazolidine-4-carboxylic acid via 1, 3dipolar cycloaddition and evaluated for their antiproliferative activity against breast cancer cells (MCF-7, T47D and MDA-MB-231) using the MTT assay (Fig. 22) [51]. All the compounds, especially those with an amino alkyl chain, were found to exhibit the growth of breast cancer cells with an IC50 value of less than 20 mM, comparable or even better activity than that of Tamoxifen. In particular, compound 88a displayed the best antiproliferative activity against MCF-7, T47D and MDA-MB-231 cell lines with the IC50 values of 3.7, 4.7 and 4.2 mM, respectively (About 3 times more potent than Tamoxifen). Shi and coworkers reported the synthesis of the dispiropyrrolidines containing an oxazolone moiety and their in vitro antiproliferative activities against hepatic carcinoma (HepG2) cells [52]. Eight compounds showed good inhibitory effect against HepG2 cells (Table 4). Compounds 89a and 89f with a 2nitrophenyl group at the pyrrole ring showed the best antiproliferative activity with the IC50 values of 10.5 and 11.7 mM, respectively. Incorporating different bioactive scaffolds into one molecule has been recognized as the powerful strategy to construct molecules with structural novelty and biological potential. Nirup et al. designed a library of compounds containing spirooxindole and pyrrolidine/pyrrolizidine rings attached to the well known

685

bioactive andrographolide core (Fig. 23) and evaluated their in vitro cytotoxicity against a panel of cancer cell lines of different origins (CHO, HepG2, Hela, A-431, MCF-7, MDCK and Caco-2) [53]. All the spirooxindoles were efficiently synthesized with high yields and less reaction time using microwave irradiation technique. Biological evaluation showed that the sarcosine series 90 and 93 had weak inhibition against the tested cell lines (IC50 > 50 mM). The proline series 92 and 95 showed slightly improved cytotoxicity with the IC50 values ranging from 9.8 to >50 mM and some of them were more potent than andrographolide. However, the N-benzyl glycine series 91 and 94 showed significantly improved inhibitory effect. Almost all the compounds had better cytotoxicity than andrographolide. Compound 91a (R1 ¼ R3 ¼ Me, R2 ¼ H) was about 6 times more potent than andrographolide against CHO cells with the IC50 values of 8.1 and 46 mM, respectively. For the proline series 92, compound 92a (R1 ¼ Br, R2 ¼ R3 ¼ H) was the most potent derivative with the GI50 value of 10.5 mM against HCT116 cells [54]. Further mechanistic studies showed that compound 92a induced death of HCT116 (GI50 ¼ 10.5 mM), MiaPaCa-2 (GI50 ¼ 11.2 mM) and HepG2 (GI50 ¼ 16.6 mM) cells, which was associated with cell rounding, nuclear fragmentation and increased percentage of apoptotic cells, cell cycle arrest at G1 phase, ROS generation, and involvement of mitochondrial pathway like andrographolide. Upregulation of Bax, Bad, p53, caspases-3,-9 and cleaved PARP, downregulation of Bcl-2, cytosolic NF-kB p65, PI3K and p-Akt and translocation of P53/P21, NF-kB p65 were observed in compound 92a treated HCT116 cells. Steroids are a family of molecules that play an important role in a wide range of biological processes and in human physiology. Some interesting steroidal heterocycles with excellent anticancer properties were discovered before in our group [55e60]. In continuation of our effort towards finding novel steroidal heterocycles with anticancer potentials, a series of novel steroidal spirooxindoles were designed and synthesized via 1, 3-dipolar cycloaddition between (E)-3b-hydroxy-5-en-16-arylidene-17ketosteroids and azomethine ylides generated in situ from substituted isatins and sarcosine [61] (Fig. 24). Most of the synthesized compounds showed moderate to good antiproliferative activity against four human cancer cell lines (EC109, MGC-803, SMMC-7721 and MCF-7) with the IC50 values of 0.7 and 43 mM and some of them were more potent than 5-fluorouracil. From the biological data of these compounds, the general trend of structureeactivity relationship is: (a) Compounds with heteroaryl groups (Ar) had similar antiproliferative activities against the tested cell lines with those having substituted phenyl groups; (b) Substituents (R) on the isatin nucleus had remarkable effect on their antiproliferative activities against SMMC-7721 cells; (c) Substituents on the phenyl group (Ar) had different effect on different cancer cells; (d) Steroid dimers 96aec did not represent significant improvement on their antiproliferative activities toward the cancer cell lines. Among them, compound 97a (Ar ¼ 4-chlorophenyl, R ¼ H) showed good antiproliferative activity against SMMC-7721 cells

Fig. 22. Spiropyrrolidine oxindoles containing thiazolidine ring.

