Synthesis and Biological Activity of Simplified Belactosin C Analogues

Successful biochemical studies of the natural products belactosin A and C and their acylated congeners have shown a β-lactonecarboxamide moiety to be a possible core structure of powerful proteasome inhibitors. As a part of further investigations, variously decorated simplified β-lactonecarboxamides have been synthesized in order to understand structure-biological activity relations in detail, to find ways of improving their biological activity and stability and to reduce the complexity of their preparation. Biological tests showed that the best compounds possess a high potential against phytopathogenic fungi in the greenhouse.


Introduction
Since their isolation, members of the belactosin family of proteasome inhibitors have attracted interest from both biological and synthetic points of view.In order to better understand their proteasome inhibitory power and the important structure-activity relationships, the prevailing interactions between such molecules and the proteasome should be known.Towards this end, a protected homobelactosin C was initially cocrystallized with the 20S proteasome from Saccharomyces cerevisiae, and the structure of this crystal was elucidated by X-ray diffraction. 1 On the basis of this analysis, it was decided which groups in the inhibitor should be modified in order to improve potentially favorable interactions with the proteasome.With this in mind, several belactosin C congeners bearing various protective groups have meanwhile been synthesized, some of them cocrystallized with the 20S proteasome, and their structures investigated. 2As those compounds showed interesting biological activities against HeLa cells, further modifications on the β-lactone nucleus and the side chain were initiated in order to achieve two purposes: to increase the biological activity of the molecules and simultaneously decrease the complexity of their preparation.Those parts of the belactosin structure that were considered for modification are marked in Fig. 1.
The most drastic modification would be to replace the whole dipeptide residue on the β-lactonecarboxamide by a relatively small and easily accessible amide moiety which, at the same time, would considerably decrease the cost of the synthesis.Of course, any such modification should not go along without a loss, but with an increase of biological activity.Looking at the structure of the first cocrystallisate, a 3,5-dimethoxybenzylamido group should be a good mimic of the dipeptide residue.Therefore, the [2R,3S(1S)]-N-(3,5-dimethoxybenzyl)-3-(1-sec-butyl)-4-oxooxetane-2-carboxamide (2) was targeted, and in order to determine the influence of the sec-butyl group on the biological activity, the analogue 3 bearing an isopropyl residue at the C-4 position of the β-lactone moiety was considered as well (Fig. 2).
As detailed investigations of the biological activities of salinosporamide A had shown, an additional substituent at the C-2 position of the β-lactone unit can be favorable. 3Accordingly, simplified belactosin analogues bearing an additional methyl group at C-2 were targeted as well.For a better understanding of the structure-activity relationship, the β-lactonecarboxamides both with the (2R,3S)-configuration as in the natural product as well as the derivatives (2S,3R)-4 and 5-7 with the opposite configuration (Fig. 2) were approached.Another worthwhile simplification of the belactosin molecule would be the removal of the stereogenic center in the side chain of the β-lactone moiety, and this would be achieved by introducing a 1-ethylcyclopropyl residue instead of the sec-butyl group.This looked particularly promising as the structure of the cocrystallized homobelactosin C 1 revealed enough space for a sterically more demanding side chain.

Synthesis of simplified belactosin analogues
The β-lactonecarboxamides 2 and 3 were prepared adopting the established protocol for the condensation of the malic acid derivatives 9 and 10 with the belactosin dipeptide fragment 1 to the condensation with 3,5-dimethoxybenzylamine, which also occurred with immediately ensuing β-lactonization (Scheme 1).
Since the yields of 22 and 38%, respectively, were unsatisfactory, another approach to 2 was developed.Towards that, the free malic acid 12 was prepared by a two-step hydrolysis of the phenylthio ethyl ester 11, and the resulting 12 was regioselectively converted to its 3,5-dimethoxybenzylamide by treatment with trichloroacetic acid anhydride, then 3,5-dimethoxybenzylamine and finally sodium hydroxide.The monoamide of the β-hydroxy acid was eventually lactonized by treatment with benzotriazol-1yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) in the presence of triethylamine.The overall yield of 2 in this sequence was 41% (Scheme 2).
Both the cis-and trans-3-(1-ethylcyclopropyl)-2-oxooxetanecarboxamides cis-8 and trans-8 were synthesized as racemates from the well-established versatile building block tert-butyl 2-chloro-2-cyclopropylideneacetate (17). 4The sequence started with a one-pot transformation involving Michael addition of ethylmagnesium bromide and ensuing aldol reactions with ethyl glyoxylate that led to an inseparable mixture of two diastereomers of 18 (ratio 4 : 1).Then the chlorine substituent was reductively removed by treatment of 18 with ammonium formate in the presence of palladium on charcoal to give 19 (a 4 : 1 mixture of diastereomers).The ethyl ester was selectively hydrolyzed with lithium hydroxide in aqueous tetrahydrofuran, and the free carboxyl group was condensed with 3,5-dimethoxybenzylamine.The diastereomeric carboxamides (R*,R*)-and (R*,S*)-20 could be separated by column chromatography, and the individual diastereomers were converted to the oxooxetanecarboxamides cis-8 and trans-8 by acid-catalyzed cleavage of the tert-butoxycarbonyl groups and subsequent HATU-mediated cyclization of the β-hydroxy acids (Scheme 4).

