Click chemistry on diterpenes: anti-inflammatory activity of the acetylenic derivatives of levopimaric acid and products of their transformations

Anti-inflammatory activity of amides of levopimaric acid (LpA) with acetylenic moiety and its esters with 4-R-triazolic substitutions was investigated using histamine-induced mice paw edema model. The synthesized LpA derivatives possess anti-inflammatory activity comparable to the activity of indomethacin


Introduction
The rational use of plant metabolites as a platform for the discovery of new medicinal agents takes advantage of the structurally unique building blocks provided by nature for the rapid increase of molecular complexity.Diterpene resin acids (DRA), produced by conifers, combine an unusual stereochemically defined hydrocarbon scaffold with the possibility for possible functionalization via reactions at the carboxyl group.Levopimaric acid (LpA), the main component of pine oleoresin, is one of the most important in this family.
The relative content of Levopimaric acid in the resin of Scots pine Pinus sylvestris and its relative Рinus hamata reaches 27% and 36%, respectively. 1,2Along with the other diterpene resin acids, it plays a key role in providing defense against insects and microbial pathogens. 3In general, the abietene types of diterpene resin acid display a broad spectrum of biological activity.In particular, derivatives of Levopimaric acid show antibacterial, cardiovascular and antioxidant activities. 1 The general mode of biological activity of abietene derivatives is related to their antiinflammatory properties.These properties are based on the suppression of inflammatory cytokines production and COX-2 expression. 4Dehydroabietic acid significantly decreases the production of pro-inflammatory mediators (MCP-1, TNFά, NO) in LPS-stimulated RAW264 macrophages and in mixed cell line of macrophages and adiposytes. 5Abietic acid was used as a building block for the preparation of heterocyclic derivatives close in activity to prednisolone but less toxic. 6ynthetic transformations based on the natural compounds expand medicinal potential of the nature's molecular treasury.For compounds of levopimaric acid family, an important role is played by the Diels-Alder reaction, usually with various benzo-and naphthoquinones.This efficient transformation provides a very convenient method for the isolation of LpA from complex mixture of product derived from the pine oleoresin.Furthermore, the recently reported adducts of LpA with benzoquinones display activity comparable with orthophen. 7The derivatives of dihydroquinopimaric acid show a particularly attractive combination of high antiinflammatory activity with low toxicity. 8hese examples illustrate the potential of abietic diterpenoids as a structural scaffold in the design of anti-inflammatory agents.In this aspect, LpA remains the less studied in this family of compounds.As the part of our ongoing research program dedicated to the investigation of plant metabolites produced by trees and herbs of Siberia, 9,10 we present the first synthesis of acetylenic derivatives of LpA, selected transformations of these compounds, and studies of the antiinflammatory activity with the aim of better understanding of the structure-activity relation.
Presence of acetylenic moiety into natural 11,12 and designed 13 medicinal agents can play important role in the mechanism of their biological activity.Furthermore, it introduces possibilities for the further structural modifications.This strategy has been successfully applied to natural compounds of triterpenoid family. 14,15However, in the latest monograph on the properties of DRA, where synthesis and properties of levopimaric acid are described in a chapter with 142 references, 1 data on acetylenic derivatives of this diterpene are absent.