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Table 4 Antiproliferative activities of selected dispiropyrrolidines against HepG2 cells.

Structures

Compounds

Ar

IC50 (mM)

89a

2-Nitrophenyl

10.5 ± 0.3

89b 89c 89d

4-Methylphenyl 4-Bromophenyl 4-Fluorophenyl

>30 >30 21.9 ± 2.7

89e 89f 89g 89h

4-Nitrophenyl 2-Nitrophenyl 3, 4-Dichlorophenyl 4-Fluorophenyl

16.4 11.7 14.8 24.3

(IC50 ¼ 0.71 mM). Steroid dimer 96a (R ¼ 5-F) showed significantly improved antiproliferative activities against SMMC-7721 and MCF7 cells with the IC50 values of 4.30 and 2.06 mM, respectively. Further evaluation showed that compound 97b (Ar ¼ 4-t-butylphenyl, R ¼ H) induced the cellular early apoptosis and G2/M arrest of MGC-803 cells in a time- and concentration-dependent manner. 3.2. Spiro-pyran oxindoles Pyrans and oxindoles have been extensively studied before because of their biological potentials and synthetic utilities. However, the reports about the pharmaceutical evaluation of spiropyran oxindoles are really rare. The first report that described the I2-catalyzed three-component one-pot synthesis of spiro-pyran oxindoles (Fig. 25) and their in vitro cytotoxic activities against U87 human glioma cells was published in 2012 [62]. Unfortunately, almost all the compounds had weak inhibition against U87 cells (IC50 > 19 mg/mL), only compound 99a (R1 ¼ H, R2 ¼ Me, R ¼ CH2 (CH2)9Br) had comparable cytotoxic activity (IC50 ¼ 2.5 mg/mL) with the control drug Carmustine (IC50 ¼ 3.9 mg/mL), about 40 times more potent than compound 99b (R1 ¼ H, R2 ¼ Me, R ¼ H) (IC50 ¼ 40 mg/mL). This finding indicated that the lipophilic moiety attached to the nitrogen atom of oxindole nucleus was beneficial to the anticancer property probably due to the increased lipophilicity of molecules.

± ± ± ±

3.5 1.4 0.2 1.9

5-Hydroxy-2-(hydroxymethyl)-4H-pyran-4-one (also known as kojic acid) has been reported to possess diverse pharmaceutical activities. Considering its biological potentials, Perumal et al. reported the Cu(OTf)2 catalyzed one-pot synthesis of kojic acid tethered spirooxindoles (Fig. 26) and evaluated their anticancer potency towards A549 human lung cancer cell lines using MTT assay [63]. All these compounds showed moderate cytotoxicity against A549 cells (IC50 > 50 mM). Compounds 101a (R1 ¼ Cl, R ¼ H, X ¼ CN) and 101b (R1 ¼ H, R ¼ Me, X ¼ CN) exhibited the best cytotoxicity with the IC50 values of 51.4 and 51.1 mM, respectively. Molecular docking studies revealed that compound 101b had strong affinity towards anaplastic lymphoma kinase (ALK) protein (Free energy binding is 5.99 kcal/mol) (Fig. 27). Tao et al. reported catalyst-free one-pot synthesis of novel 4Hpyran-fused pyrrolidinones and evaluated their cytotoxicity against Raji cells with Cisplatin as a positive control (Fig. 28) [64]. Most of these compounds (102e105) showed remarkable cytotoxicity against Raji cells with the IC50 values ranging from 17 to 75 mM. Among them, compound 102a (R1 ¼ Bn, R2 ¼ H, R3 ¼ 6-Br) exhibited the most potent cytotoxicity (IC50 ¼ 17.56 mM), comparable to that of Cisplatin (IC50 ¼ 17.32 mM). Different from Mazaahir's finding [62], substituents (R2) attached to the nitrogen atom of the oxindole ring did not affect the cytotoxicity markedly. When R1 was changed from benzyl group to the phenylethyl group, the cytotoxicity decreased significantly. The electronic characteristics

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Fig. 23. Andrographolide-based spirooxindoles.

of substituents and their position on the benzene ring had remarkable effect on their bioactivity. Except for trifluoromethyl substituted derivatives, compounds with an electron-withdrawing group exhibited decreased cytotoxicity against Raji cells. For

halogenated derivatives, the bioactivity depended on the position of halo atom on the benzene ring and brominated derivatives had better cytotoxicity than chlorinated ones. Generally, the trends in cytotoxicity followed this sequence (6-X > 5-X > 4-X; X ¼ Br or Cl).

