Synthesis and Biological Evaluation of Simplified Pironetin Analogues with Modifications in the Side Chain and the Lactone Ring

a Depart. de Q. Inorgánica y Orgánica, Univ. Jaume I, Castellón, E-12071 Castellón, Spain. E-mail: jmurga@uji.es b Current address: Depart. de Química. Univ.de Girona, E-17003 Girona, Spain. E-mail: steven.roldan@edg.edu c Current address: Depart. de Q. Analítica y Q. Orgánica. Univ. Rovira i Virgili, E-43007 Tarragona, Spain. E-mail adrian.cardona@urv.cat d Depart. de Q. Orgánica, Univ. de Valencia, E-46100 Burjassot, Valencia, Spain. E-mail: alberto.marco@uv.es


Introduction
It is widely known that cancer, one leading cause of death in developed countries, may be induced by a plethora of both external and internal factors, including genetic mutations.Accordingly, a number of types of therapeutic attack has been investigated. 1One of these involves the use of cytotoxic drugs, which exert their effect in many cases by means of inducing various mechanisms of cell death. 2 As a matter of fact, many of such drugs owe this property to interaction with the microtubule network.Microtubules are dynamic polymers that play a central role in a number of cellular processes, most particularly cell division, as they are key constituents of the mitotic spindle. 3Microtubules are constituted of a protein named tubulin, the functional form of which, and the most abundant component, is a heterodimer formed through noncovalent binding of two monomeric constituents, called and -tubulin.For cell division to occur in a normal way, microtubules must be in a constant state of formation and disruption, a process named microtubule dynamic instability. 4olecules which influence microtubule instability will also influence the cell division process, not only of normal cells but also of tumoral ones.Therefore, it is not surprising that tubulin-binding molecules (TBMs) constitute a very important class of anticancer agents. 5M are able to interfere with microtubule assembly and functions, either by causing disruption of the microtubules or else through their stabilization.Most of the hitherto described active drugs are natural products or derivatives thereof. 6ajor drugs can already be found on the market and many other promising compounds are in clinical trials.
TBM may be divided in two broad categories, those that bind to -tubulin and those that bind to -tubulin.The latter group is presently by far the most numerous and contains products which cause either disruption 7 or stabilization 8 of microtubules.The number of products that bind to -tubulin is, however, very small, 9 the naturally occurring 5,6-dihydro--pyrone pironetin (Figure 1) being the first-reported example.Pironetin is a potent inhibitor of tubulin assembly and has been found to arrest cell cycle progression in the G2/M phase. 10This feature has motivated a number of groups to undertake total syntheses of this natural compound. 11It is worth mentioning here that 5,6-dihydro--pyrones constitute an ample group of natural products endowed with a broad variety of pharmacologically useful properties, most likely related to the presence of the Michael acceptor moiety in the pyrone ring.Some structure-activity (SAR) studies on pironetin have been reported. 10These studies have shown that the presence of the conjugated double bond in the lactone ring and of the hydroxyl group at C-9, either free or methylated, are essential for the biological activity. 9The epoxidation of the C12=C13 double bond has been shown to cause a decrease in the activity but this may perhaps represent a negative feature of the oxirane ring, rather than a strict need of this C=C bond.
As a member of the up to now small group of products that bind to -tubulin, pironetin constitutes a pharmacologically interesting target.Thus, a key purpose of our research is the preparation of pironetin analogues that retain a substantial proportion of the biological activity of the natural metabolite while displaying a more simplified structure.In order to develop SAR studies based upon the pironetin framework, we designed several years ago 13 a simplified model structure where all elements that had not yet proven to be essential for the biological activity were removed.The target structures I/II are schematically shown in Figure 2. The elements that were maintained are the conjugated dihydropyrone ring and the side chain with the methoxy group at C-9.The hydroxyl group at C-7 was removed in some substrates (I) and retained in others (II), in order to see its influence on the activity.All alkyl pendants (methyl groups at C-8 and C-10, ethyl at C-4) and the isolated C12−C13 double bond were removed.The configurations of the two/three remaining stereocentres were then varied in a systematic way.Thus, all four possible stereoisomers with general constitution I, with no hydroxyl group at C-7, were prepared.Likewise, all eight stereoisomers exhibiting general structure II, with a hydroxyl group at C-7, were synthesized.Subsequently, the cytotoxic activity of these analogues and their interactions with tubulin were investigated. 13gure 2 General structures of the first generation of simplified pironetin analogues (ref.13).
In continuation of this line of research, we concentrated our attention on the importance of the alkyl pendants in the pironetin molecule for the biological properties of the natural compound.In line with this reasoning, we prepared the six pironetin analogues III-VIII (Fig. 3).In all these compounds, the configurations at the oxygenated carbons C-5, C-7 and C-9 are as in natural pironetin.With respect to general structure II (Fig. 2), compounds III and IV contain an additional methyl residue at C-10 with either configuration, whereas in compounds V and VI, the extra methyl pendant is allocated at C-8.Finally, compounds VII and VIII display an extra alkyl residue (methyl or ethyl) at C-4, in both cases with the same configuration as in natural pironetin. 14he cytotoxic activities of pironetin analogues III-VIII were then investigated.Most compounds proved cytotoxic in the low micromolar range, therefore about two-three orders of magnitude less active than pironetin itself. 14These results suggest that all alkyl pendants are important for the full biological activity, this being most likely due to the fact that the alkyl groups restrict the conformational mobility of the molecule and reduce the number of available conformations. 15,16This in turn makes more probable that the molecule adopts a shape that fits better into the active site of -tubulin.

