Natural and Semi-Synthetic Pseudoguaianolides as Inhibitors of NF-κB

Damsin (1) is a natural pseudoguaianolide sesquiterpene that inhibits NF-κB, a protein complex that controls the transcription of DNA in mammalian cells, and has a potential for standing model for the development of new anti-cancer lead structures. In order to do a preliminary structure-activity study and improve the anti-cancer activity, fourteen derivatives and analogs were prepared and evaluated. These were chosen to represent both structural diversity and structural novelty. The importance of α methylene-γ-lactone moiety for the anti-cancer activity was confirmed, even though other features in the scaffold were shown to be important for the activity. In some cases a new substitution negatively affected the initial activity, however, two analogues, indolo [3,2-c]-4desoxydamsin (5) and ambrosin (6), were found to be more potent.


Introduction
NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells, is a family of protein complexes that control the transcription of DNA as transcription factors.They are involved in the cellular responses to various stimuli, an important part of the immune system and responsible for cytokine production and cell survival.Poor regulation of NF-κB is linked to several serious conditions, including cancer.The NF-κB complexes include the subunits RelA (p65), NF-κB1 (p50; p105), NF-κB2 (p52; p100), c-Rel and RelB.The amino-terminal Rel-homology domain (RHD) is conserved within the family, containing the subdomains for dimerization, nuclear localization and DNA-binding.Therefore, homo-and heterodimers between these proteins can be found in vivo, except for RelB, which just forms heterodimers.A dimer is normally associated with the inhibitory protein IκB, and the complex (NF-κB-IκB) remains inactive in the cytoplasm.A broad number of stimuli are responsible for the activation of such complexes, e.g.pathogens, stress signals and pro-inflammatory cytokines, by activating IκB kinases (IKK) that initiate a process that eventually degrades IκB.Free and activated NF-κB can then be translocated into the nucleus and start the transcription of target genes.The most well-known heterodimer, and present in most mammalian cells, is RelA/p50 [1]- [3].The main biological functions of NF-κB are the activation of immune response and inflammation via the expression of genes encoding for cytokines, cytokine receptors, and cell-adhesion molecules [1].
In recent years, a deeper understanding of the NF-κB pathway and its role in inflammatory processes has been established, creating a molecular link between the chronic inflammatory diseases and cancer [4] [5].Specifically, this transcription factor has been shown to be crucial in oncogenesis and tumor progression, inhibiting apoptosis by transcribing genes for anti-apoptotic proteins or suppressing genes for apoptotic proteins.In a state of constant activation of NF-κB malignancies may progress even if apoptosis is induced by anti-cancer treatments, leading to chemoresistance [6].For these reasons, the NF-κB family is considered to be an interesting target for the development of new anti-cancer drugs [7]- [9], although it should be noted that pro-apoptotic and tumor suppression functions of NF-κB have been reported in some cancer cell lines [10]- [15].Nevertheless, new inhibitors of NF-κB are required in order to study and understand the following steps in the signal transduction: a) the blocking of the binding of NF-κB to DNA; b) the inhibition of the nuclear import system to prevent NF-κB from reaching the nucleus; c) the inhibition of the IκB degradation ensuring that NF-κB remains inactive; d) the inhibition of the IKK kinases that initiate the degradation of IκB [16].
Sesquiterpenes with an α-methylene-γ-lactone moiety (e.g.damsin (1), see Scheme 1) constitute a class of natural products with potential as anti-cancer agentsby inhibiting NF-κB [17]- [21].Structure-activity relationship studies have demonstrated the importance of the rigid skeleton of pseudoguaianes and/or guaianes with an α-methylene-γ-lactone moiety, as it appears to contribute significantly to both the anti-cancer activity and the NF-κB inhibition [22] [23].The critical property of these compounds is the ability of the α-methylene-γ-lactones to react as Michael acceptors with thiol groups of cysteines in proteins, modifying the protein covalently [17] [19] [21] [22] [24]- [26].The reaction of the thiol group ofcysteine-38 in RelA with a Michael acceptor is considered to be the mechanism by which the α-methylene-γ-lactone sesquiterpenes prevent the binding of NF-κB to DNA.This cysteine is considered crucial for the interaction between NF-κB and DNA, association with the coactivator RPS3 and antiapoptotic gene expression [27].The importance of this cysteine residue was also demonstrated by studying mutants of RelA/NF-κB and measuring their ability to bind to DNA in the absence or presence of α-methylene-γ-lactone sesquiterpenes, showing that this site is affected by this class of natural products [28]- [30].
We have focused our interest on damsin (1), a pseudoguaiane sesquiterpene that can be isolated in large amounts from the plant Ambrosia arborescens Mill.[31].This natural product is cytotoxic towards Hep-2 cells (epidermoid carcinoma of larynx) [32] and Eagle's ΚB cells (nasopharynx carcinoma) [33].In addition, antiproliferative activity was reported in U937 cells (monocytic leukaemia), Jurkat cells (leukaemia T) and Molt 4 cells (acute lymphoblastic leukaemia) [34].Recently we reported the antiproliferative activity of damsin (1) towards Caco-2 cells (epithelial colorectal adenocarcinoma) and its inhibitory effect on NF-kB and STAT3 (a signal transducer and activator of transcription) [35].In an effort to prepare new derivatives at least retaining the NF-kB inhibitory capacity, a broad range of chemical transformations were carried out with damsin (1).As 1 is chiral, all derivatives obtained from it (see Scheme 1) are consequently pure enantiomers with the absolute configuration as shown.The transformations were partly inspired by the reports from the 1960s, when the structure elucidation of sesquiterpene lactones was based on functional group interconversions [36] [37].In the later years, the derivatisation studies have focused on the improvement of their biological activities, such as cytotoxicity [38] [39], and the development of pro-drugs as anticancer agents [40] and anti-malarial [41].Such studies usually only utilized specific chemical reactions, but the aim of this study was to use a broad scope of reactions in order to generate novel structures that are not found in plants.