Fig. 24. Synthesis of antiproliferative steroidal spirooxindoles.

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Fig. 25. Four types of spiro-pyran oxindoles.

Fig. 26. Cu-catalyzed synthesis of kojic acid tethered spirooxindoles.

Interestingly, compounds 103 and 104 having different heterocycles fused to the 4H-pyran ring had decreased cytotoxicity with the IC50 values of 35.29 and 42.68 mM, respectively, compared to most of the compounds 102aer. It should be noted that the

stereochemistry of compounds 105 had no effect towards the cytotoxicity against Raji cells. Soliman et al. reported the synthesis of phosphanylidene indole pyranones via the [4 þ 2] cycloaddition and their anticancer

Fig. 27. Molecular docking of compound 101b with ALK protein.

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Fig. 28. Four types of 4H-pyran-fused pyrrolidinones.

activities against colon (HCT116) and liver (HEPG2) cancer cells (Fig. 29) [65]. As shown in Fig. 28, compound 106a was the most active against HEPG2 cells (IC50 ¼ 5.55 mg/mL) when compared with the known anticancer drug Doxorubicin (IC50 ¼ 3.73 mg/mL). Other compounds showed decreased cytotoxicity. The synthesis of chiral molecules with structural complexity and diversity has played a critical role in the discovery of drug candidates. Inspired by the diverse and excellent bioactivities of pyranopyrimidines, wang and co-workers developed an intramolecular asymmetric Michael/cyclization reaction of coumarins with isatylidenemalononitriles in the presence of the rosin-derived thiourea catalyst, leading to the generation of pyran-fused spirooxindoles. These intermediates were subjected to further transformation, giving chiral spirooxindoleepyranopyrimidines 107aeb, 108aeg and 109aeb (Fig. 30) [66]. Anticancer evaluation of these compounds against MDA, U937, Jurkat, Hela and EJ was carried out using MTT assay. Compounds 107aeb and 109aeb showed weak or no cytotoxicity. In contrast, most of compounds 108aeg showed good

cytotoxicity against the tested cancer cells (only the IC50 values for 108a and 108g are listed in Table 5). Compound 108a (R1 ¼ H and R3 ¼ H) was first screened out with good cytotoxicity against five different cancer cell lines. In particular, compound 108a was about twice more potent than Camptothecin (IC50: 11.004 mM vs 23.401 mM). However, the other enantiomer (S)-108a had slightly decreased cytotoxicity (data not shown here). Encouraged by this finding, compound 108a based further modifications focusing on variations of substituents on the phenyl rings (R1 and R3) were carried out, it turned out that compound 108g (R1 ¼ 7-F and R3 ¼ 6-F) had better and broad-spectrum anticancer activity in comparison with Camptothecin, especially to MDA and Hela cells. Other compounds in this series showed decreased activities against the tested cancer cell lines to varying degrees. Besides, compound 108g had an excellent selectivity between normal human peripheral blood lymphocytes (hPBLs) and human T-cell leukemia cell lines (Jurkat) (IC50: 63.824 mM vs 6.373 mM). A good selectivity for compound 108g was also observed (IC50: 29.3 mM vs 8.255 mM).

Fig. 29. Synthesis of phosphanylidene indole pyranones.

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Fig. 30. Rosin-thiourea catalyzed asymmetric synthesis of pyran-fused spirooxindoles and pyranopyrimidines.

