Identification of rapid access to polycyclic systems via a base-catalyzed cascade cyclization reaction and their biological evaluation

Abstract

A base-mediated cascade reaction between malonate esters and acrolein was developed to access complex polycyclic systems. This novel tandem reaction enables the simultaneous generation of up to seven new bonds and at least three new stereogenic centers. Mechanistic studies indicate a series of nucleophilic 1,4 and 1,6 Michael addition reactions occur, followed by an aldol condensation reaction, culminating in the formation of three fused rings. The compounds were characterized by NMR studies and the stereochemistry was confirmed by X-ray analysis. The ability to generate multigram quantities of such complex molecular scaffolds renders the method promising for medicinal chemistry campaigns. Herein, we also demonstrate that the lead compounds display promising anti-proliferative activities against human cancer cell models.

Graphical Abstract

Introduction

Synthetic methodologies that enable rapid generation of molecular complexity from simple building blocks significantly advance the field of organic chemistry. Nucleophilic addition reactions including the Michael-addition reaction remain some of the most widely used methods to form carbon-carbon bonds due to its readily accessibility to reagents and favourable energetics.^1–3 The nucleophilic addition can proceed via either conjugated 1,4- or direct 1,2- addition to α, β-unsaturated systems. Since the 1990s, the chemistry of aldehydes has been widely explored in the presence of metal catalysts, Brønsted base and various methods to capable of catalyzing proton transfer reactions.⁴ Our group has continuously focused on the development of feasible transformations of simple synthons to access the core of complex natural products to undergo derivatisation for biological evaluation against drug resistant or high-risk cancer cell models.^5–7 In those chemical transformations, we aim at generating molecular libraries using efficient and diversity oriented synthetic approaches. We became particularly interested in the isodon diterpenes, which feature polycyclic complex molecular scaffolds (Figure 1) with interesting biological properties. For instance, the isodon family members have been reported to display promising in vitro and in vivo anticancer activity against various cancer cell models.^8–10 Their biological activity against methicillin-resistant Staphylococcus aureus has also drawn significant interest from the synthetic community.¹¹ Recently, green chemistry featuring cascade reactions to minimize the number of reaction steps and/or purification protocols have gained great interest in the medicinal chemistry field to establish diverse compound library platforms.¹² More recently, domino one-pot reactions using inexpensive or readily available reagents have been reported.¹³ In line with those efforts, herein we describe a cascade reaction sequence between malonate esters and unsaturated aldehydes to access enmein-type natural product mimetics with high-order molecular complexity in a carbon economic manner.

Figure 1.

Selected polycyclic Isodon diterpenes.

Thus, we initiated a synthetic plan to access to the core of this molecular scaffold for the rapid synthesis of analogue libraries to harvest their biological potential, particularly their anti-cancer properties. Li and co-workers described a copper-catalysed cascade reaction of α, β-unsaturated esters with keto-esters,¹⁴ and we questioned whether a less constrained system under basic conditions would proceed through the less favourable diester diene 5, the Knoevenagel product (Figure 2A) as a starting point for a Robinson annulation reaction, or whether it would instead proceed onward to a more complex cyclic system. Indeed, our unique combination of these highly reactive materials under controlled conditions generated a new complex molecular scaffold of enmein-type diterpenes (Scheme 1). This cascade reaction design represents one of the most efficient approaches to realize atom-economic synthetic processes.

Figure 2.

Plausible mechanism for formation of compounds 1 and 2.

Scheme 1.

Synthesis of 1 and 2.

The structural analysis and characterization of these new compounds was achieved mainly by 2D NMR spectroscopy and X-ray diffraction analysis. A focused library for compound 1 and 2 was generated and the resultant compounds inhibited cell proliferation of several cancer cell lines yet displayed no cytotoxicity against normal tissue at the tested concentrations.