Results
Damsin (1) and coronopilin (2) are known pseudoguaiane sesquitepenes that can be isolated from Ambrosia arborescens Mill.[34] [36].As the isolation of 1 in larger amounts was achievable, it was selected as the starting material for all the chemical modifications.Our ambition was not only to modify the Michael acceptor moiety and consequently the reactivity, but also to introduce new groups into the pseudoguaiane skeleton in order to investigate the effect of non-covalent interactions.The compounds prepared can be divided in three categories (Scheme 1): Group A, compounds retaining the α-methylene-γ-lactone moiety; Group B, compounds with a modified Michael acceptor in the γ-lactone moiety and Group C, compounds lacking a carbon-carbon double bond in the γ-lactone ring.
Group A. New hydrogen donors/acceptors in 1 were introduced in compounds 3 and 4. A Rubottom oxidation provided 3 that is hydroxylated in position 3 (Scheme 1) [42], while a selective reduction of the keto function through a Luche reduction with NaBH 4 in presence of CeCl 3 afforded 4 [43].A Fischer indole synthesis with phenylhydrazine gave 5 that has a planar and rigid structure.To avoid competitive reactions of the hydrazine with the α methylene-γ-lactone moiety, the thiophenoxide-protected adduct (17) was used as starting material (Scheme 2(a)).The molecule was deprotected by a selective oxidation of the thioether to a sulfoxide with m-CPBA at −20˚C, followed by a thermal elimination.Ambrosin (6), a natural product, was prepared via a Saegusa-Ito oxidation [42].6 is unique in this investigation by containing a second Michael acceptor function.Group B. The isomerization of 1 to 7 was achieved by treatment with RhCl 3 [42], providing a derivative that still is a Michael acceptor but less available for nucleophilic attack and consequently less reactive [40].8 was designed to facilitate a Michael addition by providing an intramolecular basic catalysis for an incoming thiol [44].Several strategies towards 8 were investigated, including metathesis and a Wittig olefination reaction, but the procedure that eventually succeeded (see Scheme 2(b)) starts with a Michael addition of a cyanide ion to the acetal 20 to give the nitrile 21.After the reduction of 21 to the tertiary amine 22 and the oxidation of the lactone ring of 22 via the bromine 23 to the olefine 24.Finally, 8 was obtained by the hydrolysis of the acetal protection group.Compound 9 was prepared from 7 by a procedure similar to the Rubotton oxidation [45].Firstly, 7 was protected as the acetal 25, which wasoxidized to 27 via the 2-hydroxyfuran silylether 26 (not isolated), and finally deprotected by hydrolysis to yield 9 (Scheme 2(c)).Compound 10 was prepared from 1 by a Heck coupling reaction, following procedures reported for α-methylene-γ-lactones [46] [47].However, the isomerization could not be avoided, yielding the endocyclic α, β-unsaturated-γ-lactone 10.
Group C.Even though it has been stated that the absence of the α-methylene-γ-lactone moiety renders this class of terpenes inactive as inhibitors of NF-κB [29], we wanted to include also such compounds in this investigation.The reduction of the α-methylene-γ-lactone of 1 with NaBH 4 gave the two epimers 11 and 12 [42].The Claisen-Schmidt condensation of 1 with benzaldehyde in ethanol gave 13 [48].The hydroxylated compound 14 was prepared following an iterative procedure [49] with the hydrogen peroxide and catalyst 16 (Scheme 2).Although this catalyst hydroxylated the desired tertiary position, the epoxidation of the double bond was unavoidable.Finally, the reaction of 1 with dimethyl amine in ethanol yielded the amine adduct 15 (as an epimeric mixture 3:1 11α:11β) [50], for evaluation of its possible usefulness as a pro-drug.
The configuration of the products and intermediates was determined by comparing the 1 H NMR coupling constants and correlations observed in the NOESY spectra, with computational models.The conformational search was carried out using the MMFFs force field and the low energy conformers were optimized using B3LYP/6-31G** basis set.In several cases a second order system was resolved in order to determine the coupling constants, and this was done by approximation using spin simulation.For compound 4, for example, the coupling constants and atomic distances of the 4β-hydroxyl group were experimentally and computationally correlated as follows.While the experimental coupling constants of H-4α with H-3α and H-3β both are 9.0 Hz, the calculated coupling constants were 8.0 Hz for H-3α and 8.1 Hz for H-3β.In addition, a NOESY correlations was observed from H-4α toH-1, corresponding with the calculated distance (2.40 Å), as well as to H-6 (calculated distance 2.26 Å).The chiral centers as well as the double bond configurations were assigned in the corresponding way for all the derivatives.The structural elucidation of 3, 11 and 12 has previously been reported by us [42].
The coupling constants and atomic distances of each compound with a new chiral center are experimentally and computationally consistent as follows: Compound 9. A NOE correlation was observed between OH-6 and H-1, corresponding with the calculated distance of 3.04 Å.
Compound 13.The configuration of C-11 was assigned considering the experimental coupling constant of H-11β with H-7α (7.2 Hz), compared with the calculated coupling constant (11.9 Hz).In addition, NOESY correlations were observed between H-11β andH 3 -15, H-8α, H-8β and H-9β corresponding to the calculated interatomic distances 2.36, 3.01, 3.86 and 2.16 Å, respectively.NOESY correlations between H 2 -13 to-OCH 2 CH 3 (weak), H-7, -OCH 2 CH 3 and H-6 correspond to the interatomic distances 4.60, 2.70, 2.40 and 2.54 Å, respectively.A second order system (ABM) was resolved for H-13a and H-13b, to discriminate their coupling constants with H-11β.The error is small enough to confirm the proposed stereochemistry, even though the contribution of more than one conformer could explain the divergence.In addition the E isomerism of the 3-phenylethenyl, was confirmed trough NOE correlations of H-2' with H-2a and H-2b (2.224 Å and 2.449 Å, respectively).
Compound 15.The relative amounts of the two diastereomers in the epimeric mixture obtained was made using the integral of H-6 for each epimer.The complete assignation was done by a careful analysis of the 2D spectra.The configuration of C-11 in the two epimers was determined by the NOESY correlation observed between H-11 andH 3 -15 in the spectrum of the major epimer (11β, 13-dihydro-13-(N, N-dimethylamino) damsin)),and the correlation between H-11 and H-6 for the minor epimer (11α, 13-dihydro-13-(N, N-dimethylamino) damsin.
Compound 17.The configuration of C-11 in 17 was suggested by the experimental coupling constant between H-11β and H-7 (7.7 Hz) compared with the calculated coupling constant (11.4 Hz).This was confirmed by the NOESY correlations between H-11β and H-8β (weak), H-9β and H 3 -15 corresponding to the interatomic distances 3.01, 2.23 and 2.37 Å, respectively.
Compound 18.The configuration of C-11 in 17 was suggested by the experimental coupling constant between H-11α and H-7(6.0Hz) compared with the calculated coupling constant (6.4 Hz).A second order system (AMNX) was resolved for H-11α, H-7, H-13a and H-13b, in order to determine their coupling constants and chemical shifts.In addition, a NOESY correlation was observed between H-11α and H-6 corresponding to the interatomic distance of 2.51 Å.
Compound 22. NOESY correlations were observed between H-11β and H-8β as well as H 3 -15, corresponding with the calculated distance of 2.42 and 2.44 Å, respectively.