Table 5 In vitro anticancer activities of compounds 108a and 108g. Compounds

108a 108g Camptothecin

IC50 (mM) MDA

U937

Jurkat

Hela

EJ

11.004 ± 2.677 6.952 ± 2.654 23.401 ± 3.210

7.711 ± 3.444 6.296 ± 0.460 0.015 ± 0.004

8.225 ± 2.854 6.548 ± 1.228 0.021 ± 0.002

20.394 ± 7.143 6.373 ± 0.652 25.226 ± 3.987

11.844 ± 0.987 10.381 ± 3.008 0.026 ± 0.013

Pyranophthoquinone motif exists in some natural products such as dilapachone, adenophyllone, etc. Derivatives with pyranophthoquinone motif are endowed with diverse biological activities [67]. Wang et al. [68] reported the organocatalytic asymmetric cascade Michael cyclization of 2-hydroxynaphthalene-1, 4-diones to isatylidenemalononitriles in the presence of the dihydroquinine-derived bifunctional thiourea catalyst, generating a series of chiral spiro[benzo[g]chromene-oxindole] derivatives (Fig. 31). All the final products 110aew were obtained with up to 99% yield and 99% ee. Further modification was conducted to give compound 111 without any loss of enantioselectivity. Interestingly,

all these compounds showed over 93% inhibition rate against HEPG2, MDA-MB-231 and Du145 cells at the concentration of 50 mM. Even at 10 mM, compound 111a (R2 ¼ allyl and R1 ¼ R3 ¼ H) had excellent inhibition against these three cell lines with the inhibition rate of 88%, 78% and 93%, respectively. Among these compounds, compound 110a (R1 ¼ 7-CF3 and R2 ¼ R3 ¼ H) exhibited the best cytotoxicity against MDA-MB-231 cells at the concentration of 10 mM. A recent patent by Cui and Tanaka [69] reported that spiropyran oxindoles (Fig. 32) exhibited antiproliferative activity against K562 cells probably through the inhibition of the

Fig. 31. Chiral spirooxindoles with pyranophthoquinone moiety.

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Fig. 32. Spiro-pyran oxindoles reported by Cui and Tanaka.

hexokinase-Ⅱ (HK-Ⅱ), a key enzyme for glycolysis. It is evident that the introduction of a halo atom (Br or Cl) into the oxindole ring was beneficial to the activity. Compared to compounds 112aed, compounds 112eeh showed good inhibition with the IC50 values ranging from 15 to 21 mM. Compounds 112iej showed 37.7% and 33.8% inhibition towards HK-Ⅱ, respectively.

3.3. Spiro-thiazolidine oxindoles It is well documented that some vegetables from Cruciferous family can lower the risk of various cancers. In 2005, Mojzis et al. [70] examined the antiproliferative effect of cruciferous phytoalexins 113e115 (Fig. 33) against T-Jurkat leukemic cells using MTT assay. These phytoalexins caused 45e56% reduction in Jurkat cells survival at the concentration of 100 mM. Besides, (±)-spirobrassinin (113) induced G2/M arrest of Jurkat cells. However, these phytoalexins (113e115) had little effect on apoptosis. As a follow-up, they further explored the antiproliferative effect of such compounds against several human cancer cell lines (Jurkat cells, MCF-7 and Hela) [71]. The natural (2R, 3R)-()-(117) remarkably inhibited the growth of Jurkat cells, about twice more potent than its (2S, 3S)enantiomer at the concentration of 100 mM (Cell viability: 36.9% vs 79.8%). The cell viability was 72.3% when treated with racemic 117 at the same concentration. However, weak and similar cytotoxicity for both enantiomers of 116 and the racemic form was observed.

Besides, both phytoalexins (116 and 117) also displayed weak cytotoxicity of MCF-7 and Hela cells. Spirotryprostatin A, a natural oxindole alkaloid isolated from Aspergillus fumigates, could cause G2/M arrest of tsFT210 cells. The excellent activity of Spirotryprostatin A has inspired medicinal chemists to design more analogs with biological potentials. GomezMonterrey et al. designed a series of Spirotryprostatin A analogues (118, Fig. 34), where the pyrrolidine nucleus was replaced by a thiazolidine moiety and the oxindole and diketopiperazine rings were maintained [72]. Unfortunately, all these compounds with different substitutions (R and R1) and configuration (R or S-enantiomer) did not show significant cytotoxicity against three human colon carcinoma cell lines (MCF-7, T47D and A-431) at concentrations below 100 mM and failed to inhibit the cell cycle of MCF-7 cells. With the spirooxindole-3, 30 -thiazoline having a structural similarity with spirooxindole pyrrolidine as the template, GomezMonterrey et al. further designed and synthesized a series of novel spiroimidazothiazoloxoindole derivatives (119, Fig. 35) and evaluated their cytotoxicity against three human cell lines (human embryonic kidney HEK, human melanoma M14 and human leukemia monocyte lymphoma U937) using the Doxorubicin as the control [73]. The general structureeactivity relationship is listed in Fig. 35. Within this series, compound 119a showed the best cytotoxicity against HEK, M14 and U937 with the IC50 values of 0.44, 0.53 and 0.87 mM. Compound 119b also had better activity against

Fig. 33. Several natural cruciferous phytoalexins.