Materials and Methods

All chemical manipulations were carried out under inert gas atmosphere unless otherwise noted. Anhydrous tetrahydrofuran (THF), diethyl ether (Et₂O), dichloromethane (CH₂Cl₂), toluene (PhCH₃), acetonitrile (CH₃CN), methanol (MEOH), and dimethylformamide (DMF) were obtained from solvent drying system. Reagents of the highest available quality were purchased commercially and used without further purification unless otherwise stated. Title compounds were purified by flash column chromatography using E. Merck silica gel (60, particle size 0.040–0.063 mmol) or Biotage Isolera Four with normal-phase silica gel. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mmol E. Merck silica gel plates (60F-254), using UV light for visualization and an ethanolic solution of anisaldehyde, or PMA, CAM solutions and heat as developing agent. Reactions were also monitored by using Agilent 1100 series LCMS and low-resonance electrospray ionization (ESI) model with UV detection at 25 nm. Reactions were also monitored by using Agilent 1100 series LCMS and a low-resonance electrospray model (ESI) with UV detection at 250 nm. The structures of the synthesized compounds were confirmed by ¹H and ¹³C-NMR that were recorded on 400/or 500 MHz Bruker AVANCE III HD NMR. Chemical shifts were reported as ppm relative to the solvent residual peak (CHCl₃: 7.26 ppm for ¹H, 77.2 ppm for ¹³C; acetone-d₆: 2.05 ppm for ¹H, 29.9 ppm for ¹³C; D₂O: 4.80 ppm for ¹H; DMSO d₆: 39.5 ppm for ¹³C). Data are reported as follows: chemical shifts, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br = broad), coupling constant (Hz), and integration. High resolution mass spectra (HRMS) were recorded on an Agilent ESI-TOF (time of flight) mass spectrometer using MALDI (matrix-assisted laser desorption ionization) or ESI (electrospray ionization) or on a Waters Xevo G2 Q-ToF mass spectrometer. Compounds were analyzed by using electrospray ionization in positive-ion mode. The purity of the synthesized compounds was determined on a Waters ACQUITY UPLC-PDA-ELSD-MS system using a C₁₈ reverse phase column and 0.1% formic acid/water - 0.1% formic acid/acetonitrile as the solvents. All synthesized compounds were at least 95% pure based on analytical HPLC and NMR. Chemical yields refer to purified compounds (¹H-NMR).

Cell lines and reagents

All cell lines were incubated at 37°C and maintained in a 5% CO₂ atmosphere according to proper sterile cell culture practices.¹⁵ Cells were tested for mycoplasma contamination with Mycoplasma Detection Kit (# LT07–318, Lonza) using the manufacturer’s conditions and were deemed negative. Cell lines were purchased from American Type Culture Collection (ATCC®) and cultured without antibiotics. The human leukemia cell lines used to test general cytotoxicity by the methods described were KOPN-8 (ACC 552, infant human B cell precursor acute lymphoblastic leukemia with MLL-MLLt1/ENL fusion, 4600 cells/well for 96 well plates), SUP-B15 (ACC389, human B cell precursor acute lymphoblastic leukemia of pediatric second relapse carrying the ALL-variant (m-bcr) of BCR-ABL1 fusion gene (e1–a2), 4800 cells/well for 96 well plates Corning #3917), MV411 (ATCC CRL-9591, biphenotypic B myelomonocytic leukemia, 5000 cells/well for 96-well plates). Cells were grown in RPMI supplemented with 10% fetal bovine serum (FBS, Hyclone). Adherent cells HCT116 (colorectal carcinoma ATCC® CRL-9591), and BJ (ATCC® CRL-2522) were grown to 80%−90% confluence to densities recommended by ATCC before use, these cells were cultured in McCoy’s 5a, (ATCC®, 30–2007) and EMEM (ATCC® 30–2003) respectively, and supplemented with 10% fetal bovine serum (FBS, Hyclone). HCT116 (4600 cells/well for 96 well plates), and BJ (CRL-2522, normal human foreskin fibroblast cells, 400 cells/well for 384 well plates, corning #8804BC).

Cytotoxicity assay was performed using the CellTiterGlo (CTG) Luminescent Cell Viability Assay kit (G7570, Promega, Madison, WI), performed according to the manufacturer’s instructions. Luminescence was recorded with an Envision plate reader (Perkin Elmer). Cells were then seeded in 96-well white polystyrene flat-bottomed plates (Corning #3917 in 100 uL/well or 30 μL/well for 384-well plates) at concentrations experimentally determined to ensure logarithmic growth during the duration of the experiment and prevent adverse effects on cell growth by DMSO exposure. The plates were incubated at 37°C in a 5% CO₂ atmosphere for 24 h before treatment. Stock solutions of test compounds (10 mmol in DMSO) in nine 3-fold serial dilutions were dispensed via pintool. The final concentration of DMSO was 0.3 % (v/v) in each well. Positive controls included staurosporine (25 μM), and gambogic acid (10 μM). The plates were incubated for 72 h at 37°C in a 5% CO₂ atmosphere, and then quenched with CTG, 50 μL per well (96-well plate and 30 uL for 384-well plate) at RT. Plates were then incubated at RT for 20 min and centrifuged at 1000 rpm for 1 min. Luminescence was read on an Envision plate reader (Perkin Elmer). The mean luminescence of each experimental treatment group was normalized as a percentage of the mean intensity of untreated controls. EC₅₀ values were calculated by Pipeline Pilot software (Accelrys, Enterprise Platform, CA, USA) and from dose response curve-fitting via non-linear regression.