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In view of these results, we decided to prepare a new group of pironetin analogues with a higher degree of alkylation in the side chain but still retaining a simplified structure.Figure 4 shows the eight compounds we have prepared and evaluated for their biological properties.

Figure 4 Structures of compounds of the third series of pironetin analogues (this work).
In comparison to pironetin, pyrones 1, ent-1, 2 and ent-2 in Fig. 4 display a shorter carbon chain, two stereocentres less (C-4 and C-10 in pironetin numbering) and an additional gemdimethyl moiety (at C-6 in pironetin numbering).Furthermore, and in order to investigate the importance of the lactone ring size, analogues 3, ent-3, 4 and ent-4 having a furanone system were also prepared.
In recent times, we have not limited our biological investigations on bioactive molecules to solely measurements of their cytotoxic activity, expressed as IC 50 values.Indeed, while mechanisms of anticancer activity are often related to interference with microtubule assembly and functions, other mechanisms may also be operative.In most solid tumors, for example, angiogenesis is an important process for tumor growth and metastasis.Many different mediators are involved in this process, including the vascular endothelial growth factor (VEGF), which has been shown to play a critical role in pathological angiogenesis. 17other relevant mechanism in cancer genesis is related to the role of the chromosomal telomers.Most cancer cells exhibit telomerase activity.The latter mantains the length of the telomeres, thus preserving genomic stability. 18Telomerase is a ribonucleoprotein composed of two main subunits which, in the case of human beings, are called human telomerase RNA (hTR) and human telomerase protein (hTERT).Many studies have demonstrated that interference in the expression of the hTERT gene can efficiently inhibit the growth and tumorigenicity of cancer cells, as the hTERT gene is a rate-limiting factor in telomerase synthesis and activity.Equally important is the c-Myc gene, which has been found to be amplified in various types of human cancers.The result of the expression of this gene, the c-Myc protein, is a transcriptional factor with an important role in cell proliferation, differentiation, invasion and adhesion of tumor cells. 19It is also involved in the activation of hTERT gene transcription.
Since on one hand tumoral cell secretion of VEGF is an important factor in metastasis and, on the other hand, telomerase is responsible for the inmortality of tumoral cells, the potential multiple ability 20 of some compounds to perturb microtubule dynamics and, at the same time, to inhibit VEGF secretion by tumoral cells and the expression of the VEGF, hTERT and c-Myc genes was considered a goal worth pursuing.For that reason, we have also included the last types of biological activities in our unvestigation of the general pharmacological profile of our compounds.