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Fig. 34. Spirotryprostatin A inspired design of spiro-thiazolidine oxindoles.

these three cell lines with the IC50 values of around 2.0 mM. Furthermore, compound 119a induced the apoptosis of M14 cells while compound 119b failed to induce the apoptotic death. Compound 1b markedly induced G2/M arrest but compound 119a had no effect on the normal cell cycle. However, both compounds timedependently induced p53 expression. NMR analysis showed that both compounds could block the p53eMDM2 interaction but were not as efficient as Nutlin-3 as indicated by the release of p53. 3.4. Spirooxindole-based 2, 5-dihydropyrroles Considering the versatile bioactivities of spirooxindole and 2, 5dihydropyrrole, Tan et al. designed and synthesized a series of novel spirooxindole-based 2, 5-dihydropyrroles with structural diversity via an efficient three-component reaction of isatin, amino ester and alkyne (Fig. 36) [74]. In vitro cytotoxicity of these spirodihydropyrroles against MCF-7 cells was carried out at three different concentrations (1, 10, 100 mg/mL) using doxorubicin hydrochloride as the control. Six compounds showed slightly inhibition against MCF-7 cells at 1 mg/mL, while doxorubicin hydrochloride had no effect towards MCF-7 cells at this concentration. At 100 mg/mL, all 16 compounds showed remarkable cytotoxicity against MCF-7 cells. Among them, compound 120a was the most potent to inhibit the growth of MCF-7 cells to 28.0%, which was comparable to the positive control. Further mechanism study showed that compound 120a induced the apoptosis of MCF-7 cells

by the MAPK pathway as indicated by the increase of phosphorylated ERK1/2, p38 and JNK levels. 3.5. Spiroisoxazoline oxindoles As shown in the known inhibitors of p53eMDM2 interaction (Nutlin, TDP665759, MI-888 and the recently discovered AM8553), all these molecules detain a rigid heterocyclic scaffold, from which three lipophilic groups are projected into p53 pocket in MDM2 mimicking the three pivotal residues (Phe19, Trp23 and Leu26) [75]. With this in mind, Santos et al. designed and synthesized a library of novel spiroisoxazoline oxindoles (121, Fig. 37) as inhibitors of p53eMDM2 interaction and evaluated their antiproliferative effect towards human hepatocellular carcinoma cell line (HepG2) with wild type p53 [76]. The SARs study showed the substituents on the three phenyl rings played an important role in the activity, compounds with no substituents on the phenyl rings were inactive. Any substituents introduced at position R2 and R4 yielded more active compounds. Introducing a methoxyl group at R3 abrogated the cytotoxicity, regardless of the pattern of substituents in other positions. Different substituents at R4 in para position (methoxy, nitro and methyl) and ortho-methoxy were well tolerated, not affecting substantially potency. A halogen atom (Br or Cl) at position 6 of the oxindole moiety was beneficial to the activity, as exemplified in compound 121d, which had the best cytotoxicity against HepG2 cells (GI50 ¼ 29.11 mM). Compounds

Fig. 35. Spirooxindole pyrrolidine-based design of spiroimidazothiazoloxoindole derivatives.

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Fig. 36. Novel spirooxindole-based 2, 5-dihydropyrroles with structural diversity.

121a, 121b and 121d can inhibit p53eMDM2 interaction in a cellbased bimolecular fluorescence complementation assay. Compounds 121a and 121d induced a significant dose-dependent increase of cleaved PARP and active caspase-3, which are reliable markers of the apoptotic process. Moreover, compounds 121b and 121g showed good stability in 7.4 phosphate buffer and plasma, and had moderate susceptibility towards NADPH-dependent rat microsomal enzymes. Similarly, Ung and co-workers reported that isoxazolidine 121h had appreciable cytotoxicity against H460, MCF7 and SF-268 cell lines (GI50 ¼ 2.6 mM against MCF-7 cells) [77]. However, no further mechanism was studied. 3.6. Spiropyrazoline oxindoles Based on the previous report about spiroisoxazoline oxindoles [76], Santos et al. [78] designed a new series of spiropyrazoline