Propidium iodide (PI) assay was performed to complement the viability assay. The assay was conducted with HTC116 cancer cell models seeded at 5 × 10³ cells/well (96 clear, Corning#353072) and cultured for 12 h, then treated for 72 h. Cells were fixed with cold methanol, and stained (0.4% PI, Sigma-Aldrich). Fluorescence was measured and analyzed with Lionheart FX (BioTek, Winooski, VT, USA). Cell viability was calculated/plotted as percent of surviving cells after treatment relative to vehicle wells (GraphPad Prism 7.0).

Annexin V-FITC Apoptosis and Cell Cycle:

The samples were probed with AnnexinV-FITC (Roche/Boehringer Mannheim) according to the manufacturer’s instructions. SUB-P15 and HCT116 cells were plated (1.00 × 10⁶ cells/plate) and incubated for 12 h, at 37°C. Then, cells were treated with compounds or control reagents for 24 h. Cells were stained with AnnexinV-FITC, PI and the staining profiles were determined with FACScan and Cell-Quest software. For cellular DNA content, the same cell treatment as above was performed. Then, cells were fixed in cold 75% ethanol, treated with RNase and then stained with PI solution (50 μg/mL). Cell cycle distribution was analyzed with the FACSCalibur analyzer (BD Biosciences, Franklin Lakes, NJ) and Cell-Quest software. The percentage of DNA content at different phases of the cell cycle was analyzed with ModFit-software (Verity Software House, ME, USA).

Statistical analysis and data availability:

For the CTG assay, three two-replicate assays were conducted for each experimental condition and a minimum of three independent experiments for validation were conducted. The mean luminescence of each experimental treatment group was normalized as a percentage of the mean intensity of untreated controls. EC₅₀ values (μM) were calculated by Pipeline Pilot Software (Accelrys, Enterprise Platform, CA, USA) or were calculated from dose response curve fitting using non-linear regression GraphPad (Version 7.0 San Diego, CA). For cell viability, cell cycle progression, and cell death assays, the analysis was conducted via one-way or two-way ANOVA with Tukey’s HSD test for each condition (concentration, cell cycle stage, apoptotic event, cell line) using GraphPad (Version 7.0 San Diego, CA). Additional details and data are included in Supplementary Information.

Results and Discussion

Given the challenges in modulating the high reactivity of aldehydes and diesters, solvent and additives were first evaluated, supported by the existing extensive nucleophilic addition reaction studies, which have previously surveyed regarding the reaction scope, solvent efficiency, and the base (including organocatalysts, transition metal¹⁶ and lanthanide mediated reactions¹⁷). First, we investigated phase-transfer-catalysis (PTC).¹⁸ PTC systems contain two immiscible liquid phases with a heterogeneous PTC promoting the conversion of the reactant to the desired product. These reagents are easy to operate, lead to higher yields, and eliminate the use of expensive solvents. Tetra n-butylammonium bromide (TBAB), methyl trioctylammonium chloride (MTOAC) and triethylbenzylammonium chloride (TEBAC) were evaluated in toluene/benzene/water systems. First, one equivalent of ethyl malonate and two equivalents of acrolein were evaluated with varying catalytic loads of PTC, base and 2 mol% TEBAC produced the desired product compound 1 (Scheme 1), when using aprotic solvent systems. Because the aldol reaction involves the nucleophilic addition of a ketone enolate to an aldehyde to form a β-hydroxy ketone, or aldol product followed by a dehydration—an elimination reaction, the base and solvent were then investigated. The type of base used can modulate the reaction direction to provide synthon 4 or 5 (See SI). Most of the tested organic bases such as DBU, 2,6-di-tert-butylpyridine and Hünig’s base failed to afford the expected synthons or traces of compound 1, except tert-BuOK.

In screens of inorganic bases, K₂CO₃ or K₃PO₄ performed better, whereas Li₂CO₃, Na₂CO₃, and Na₃PO₄ failed to produce the desired cyclized product 1, only traces of compound 4, 5 and acrolein were recovered along with polymerized by-products. Thus, the potassium cation was able to stabilize the proposed intermediates and enable the cascade reaction to proceed favourably toward compound 1 and 2, whereas smaller cations were not.