Synthesis of compounds 1-4 and their enantiomers
For our purposes, we aimed at performing a simple synthetic sequence in which stereochemical complexity is rapidly achieved through a convergent methodology.Thus, the synthesis of compounds 1-4 was carried out as depicted in Scheme 1. Creation of chirality was achieved by means of an adaptation of a published organocatalytic procedure. 21Thus, propionaldehyde and isobutyraldehyde were allowed to react in DMF in the presence of D-proline.This gave a crossed aldol product which was subjected in situ to Barbier-type, indium-mediated prenylation to yield diol 5 in fair yield and high enantio-and diastereoselectivity. 21 Methylation to 6 was followed by ozonolysis to yield an unstable intermediate aldehyde which, without isolation, was allowed to react with allylmagnesium bromide in THF.This sequence furnished a mixture of diastereoisomeric alcohols 7 and 8 (d.r.2:1), which proved amenable to chromatographic separation.Both compounds were then esterified with acryloyl chloride, and the resulting acrylates, 9 and 10, were subjected to rutheniumcatalyzed ring-closing metathesis 22 to afford the target dihydropyranones 1 and 2, respectively.Their enantiomers ent-1 and ent-2 were obtained by means of an identical synthetic sequence with the only difference of using L-proline as the organocatalyst (see Experimental).Furanones 3 and 4 were prepared by means of a similar reaction sequence starting from olefin 6 (Scheme 2).Thus, the latter compound was subjected to ozonolysis followed by treatment of the crude unstable aldehyde with vinylmagnesium chloride to yield alcohols 11 and 12.These were then separated and subjected to esterification to acrylates 13 and 14, respectively.Ring-closing metathesis of the latter compounds required the use of a second generation Grubbs ruthenium catalyst 22 in hot toluene as the solvent, and provided the target furanones 3 and 4. Their enantiomers ent-3 and ent-4 were obtained from ent-6 alongside the same reaction sequence (see Experimental).In order to check the influence of having hydroxy instead of methoxy groups in the side chain (pironetin and compounds in Fig. 3 have one methoxy group and one hydroxyl group), we also tried to prepare analogues of compounds 1-4 with two hydroxy groups.To that purpose, diol 5 was doubly silylated to 15, and the latter subjected to the same ozonolysis/allylation or alternatively ozonolysis/vinylation sequence to yield the diastereoisomeric pairs 16/18 and 24/26, respectively (Scheme 3).After esterification with acryloyl chloride to 17/19 and 25/27, ring-closing metathesis using in this case a Hoveyda-Grubbs-type ruthenium catalyst 22 afforded 20/21 and 28/29, respectively.Unfortunately, all attempts at desilylation of the latter compounds under many different conditions to the desired lactones 22, 23, 30 and 31 only led to either no reaction, decomposition or formation of complex inseparable mixtures. 23

Biological properties of pironetin analogues 1-4 and their enantiomers
Cellular effects of the compounds.We have determined the IC 50 values for pironetin analogues 1-4, and ent-1/ent-4 on five tumoral cell lines: human colorectal adenocarcinoma HT-29 and HTC-116, human breast adenocarcinoma MCF-7, human cervical cancer HeLa and human promyelocytic leukemia HL-60, and compared these values with that of pironetin.Table 1 shows the cytotoxicity values for pironetin (nM) and pironetin analogues expressed as the compound concentration (µM) that causes 50% inhibition of cell growth (IC 50 ).The first conclusion that can be drawn from the IC 50 values is that furanones 3 and 4 and their respective enantiomers ent-3 and ent-4 show almost no cytotoxicity.Pyranones did prove cytotoxic in the low micromolar range, thus about two-three orders of magnitude less active than pironetin itself.Among pyranones the most active compounds are 1 and ent-2.These two compounds share a common structural feature, the configuration of the lactone stereocenter, which is the same as in pironetin.
Effect of pironetin derivatives on the in vitro microtubule assembly.As pyranone derivatives showed to be cytotoxic we selected them to study their effect on the in vitro microtubule assembly.Figure 5 shows the effects of pyranones 1, 2, ent-1 and ent-2 on the microtubule formation studied by time-resolved turbidity measurements.Blue line shows the effects on the microtubule assembly when 25 µM of tubulin was reacted in the presence of 20 mM sodium phosphate (NaPi), 10 mM MgCl 2 , 1 mM EGTA, 3.4 M glycerol and 0.1 mM of GTP at pH=6.5.It can be seen that the nucleation phase takes aproximately 18 min.Then elongation phase starts and after 20 min the steady state is reached.When tubulin assembly is carried out in the presence of 27.5 µM of paclitaxel, absorbance is immediately increased (orange line) which is in accordance with the behaviour of a drug that promotes tubulin polymerization.Conversely, when tubulin assembly is carried out in the presence of 27.5 µM of pironetin, no absorbance is measured throughout the reaction time (green line), which is in accordance with the behaviour of a drug that inhibits tubulin polymerization.When tubulin assembly is carried out in the presence of 27.5 µM of each pyranone the dinamic of microtubule formation is quite similar to the one in the ausence of any compound (blue line).It can be inferred from these experiments that, in contrast to pironetin itself, pyranones 1, 2, ent-1 and ent-2 have little influence on the process of tubulin polymerization.