oxindoles (122) containing a pyrazoline ring with one more aromatic substituent (R4), in which the oxygen atom in isoxazoline ring was replaced by a N-Ar group (Fig. 38). In vitro cytotoxicity against MCF-7 cells was evaluated using MTT assay. The general SARs are listed as below. (a) No activity against MCF-7 cells was observed when R4 ¼ H (GI50 > 100 mM); (b) Compared to the spiroisoxazoline oxindoles (121d: GI50 ¼ 29.11 mM, Fig. 37), six compounds from the library of spiropyrazoline oxindoles had GI50 values of less than 12 mM, indicating that the substitution of an isoxazoline ring by a pyrazoline ring led to an increased cytotoxicity; (c) The inhibitory effect towards MCF-7 cells significantly depended on the nature of R2 group. When R2 ¼ Ph, the activity against MCF-7 cells depended on the nature of R1 and varied in the following order (Br > Cl > H); (d) For 6-Br spirooxindoles containing phenyl groups at R2 and R4, the activity against MCF-7 cells was almost the same regardless of the R3 group. However, for 7-Cl spirooxindoles containing phenyl

Fig. 37. Novel spiroisoxazoline oxindoles.

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Fig. 38. Spiroisoxazoline oxindoles based design of spiropyrazoline oxindoles.

Fig. 39. Spirothiadiazole oxindoles.

groups at R2 and R4, the activity varied with the R3 group. Compounds with the GI50 values of less than 30 mM against MCF-7 cells were further tested on human MDA-MB-231 cell line and normal human HEK293T cell line. Interestingly, all these compounds showed low or no cytotoxicity against HEK293T cells at concentration up to 100 mM. Among them, compound 122a with the most potent activity against MCF-7 cells (GI50 ¼ 7.0 mM) also had good cytotoxicity against MDA-MB-231 cells (GI50 ¼ 28.6 mM) and no cytotoxicity against nHEK293T cells (GI50 > 100 mM), respectively. 3.7. Spirothiadiazole oxindoles Indolin-2-ones (e.g. SOID8) have been reported as inhibitors for anti-apoptotic Bcl-2 proteins [46]. 1, 3, 4-thiadiazoles could also induce apoptosis and inhibit the expression of Bcl-2 [79]. Inspired by the anticancer potentials of indolin-2-one and 1, 3, 4-thiadiazole derivatives, Wang et al. designed and synthesized a series of 50 phenyl-30 H-spiro-[indoline-3, 20 -[1, 3, 4] oxadiazol]-2-one analogs (123, Fig. 39) that combined these two scaffolds and evaluated their Bcl-2 protein inhibitory activities using a fluorescence polarizationbased binding assay [80]. Among these compounds, compound 123a (R1 ¼ H, R2 ¼ Bn) showed good binding affinities to Bcl-xL and Mcl-1 with inhibition constants of 8.9 mM and 3.4 mM, respectively. While compound 123b (R1 ¼ 5-Cl, R2 ¼ H) achieved tight binding affinity to Bcl-xL (Ki ¼ 0.16 mM). The general SARs are listed in Fig. 39.

synthesized via a 1, 3-dipolar cycloaddition of acrylates with nitrones. Unfortunately, these two compounds had no activity against H460, MCF-7 and SF-268 cell lines. 3.9. Spiro furo/thieno oxindoles Incorporating boron, silicon, selenium or germanium into small molecules has been recognized as a powerful strategy to provide new structures where the unique properties of these elements may contribute to new biological activities [81]. Schreiber et al. synthesized diverse organosilicon small molecules (Fig. 41) via the Lewis acid-mediated stereoselective annulation of isatins with allylsilanes and evaluated the potential of these molecules to modulate cellular processes using multidimensional screening [82]. Among 90 compounds, compound (R)-125 showed an excellent inhibition against hepatocellular carcinoma (HepG2) cells with an EC50 value of 16.8 mM. The conversion of the arylsilane afforded compounds with induced activity.

3.8. Spirooxazinane oxindoles Inspired by the excellent cytotoxic activity of spiroisoxazolidine oxindole 121h (Fig. 37) against H460, MCF-7 and SF-268 cell lines [77], spirooxazinane oxindoles 124aeb (Fig. 40) were designed and

Fig. 40. Spirooxazinane oxindoles 124aeb.