Results and Discussion
Based on the above, we had chosen double modification of LpA using the Diels-Alder reaction with 1,4-naphthoquinone 1 with following introduction of alkyne groups via the LpA carboxyl.
The first step of the sequence proceeds under very mild conditions (one week at the room temperature).The precipitated naphtholevopimaric acid 1 (NqLpA) is filtered to provide the 93% of the product. 16The NqLpA amides 4a-e with alkyne substituents were prepared by condensation of NqLpA acid chloride 2 with the library of aminoacetylenes 3a-e (see the SI part).We had used amides as the first set of targets based on our earlier reports of promising anti-inflammatory and anti-ulcer properties of related NqLpA amides. 17The NqLpA acid chloride 2 was synthesized by the reaction of acid 1 with oxalyl chloride in DCM at the room temperature.After removal of solvent from the reaction mixture, it was diluted with toluene and introduced in the reaction with the respective acetylenic amine 3 in triethylamine.
We had also explored the possibility of modifying the acetylenic derivatives of LpA via 1,3dipolar cycloaddition with aryl and alkyl azides.The Cu-catalyzed version of this process works very well for terminal alkynes. 18,19The high yields, attractive regioselectivity and mild conditions account for the growing popularity of this reaction (the most popular of the "click" reactions) in the preparation of bioconjugates, [20][21][22][23][24][25][26][27] including those of triterpenoid family. 15This remarkable selective and cost-effective process, leading to 1,2,3-triazoles, possessing antiinflammatory activity 28 prompted us to synthesize the levopimaric acid-1,2,3-triazole hybrid.
In the following steps, the terminal alkyne 5 was introduced in the "click" reactions with alkyl and aryl azides 6a-e in the presence of Cu(I) salts.Interestingly, alkyl azides are more reactive in comparison to their aromatic analogues.Whereas the reactions with alkyl azides proceed at the room temperature in 84-96 % yields, the reactions with aromatic azides require reflux in toluene (1H-1,2,3-triazole yields of ~70%).
The structure of all new acetylenes and the products of their transformations was established on the basis of analytical and spectroscopical methods (IR, 1 H and 13 C NMR)

Anti-inflammatory activity of levopimaric acid derivatives
Two groups of levopimaric acid derivativesamides with acetylenic moiety (4-5) and triazolyl esters (7)were tested for their anti-inflammatory activities and structure-activity relationships using the histamine-induced paw edema model.Data in Table 1 illustrate that all synthesized derivatives injected intraperitoneally in mice (20 mg/kg dose) decrease paw edema by 23-47 percent relatively to control group.The same effect of indomethacin is 49 percent.The majority of the levopimaric acid derivatives (4a-c, 4e, 7a-d) have moderate anti-inflammatory activity -1.5-2.0-foldlower than that of indomethacin.However, the activity of several esters (4d, 5, 7e) compares favorably with that of reference drug.Comparative analysis of anti-inflammatory potencies of derivatives with acetylenic moiety in side residue (4a, 5, 4e) indicates that it depends on the structure of the linker at carbon group.Exchange of amide moiety to ester in the side residue causes significant 1.8 fold-increase of the anti-inflammatory effect (compound 5).Introduction of a methoxy group in the diphenylacetylenic residue causes 1.7-fold increase of activity (4d4b).The activity of triazolic esters is also modulated by the substitution.For example, both change from an aliphatic to an aromatic substituents and fluorine introduction in the pendant aryl increase the activity.These data indicates that anti-inflammatory activity of amides and esters of LpA is sensitive to the nature of substitution and confirms the potential value of this class of biological active substances.
In conclusion, we reported the first synthesis of acetylene derivatives of LpA, their Cu-catalyzed alkyne-azide cycloadditions reactions, and anti-inflammatory activity of the new compounds.The possibility of combining the stereochemically defined polycyclic diterpene scaffold with the flexibility of structural "click" modifications should open new opportunities for the rational design of terpene-based medicinal agents.Encouragingly, the synthesized LpA derivatives possess anti-inflammatory activity comparable to the activity of indomethacin.

Pharmacological experiments
The experiments were carried out using outbred female mice in accordance with the Guidelines for the Care and Use of Laboratory Animals.The animals were given standard granulated food and water ad libitum.

Histamine-induced edema model
Inflammatory edema was induced by subplanar injection of 0.05 ml 0.01% histamine in water solution into the hind paw of female mice.The test compounds were administered intraperitoneally in 20 mg/kg b.w.dose (as aqueous-Tween-80 suspension) one hour before the histamine injection.The reference agent indomethacin ('Fluka BioChemica') was administered in the 20 mg/kg dose.The control group of animals received a water-Tween-80 mixture.The animals were sacrificed by cervical dislocation 5 hours after the phlogogenic agent injection, the mouse paws were cut off at the ankle joint and weighed.The ratio of the difference in weight between the treated and untreated hind paws to the weight of the untreated hind paw was used as an index of inflammation.The results were analyzed using "STATISTICA 6" software.The differences were significant at p<0.05.The anti-inflammatory activity was presented as a difference between 100% and percentage of inflammation index relative to control group.