Effect of pyranone derivatives on the hTERT, c-Myc and VEGF gene inhibition and on the VEGF protein secretion.
We have also studied the ability of pyranones 1, 2, ent-1 and ent-2 to downregulate the expression of the hTERT and c-Myc genes, both involved in telomerase activity, and the expression of the VEGF gene and its associated protein VEGF, both involved in angiogenic activity.The expression of hTERT, c-Myc and VEGF genes were measured upon reverse transcription quantitative PCR (RT-qPCR) analysis on HT-29 tumoral cells.The VEGF protein production was determined with the ELISA procedure and corresponds mainly to the lighter VEGFA-165 isoform that is secreted to the culture medium.Table 2 shows expression percentage of the hTERT, c-Myc and VEGF genes after 48 h of incubation of HT-29 cells.As regards the inhibition of the hTERT and c-Myc genes, compounds 1 and ent-2 are the most active ones, especially if one considers that the concentration of these two compounds is five times lower than that of compounds 2 and ent-1.Particularly appealing is the activity of compound 1 on the inhibition in the expression of the c-Myc gene, which is decreased to 18% of the control value.Regarding the VEGF gene expression compound ent-2 shows the greatest inhibition.However, this high decrease in gene expression is not accompanied by a similar decrease in VEGF protein secretion as compound ent-2 downregulates protein secretion by only 24%.In this sense, the most active compound is pyranone 1 which downregulates VEGF protein secretion by 71%.

Summary
Pironetin analogues 1-4 and their enantiomers were synthesized with the aim at exploring the influence of the alkyl pendants as well as their stereochemistry and lactone ring size in their biological activity.Pyranones showed to be cytotoxic at micromolar level while furanones showed no cytotoxicity.Among pyranones the most cytotoxic were 1 and ent-2, which have the same configuration at the lactone stereocenter as pironetin.The influence of pyranones in tubulin polymerization was also measured but, in contrast to pironetin, they seem to have little influence in the tubulin polymerization process.It thus seems that removal of the methyl pendants at the side chain by a gem-dimethylated pattern causes a strong decrease in the interaction of the compounds with tubulin.As regards the inhibition of the c-Myc and VEGF genes, pyranones 1 and ent-2 proved to be the most active compounds with 1 showing the strongest inhibition of VEGF protein secretion.