The use of polar aprotic solvents can have a significant effect on the rate of S_N2 reactions because they do not solvate nucleophiles.¹⁹ We observed that the lithium bases were ineffective, which we attribute to the small ion strongly chelating to the nucleophile. Applying crown ether under the identified conditions provided better yields. Polar aprotic solvents (ethyl acetate, dichloromethane, chloroform and tetrahydrofuran in the presence of moisture) alone or in combination with non-polar solvents (benzene, toluene, xylenes), provided the desired compound in varying yields, while polar protic solvents (methanol, ethanol, water) did not favour the desired reaction as expected. Gratifyingly, polar aprotic solvents (1,4-dioxane, DMSO) led to optimal reaction results, including shortening the time of reaction without TEBAC (scheme 1, condition b). 2D NMR studies established the general structure of the product, and x-ray analysis (See SI) confirmed the structure and established the exact stereochemistry of its three consecutive stereogenic centers of compounds 1 and 2. The malonate esters (alkyl, aryl) were tolerated, but substituted (alkyl, aryl) unsaturated aldehydes did not provide the desired cyclized products. On the other hand, 6a-6c provided the corresponding intermediates (7a-7c) with low-order molecular complexity. In addition, several nucleophiles other than diethyl malonate (8a-8i, Scheme 2) failed to produce compound 1-like cascade products presumably due to unfavourable nucleophilicity.

Scheme 2.

Evaluation of reaction substrates.

The aldol addition product is dehydrated by a strong base (e.g. K₃PO₄) in an enolate mechanism and we speculated and later confirmed that compounds 4 and 5 were being generated close to 1:1 ratio. The highly conjugated Knoevenagel product 5 is favoured by the reaction of diethyl malonate and acrolein (Figure 2A) under dehydrating conditions.²⁰ Although, the extended 1,6-Michael addition intermediate A was not isolated, we hypothesize that the reaction could proceed through intermediate B via pathway a (Figure 2B) after reacting with compound 4, which is the limiting reagent. Once intermediate B is generated, it would presumably form compound 1 irreversibly via decarboxylation. An alternative pathway is possible if intermediate B encounters unreacted acrolein via pathway b, following a similar course to form compound 2. This cascade reaction sequence is presumably driven by the highly reactive intermediates, which undergo decarboxylation in an exothermic process. Validation studies included the evaluation of compound 4 or 5 alone using the optimal reaction conditions (Figure 3). Neither compound 1 or 2 were detected after 72 h when using either compound 4 or 5 as the sole starting material, rather complex mixtures and decomposition were observed. Although, we could not isolate intermediate A, we confirmed that the combination of compound 4 and 5 undergo cyclization to produce compound 1 in moderate 16 % yield with no trace of compound 2. Finally, compound 1 was allowed to react with access acrolein for 72 h under the optimal reaction conditions and no production of compound 2 was observed, suggesting this cascade reaction takes place in a rapid stepwise manner. The scope of reactivity for this reaction is restricted primarily by steric factors, which limit the formation of the corresponding 4 and 5 intermediates in sufficient quantities to form appreciable amounts of intermediate A or B. Although the exact sequence of the reaction events is still under investigation, the cascade reaction can produce compound 1 in multi-gram quantities effectively, therefore enabling the establishment of a medicinal chemistry platform.

Figure 3.

Evaluation of proposed mechanism.

Previous biological studies of isodon diterpene compounds suggested that the α,β-unsaturated system was necessary for biological activity against cancer cell models.²¹ An exocyclic olefin is present in those systems, however, which would be more readily accessible than the endo-olefin found in compound 1 and 2. To gather further information on the structure-activity-relationship of compound 1 and 2, modifications were conducted at the C-7, C-8 olefin and C-9 of compound 1. For compound 2, the side chain at C-2 was further derivatized while maintaining the endocyclic olefin (Figure 4).

Figure 4.

Synthesis of 11–18. Reagents and conditions:(a) NaOH (aq), THF, MeOH, 25°C, 73%. (b) H₂O₂ (30% aq, w/w), NaOH, THF/MeOH, 61%. (c) OsO₄/NMO, acetone and H₂O, 82%. (d) Br₂, CH₂Cl₂, 70%. (e) Rh₂(cap)₄, t-BuOOH, K₂CO₃, CH₂Cl₂, 12%. (f) OsO₄/NMO, acetone and H₂O, 67%. (g) thionyl chloride, Et₃N, CH₂Cl₂, 87%. (h) N-Boc- piperazine, benzene, 60°C, 77%. (i) NaN₃ , DMSO, 60°C, 1h, 92%.