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Fig. 41. Lewis acid-catalyzed synthesis of spiro furo-oxindoles with silicon atom.

Lin and co-workers reported the metal-catalyzed transformation of 2-furylcarbinols and 2-thienylcarbinols into corresponding spiro furo/thieno oxindoles (Table 6) and evaluated their cytotoxicity against Du145, LNCaP and PC-3 cells [83]. As shown in Table 6, compound 126d had the best cytotoxicity against Du145 cells with the GI50 value of 3.2 mM, while compound 127b where the oxygen atom was replaced by the sulfur atom showed significantly decreased activity against Du145 cells (GI50 > 50 mM). By contrast, spirothienooxindoles 127aee had excellent inhibition against LNCaP and PC-3 cells (GI50 < 10 mM). Among them, compound 127d was the most active against LNCaP cells (GI50 ¼ 1.8 mM). However, spirofuryloxindoles 126aee had weak activity against these two cell lines but was relatively sensitive to Du145 cells. 3.10. Gelsedine-type indole alkaloids Several gelsedine-type indole alkaloids were isolated from the leaves of Gelsemium elegans and further evaluated for their

cytotoxicity against A549 cells using cisplatin as the control (Fig. 42) [84]. Indole alkaloids 128, 132, 134e135 were found to have excellent cytotoxicity with the EC50 values of 0.25, 1.3, 0.35 and 0.75 mM, respectively and were more potent than Cisplatin (EC50 ¼ 3.5 mM). However, no synthesized analogues have been reported probably due to the complex structures that make it extremely difficult to access such compounds. 4. Conclusions Due to the drawbacks of current anticancer drugs, the research for more effective and selective anticancer agents has always been the hot topic in medicinal chemistry. Spirooxindoles have emerged as privileged scaffolds because of their prevalence in numerous natural products and biologically active molecules. Structure (spirotryprostatin A and alstonisine) based drug discovery has been proved as an efficient strategy to find new anticancer agents (e.g. the discovery of MI-888). For spirooxindoles, their anticancer potency mainly depends on the type of cyclic ring fused on the C3

Table 6 In vitro cytotoxicity of spiro furo/thieno oxindoles against Du145, LNCaP and PC-3 cells.

Structure

Compounds

Ar

GI50 (mM)

R

Du145

126a 126b 126c 126d 126e

Phenyl 4-Methoxyphenyl 2-Chlorophenyl 2-Fluorophenyl 2-Bromophenyl

3, 3, 3, 3, 3,

4, 5-Trimethoxy 4, 5-Trimethoxy 5-Dimethoxy 4, 5-Trimethoxy 4, 5-Trimethoxy

127a 127b 127c 127d 127e

Phenyl 2-Fluorophenyl 1-Naphthyl 1-Naphthyl 4-Fluorophenyl

3, 3, 3, 3, 4,

4, 5-Trimethoxy 4, 5-Trimethoxy 4, 5-Trimethoxy 4, 5-Trimethoxy 5-Dimethoxy

21.5 35.7 16.7 3.2 10.4

>50 >50 >50 >50 >50

LNCaP

PC-3

21.7 28.4 18.5 18.2 19.1

>50 >50 >50 25.0 >50

2.3 7.0 5.0 1.8 2.6

6.0 8.2 10.4 7.1 16.4

696

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Fig. 42. Gelsedine-type indole alkaloids.

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Abbreviations p53: tumor protein p53 MDM2: mouse double minute 2 homolog MDMX: a homolog of MDM2 SARs: structureeactivity relationships PK: pharmacokinetics AUC: area under the curve Cmax: the peak plasma concentration of a drug after administration ADME: absorption, distribution, metabolism and excretion SRB: sulfo-rhodamine B standard method QSAR: quantitative structureeactivity relationship ALK: tyrosine kinase receptor MTT: (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) ROS: reactive oxygen species HK-Ⅱ: hexokinase-Ⅱ MAPK: mitogen-activated protein kinase PARP: poly ADP-ribose polymerase NADPH: lactaldehyde reductase Bcl-2: B-cell lymphoma 2 EC50: the concentration of a drug which induces a response halfway between the baseline and maximum after a specified exposure time IC50: the half maximum inhibitory concentration GI50: the concentration for 50% of maximum inhibition of cell proliferation