NF-κB Inhibition and Structure Activity Relationship
To analyze the NF-κB inhibitory activity of the synthesized compounds we used the stably transfected cell line 5.1, a lymphoid T cell line in which the HIV-1 LTR is activated by TNF-α through an NF-κB dependent mechanism [51].The HIV-1 LTR promoter contains two κB sites that are critically required to respond to TNFα, and it is well known that deletion of the two κB sites in the LTR promoter abolishes completely the response to TNFα.Thus, 5.1 cells represent an excellent cellular model for the screening of anti-NF-κB compounds.We have previously found that dasmin (1) and coronofilin (2) possess anti-NF-κB activity, with IC 50 values of 7.2 and 10.1 µM respectively [35].As shown in Table 1, two compounds from group A (5 and 6) have a lower IC 50 value compared to 1, suggesting that indolic system slightly favors the ability of the compound to interact with the protein.Compound 6 is the most active, probably due the presence of two α, β-unsaturated carbonyl moieties that increase the alkylating capacity of the product, and this has been noted previously [18] [23] [52].
On the other hand, compounds 2, 3 and 4 still possess theoriginal α-methylene-γ-lactone but are less potent (especially 3 and 4) compared to 1.These results strongly indicate that a number of intermolecular interactions between the compound assayed and the protein it affects modulate their effects, and that a more systematic QSAR study to understand such interactions in detail is motivated.The compounds in group B possess a modified α-methylene-γ-lactone moiety.Compound 7 is inactive at the maximal concentration tested, which could be explained by the lower reactivityof the endocyclic α, β-unsaturated-γ-lactone moiety and its inability to form stable adducts with thiols [53].However, compound 8 is also inactive although it was designed to provide support for a thiol attacking C-13.Possibly the steric hindrance posed by the C-13 substituent prevents an attack.Compound 9 is an interesting exception with an intermediate activity, possibly caused by a hydrolysis of the hemi-acetal lactonic ring forming a 1,4-enedione with a higher reactivity.Compound 10 is less potent, but the comparison with 7 and indicates that the aromatic group in position 13 slightly enhance the ability of the compound with the protein.
The compounds of group C as 11 and 12 are inactive as was predictable, confirming the crucial role of the α-methylene-γ-lactone moiety for the inhibition of NF-κB [23].The inactivity of compound 14 demonstrate that other electrophilic groups, such as an epoxide, in positions 11, 13 can not replace the α-methylene-γ-lactone moiety, even though the position is the same.Compounds 13 and 15 are weakly active, this could be explained ifthe Michael addition of etoxide and dimethyl amine to the α-methylene-γ-lactone moiety is reversible but this was not further investigated.Amines like 15 are classical pro-drugsof sesquiterpene lactones [18] [40] [50], although 15 is considerably less activecompared todamsin (1) in this study.