The rate of a saponification reaction is altered by steric and electronic effects of the substrate. Saponification processes typically involve the use of aqueous solutions of hydroxides in excess (KOH, NaOH, LiOH), in concentrations between 0.1 N and 2.0 N. Compound 1 yielded a mixture of inseparable products under such hydrolysis conditions at RT. However, using four equivalents of base in tetrahydrofuran/methanol/water (5:1:1) as solvent system at RT, resulted in 73% yield of the desired compound 11. The epoxidation of the α, β-unsaturated system in heterogeneous reaction solvent (THF/MeOH) using aqueous hydrogen peroxide as an oxidant provided the corresponding epoxylactone 12 in good yield. Catalytic osmium tetroxide in the presence of NMO mediated the dihydroxylation of the olefin to the corresponding 1,2-diol, 13, in good yield. Several conditions for the bromination of the unsaturated enone system were evaluated (NBS, Br₂/AgOTf, etc.), and none provided the desired brominated compound, but the conditions reported by Djerassi et al.²² without additives led to compound 14 as a single compound in 70% yield. Most allylic oxidation attempts failed to generate compound 15. Gratifyingly, we found that catalytic dirhodium (II) caprolactamate [Rh₂(cap)₄] (0.1 mol %) in combination with tert-butyl hydroperoxide²³ effectively provided 15 in 1 h albeit in modest 12% yield. Next, Compound 2 was treated with catalytic osmium tetroxide and excess NMO in aqueous conditions to yield compound 16 in 67% yield. To maintain the olefin systems, we used thionyl chloride to perform an allylic transposition on compound 2 in DCM at RT, compound 17 was readily formed in good yield. Next, displacement of the chloride with 4-Boc-piperazine under thermal conditions provided compound 18 in 77% yield. Finally, we turned our attention to prepare the chemical probe, azide 19 to facilitate target identification efforts. One of the major challenges in phenotypic screening is the identification of the molecular targets of bioactive compounds and the most appropriate strategy is mass spectrometry (MS)-based proteomic approaches. The corresponding typical affinity-isolation experiment (“pull-down”) involves the synthesis of a suitably functionalized derivative of a hit compound, as compound 19 which will serve in click reactions with biotin/other tags and will be incubated with cell lysates to capture bound proteins non-covalently as we have previously shown with other natural product chemical probes.²⁴

Acute lymphoblastic leukemia (ALL) is a leading cause of death among adolescents and young adults (AYA).²⁵ Although, strong evidence indicates that ALL patients with receptive prognostic cytogenetics (i.e. trisomies of chromosome 4, 10 or 17 or t(12:21)/TEL-AML1) have high survival rates, there is a need for new therapeutic agents targeting the high-risk patient cohorts, such as the Philadelphia chromosome (Ph/BCR/ABL+) lesion among other genetic abnormalities. Although, response to high-risk drug regimens varies, drug resistance and disease recurrence are frequent causes of treatment failure.^26–27 It is also a fact that AYA patients with colorectal cancer have a poorer prognosis and more aggressive disease than older adults.²⁵

Thus, we evaluated the cytotoxicity of the newly generated compounds 11–18 against cellular models of these cancer subtypes to identify new molecular scaffolds with clinical potential. The cytotoxicity of these derivatives was assessed by an established cell viability assay previously described,⁵ and cell death was validated by additional studies (Figure 5). A summary of the cytotoxicity findings is shown in Table 1. The high-risk cellular models of ALL (SUP-B15, KOPN-8 and MV411) and HCT116 for colorectal carcinoma along with the non-cancerous cell line BJ were evaluated using a dose-response proliferation assay (CTG) to determine therapeutic index.⁶

Figure 5.

Representative CTG graphs for compounds (A, D). Annexin V/Propidium Iodide (PI) staining and cell cycle analysis (B-C, E-F). Bars depict mean and SD of at least three independent experiments. ****P<0.0001, ***P<0.0006, *P<0.0201 and ns (not statistically significant) according to Tukey’s test when compared to DMSO control.

Table 1.

Cytotoxicity CTG assay of 1, 2 and a selected number of analogues against several cancer cell lines. Graphs of experiments (SI) and values indicated means ± standard error of the means (SEM) from three independent experiments.