Biological activities of simplified belactosin analogues
Since the in vivo biological activity of a given substance in the greenhouse depends not only on the in vitro biological activity at the target, but also on such parameters as solubility, cell permeability, and others that cannot be easily taken into account, it was decided to synthesize a library of compounds to be able to choose the most potent ones.Following the previously expressed hypothesis that the binding site in the proteasome is structurally rather flexible, 200 diverse amines were condensed with the hydroxy acid 9, 5 and the activities of the corresponding amides were determined against phytopathogenic fungi in the greenhouse (Table 1).Many of these amides showed activities comparable to that of the protected belactosine C at the target, but significantly better activity in the greenhouse.This effect may be related to beneficial physicochemical properties and is more strongly affected by the amines used. 6For example, the target activity appears to be independent of the substitution pattern on the phenyl ring and the spacer between the phenyl ring and the amide nitrogen.Compound 23 turned out to be by far the best compound in the greenhouse.
The α-branched side chain in the 3-position of the lactone appears to be important for good activity, compare e.g. the 1-methylpropyl analogue 2 with the 2-methylpropyl derivative (not shown) (Table 2).The isopropyl group in the same position affects the target activity slightly, but resulted in very weak greenhouse efficacy.The gem-disubstituted spirocyclopropanated analogues trans-8 and cis-8 are much weaker even at the target, with only little activity for the trans-configured lactone trans-8.Surprisingly weak were also all analogues bearing a methyl group in the 2-position of the lactone.

Conclusion
Simplified analogues of the natural dipeptide belactosin show very good activities against the 20S proteasome, 8 which are translated into promising fungicidal activities against some phytopathogenic fungi in the greenhouse.This opens up possible applications in crop protection and underlines once more the potential of natural products 9 for the identification of novel chemical classes with a novel mode of action.Based on various developed methodologies, a first structure-activity relationship has been established.This includes broad variation of the amine part as well as the side chain in the 3-position and modulation of the 2-position of the lactone ring.The target activity reacts in a very flexible way on the variation of the amine part, but has a strong effect on the greenhouse activity.Smaller residues likely help to optimize the physico-chemical properties and thereby improve the transfer of target activity to the greenhouse.Substituents in the 2-position of the lactone are not allowed, whereas the side chain in the 3-position requires an α-branching methyl group.
Silver trifluoroacetate (7.7 g, 35 mmol) was added to a solution of the malic acid derivative 9 (5 g, 17.5 mmol) in THF (20 mL) and H 2 O (5 mL).The mixture was stirred at 55 °C for 16 h.The precipitate was filtered off and washed with THF.Then the volatiles were removed from the filtrate under reduced pressure.Water (20 mL) was added to the residue, and this mixture was extracted with CH 2 Cl 2 .The solution was dried (Na 2 SO 4 ), and the solvent was distilled off to yield 3.0 g (89%) of the product.

Method B.
A mixture of [2R,3S(1S)]-3-sec-butyl-2-hydroxybutanedioic acid (11) (0.7 g, 3.7 mmol), trichloroacetic acid anhydride (2.3 g, 7.4 mmol) and dioxane (2 mL) was stirred at 75 °C for 3 h.Then the volatiles were removed under reduced pressure at r.t., the residue was dissolved in THF (10 mL) and the solution cooled to 0 °C.3,5-Dimethoxybenzylamine (1.4 g, 8.4 mmol) was added, and the reaction mixture was stirred first at 0 °C for 1 h, then at r.t. for 16 h.The volatiles were removed in vacuo, the residue was taken up in an aq.solution of NaOH (1.5 mL, 6 N), and the mixture was stirred at r.t. for 16 h.Then the solution was neutralized with conc.HCl, dichloromethane (20 mL) was added, and the mixture was washed with 1 N HCl (2 × 5 mL).The organic phase was dried (Na 2 SO 4 ).The solvent was distilled off, the residue was purified by flash chromatography on silica; by-products were removed by eluting with pentane-diethyl ether (1 : 4), and the desired product was eluted with MeOH.The solvent was distilled off in vacuo, and the crude malic acid monoamide was directly used for the next step.

Fig. 1
Fig. 1 Structure of belactosin C with possible modifications.