Experimental Chemical procedures
NMR spectra were recorded at 500 MHz ( 1 H NMR) and 125 MHz ( 13 C NMR) in CDCl 3 solution at 25 C, with the solvent signals as internal reference. 13C NMR signal multiplicities were determined with the APT pulse sequence.Mass spectra were run in the electrospray (ESMS) mode.IR data, which were measured as films on NaCl plates (oils) or as KBr pellets (solids), are given only when relevant functions (C=O, OH) are present.Optical rotations were measured at 25 C.Reactions which required an inert atmosphere were carried out under dry N 2 with flame-dried glassware.Commercial reagents were used as received.THF and Et 2 O were freshly distilled from sodium-benzophenone ketyl.Dichloromethane was freshly distilled from CaH 2 .Toluene was freshly distilled from sodium wire.Tertiary amines were freshly distilled from KOH.
(3R,4R,5R)-2,4,6,6-Tetramethyloct-7-ene-3,5-diol (5).The following reaction conditions should be strictly adhered to, with particular attention to the words highlighted in italics: D-proline (230 mg, 2 mmol) was dissolved under N 2 in dry DMF (2 mL) and placed in an ice bath.After stirring for 5 minutes, freshly distilled isobutyraldehyde (1.82 mL, 20 mmol) was added  17 (see Electronic Supplementary Information).The procedure described above represents the maximum scale at which we were able to obtain reasonable yields.Attempts at increasing the scale only led to a decrease in the yield.
The procedure was repeated under the same conditions with L- proline to yield ent-5: [] D −18.1 (c 1; CHCl 3 ).Physical and spectral data identical to those of 5.
The stereostructures of 5 and ent-5 have been secured by means of an X-ray diffraction analysis. 24R,5R,6R)-4,6-Dimethoxy-3,3,5,7-tetramethyloct-1-ene (6).Sodium hydride (60% slurry in mineral oil, amount equivalent to 16 mmol) was washed two times under N 2 with dry hexane and once with dry THF.Then, THF (50 mL) was added and the suspension was cooled in an ice bath.Alcohol 5 (801 mg, 4 mmol) was then dissolved in dry THF (10 mL) and added dropwise to the sodium hydride suspension.The mixture was then allowed to reach room temperature.Subsequently, methyl iodide (1.25 mL, 20 mmol) was added dropwise and the mixture was stirred for 24 h at room temperature.(4S,6R,7R,8R)-6,8-Dimethoxy-5,5,7,9-tetramethyldec-1-en-4-ol ( 7) and (4R,6R,7R,8R)-6,8-dimethoxy-5,5,7,9-tetramethyldec-1-en-4-ol (8).Olefin 6 (685 mg, 3 mmol) was dissolved in dry CH 2 Cl 2 (60 mL) and cooled to 78C.A stream of ozone-oxygen was bubbled through the solution until persistence of the bluish color.Dry N 2 was then bubbled through the solution for 10 min.at the same temperature.After addition of PPh 3 (1.18g, 4.5 mmol), the solution was left to stir at room temperature for 2 h.Solvent removal under reduced pressure gave a solid material, which was put on the top of a short silica gel pad and rapidly washed with hexane-EtOAc 9:1.After removal of volatiles under reduced pressure, the crude oily aldehyde was then directly used as such in the next allylation step (for weight calculations, the yield of the ozonolysis step was assumed to be quantitative).

Figure 1
Figure 1 Structure of pironetin, a highly cytotoxic natural pyrone.

Figure 3
Figure 3 Structures III-VIII of the second series of pironetin analogues (ref.13).

Figure 5 ..
Figure 5.. Effects of colchicine, pironetine and compounds 1, 2, ent-1 and ent-2, as well as paclitaxel and pironetin, on the in vitro microtubule assembly.The lines in the figure show the turbidimetric time course of polymerization of tubulin in the presence of GTP, and in the presence of 27.5 µM of each of the indicated compound.

Table 2 .
Table 2 also shows VEGF protein secretion percentage from HT-29 cells determined after 72 h of incubation of HT-29 cells.Percentages of gene expression and VEGF protein secretion a At least three measurements were performed in each case.
In a separate flask, freshly distilled propionaldehyde (722 L, 10 mmol) was dissolved in dry DMF (7 mL).The resulting solution was ice-cooled and added dropwise to the isobutyraldehyde solution by means of syringe pump at a rate of 0.10 mL/h (the needle of the pump should be just below the surface of the liquid).
The reaction mixture was then poured onto saturated ammonium chloride and extracted several times with Et 2 O.The organic layers were then dried over anhydrous Na 2 SO 4 , filtered and under reduced pressure.