#	SUP-B15 (μΜ)	KOPN-8 (μΜ)	MV411 (μΜ)	HCT116 (μΜ)	BJ (μΜ)
1	51 ± 21.21	55 ± 23.65	40 ± 11.30	40 ± 11.28	>36
2	7.4 ± 2.38	19 ± 3.64	15 ± 3.02	7.66 ± 4.22	>36
11	82 ± 35.15	>49	2.5 ± 1.07	>35	>36
12	>16.6	ND	ND	40 ± 7.09	>36
13	55 ± 2.5	155 ± 62	22 ± 5.6	22 ± 4.48	>36
14	24 ± 8.4	6.8 ± 2.29	13 ± 4.8	10.47 ± 3.49	>36
15	37.8 ± 6.89	36 ± 8.84	3.32 ± 1.63	18 ± 3.1	>36
16	66 ± 5.78	26 ± 8.06	37 ± 7.2	27.39 ± 4.8	>36
17	3.8 ± 2.21	4.5 ± 1.93	3.3 ± 1.6	3.3 ± 1.06	>36
18	3.5 ± 1.4	4.5 ± 2.9	2.5 ± 1.07	2.5 ± 1.62	>36

Compounds 17-18 show half maximal effective concentration (EC₅₀) in the low micromolar range (<10 μM) and display therapeutic index (TI>5). The data indicate that the combination of the α, β-unsaturated system (C-7/C-8) and the appended side chain at C-4 might be responsible for the observed bioactivity. Our study shows that compounds 2, 17, and 18 showed the most promise across cell line models (representative graphs for selected compounds Fig. 5, A, D). To determine whether the inhibitory effect on cell viability of these compounds was due to cell death and effects on cell cycle progression, annexin V/propidium iodide assays were performed for compound 17 using SUP-B15 and HCT116 cancer cells. The study demonstrated that both cell types (SUP-B15 and HCT116) undergo cell death in a similar pattern, with a considerable decrease in live cells after 24 h (Figure 5, B–C, E–F). DMSO and staurosporine were used as the negative and positive controls respectively. A significant increase in apoptotic cells was observed for SUP-B15 but not for HCT116, indicating longer drug-exposure time is required to capture such effects. The DMSO control indicates that both cell lines were primarily at the G1/G0, and small numbers (~10%) were entering the S and G2/M phases at the time of the experiment. Staurosporine showed cell arrest in the S phase in SUP-B15, but G2/M arrest in HCT116. Interestingly, compound 17 did not produce a significant cell arrest in SUP-B15, but rather an equal distribution between G2/M and S phases, and significant decrease in G1/G0 compared to DMSO control. Whereas it showed significant cell arrest in G2/M in HCT116 and decrease in G1/G0 and S phases. Interestingly, the antiproliferative profile of compound 17 in HCT116 was comparable to that of staurosporine, which can directly activate the mitochondrial apoptosis pathway similar to a broad-range of kinase inhibitors and clinical anticancer drugs that requires caspase-9 for apoptosis independent of death receptor signaling.²⁸ Our future studies include target (s) identification of these lead compounds to improve potency. The identified compounds offer ample opportunities for further chemical derivatization and biological evaluation.

Conclusion

In summary, our study describes a new cascade reaction application based on a series of nucleophilic addition reactions. The substrate scope survey demonstrates the efficiency of this cascade reaction sequence to afford enmein-type complex natural product mimetics in multigram scale, which exhibit important bioactivity profiles against several cancer cell models. Although the presently developed cascade reactions had a limited substrate scope, the potential of its extension to electrophiles other than acrolein as a Michael acceptor could provide a mean to achieve more complex molecular scaffolds, and an asymmetric version of this reaction is under investigation. This synthetic approach served as a rapid entry to the synthesis of complex molecular scaffold with antiproliferative activity against cancer cell models. Compounds 17 and 18 showed significant antiproliferative properties in the low micromolar range against multiple cancer cell lines with no cytotoxicity in normal cell lines at the tested concentrations. Further efforts on extending this cascade process to different reactants and trapping other reactive intermediates for the discovery of new chemical matter are currently being pursued in our laboratory.

Highlights.

• A base-catalyzed cascade reaction sequence to higher molecular complexity was discovered

• The newly generated polycyclic molecular scaffold can potentially lead to enmein-type isodon diterpenes

• Identification of new promising polycyclic compounds against multiple cancer cell lines