Conclusion
In total, fourteen derivatives were prepared, characterized and assayed.Several (7, 8, 11, 12 and 14) were inactive at the tested concentrations, confirming the importance of the α-methylene-γ-lactone moiety for the activity.Nevertheless, compound 10 is slightly active, suggesting that also endocyclic Michael acceptors may be active and that an aromatic group in position 13 can be important.Also, compound 9 was unexpectedly active, probably because the hemi-acetal can be hydrolyzed to a more reactive 1,4-enedione.The compounds 2, 3 and 4 are somewhat surprisingly less active compared to 1, indicating that hydroxyl groups on positions 1, 3 or 4, respectively, have a negative effect on the ability of the sesquiterpene to interact with the protein.For compound 15, the ability to regenerate the α-methylene-γ-lactone moiety during the performance of the assay is the probable reason for its modest activity, amine adducts (15) have previously been demonstrated to possess such a reversibility [50].This is not the case for ethers, e.g. 13, so there may be another reason for its modest activity.Finally, compounds 5 and 6 are more potent compared to 1.This was expected for 6 as it possesses two Michael acceptors, but unexpected for 5. Possible explainations are that the indol system imposes a steric strain on the molecule, including the α-methylene-γ-lactone moiety, which could be relieved if C-11 no longer is part of an unsaturation.Alternatively the indole may be involved in some specific interaction with the molecular environment of the reacting cysteine, favoring the position of the α-methylene-γ-lactone moiety in the right place.The need for a more systematic investigation is evident.

Experimental a) Chemistry General
All other chemicals were obtained from commercial suppliers of analytical grade.High Resolution Mass Spectrometry (HMRS) Electrospray Ionization (ESI) spectra were recorded with a Micromass Quadrupole-time of Flight (Q-TOF) Micro spectrometer.Nuclear Magnetic Resonance (NMR) spectra (in CDCl 3 ) were recorded with a Bruker DRX at 400 Mhz ( 1 H) and at 100 MHz ( 13 C).Chemical shifts are given in ppm relative to the residual CHCl 3 in CDCl 3 (7.25 ppm 1 H and 77.00 ppm 13 C).All flash chromatography was performed with 60 Å 30 -75 μm silica gel.Thin Layer Chromatography (TLC) analyses were come out on silica Gel 60 F254 (Merck, Darmstadt, Germany) plates.

Table 1 .
Inhibitory activity (IC 50 ) on TNFα-induced NF-κB activation.The results represent the mean of three independent experiments and the SD was less than 10%.