Introduction

Piperlotines are very important secondary metabolites which have been isolated from different Piper species (Piperaceae) [¹]; such valuable compounds have been just partially explored in search of interesting pharmacological activities, for example as antiplatelet aggregation [²] and anticonvulsant agents [³-⁴].

Nonetheless, pharmacological applications of these α,β-unsaturated amides and related derivatives still remain underexplored. For example, inflammation-related diseases as arthritis, psoriasis, lupus, allergy and asthma, are important public health problems all over the world [⁵-⁷] but actually, to the best of our knowledge no one in vivo inflammation test has been undertaken using piperlotines as potential anti-inflammatory agents.

In other instance, tuberculosis (TB) is a health problem especially in developing countries, being one of the top ten causes of death by infectious processes and affecting a quarter of the world population, in addition to the increase of multidrug-resistant and extensively drug-resistant cases [⁸] that motivate the search of new anti-mycobacterial therapies. In this respect, α,β-unsaturated amides, including amide derivatives of cinnamic acid, have been proposed as alternative or complementary anti-tubercular agents in multiple studies [⁹-¹²].

Few methods have been reported for the synthesis of piperlotines and derivatives, including: alkyne’s catalyzed aminocarbonylation [¹³], transamidation from another unsaturated amides [¹⁴], amidation of cinnamic acids [¹⁵-¹⁷] and olefination of aromatic aldehydes by means of the Horner-Wadsworth-Emmons reaction [¹⁸].

Mechanosynthesis is a solvent-free method used to generate a wide variety of compounds, including molecules of pharmacological interest [¹⁹, ²⁰], in this regard, our group has developed a simple, fast, and diastereoselective method to obtain piperlotines A, C and their derivatives through the mechanical activation of a β-amidophosphonate intermediate [²¹].

In this work, piperidine-derived α,β-unsaturated amides 5, 6 were prepared by means of mechanochemical activation of a Horner-Wadsworth-Emmons reaction (HWE) between a β-amidophosphonate 4 and an aromatic aldehyde in alkaline media under solvent-free conditions.

Then, compounds 5,6 and previously synthesized ones were then tested as anti-inflammatory agents through two in vivo acute inflammation models (Carrageenan, by oral administration and TPA through topical administration). In addition, their potential anti-tubercular activity was tested on Mycobacterium tuberculosis (MTb) H37Rv strain (ATCC 27294).

Experimental

Pharmacological assays

Results and discussion

Rational drug design is a modern strategy followed to discover new bioactive compounds and for to develop new drugs. In this regard, computer aided drug design (CADD) is a powerful tool for prediction of potential pharmacological activity. For example, the Prediction of Activity Spectra for Substances (PASS Online) [³³] database is a freely available resource successfully utilized for prediction of pharmacological activity of known or novel organic compounds. With this on mind, and according to our interest in the synthesis of Piper amides we envisaged the synthesis and pharmacological evaluation of piperlotines structurally related to anti-inflammatory natural products and commercial drugs as piperine [³⁴-³⁶], idrocilamide [³⁷], tranilast [³⁸], and E93 [³⁹] (Fig. 1) which contains: an α,β-unsaturated amide conjugated with one or more C=C double bonds and an aromatic ring. Alkoxy substituents are very common in the aromatic portion of Piper amides’ molecular structure, including the above-named anti-inflammatory compounds. In this regard, we considered to include electron-donating (R = OCH_3, EDG) substituents into aromatic ring of Piperlotine A, Piperlotine C and 1. Additionally, an electron-withdrawing (R = NO₂, CF_3, EWG) substituent was introduced in 5,6 in order to determine the effect of electronic density on pharmacological activity (Fig. 1).

Fig. 1 Anti-inflammatory α,β-unsaturated amides structurally related to proposed piperlotines. 

Next, piperlotines A, C and derivatives were predicted as potential anti-inflammatory agents (probability of activity from 0.31 to 0.47, being 1 the maximal probability of activity) through PASS prediction (Table 1). Altogether, above named derivatives obtained interesting prediction as matrix-metalloproteinase-9 (MMP-9) expression inhibitors (0.61-0.82) (Table 1). Now, it is well established a relationship between increased levels of matrix-metalloproteinases and the development of acute or chronic inflammation processes in diverse tissues [⁴⁰]. Moreover, inflammatory-related diseases as arthritis and systemic lupus erythematosus have been correlated with unusual tissular matrix-metalloproteinases concentration [⁴¹-⁴²]. In addition, target amides were predicted as tumor necrosis factor (TNF) expression inhibitors (Pa from 0.373 to 0.694), another important pro-inflammatory mediator [⁴³].

Finally, all compounds agreed with Lipinski’s rules as drug-like criteria [⁴⁴]. In view of all above mentioned, piperlotine-like amides were ideal candidates for pharmacological tests as anti-inflammatory agents.

Consequently, the reaction of piperidine and bromoacetyl bromide produced amide 3 in 86% yield, which when treated with triethyl phosphite afforded β-amidophosphonate 4 in 84% yield. Finally, 4 was reacted with an aromatic aldehyde and K₂CO₃ in a mortar and pestle under solvent-free conditions [²¹], affording products (E)-5, 6 in 70-88% yield in just 30 min (Scheme 1).

Scheme 1 Synthesis of 5-6. 

Next, synthesized compounds 5-6 and previously prepared piperlotines A, C and 1-2 were tested through carrageenan induced acute inflammation model. Structure-activity relationships (SAR) studies indicated the aromatic substitution pattern in tested derivatives was a strong determinant of anti-inflammatory activity. In this regard, presence of EDG (R = OCH₃) in phenyl ring of piperlotines A, C and compound 1 determined its low edema inhibition, compared with positive control -indomethacin- (Table 2). The extension of olefin conjugation in 2 had a markedly deleterious effect in its anti-inflammatory activity. Furthermore, the presence of an EWG (R = CF₃) in phenyl ring greatly favored edema inhibition in 6 (having most potent anti-inflammatory activity than indomethacin, being the most active). Notwithstanding, the presence of nitro group in 5 produced a pronounced pro-inflammatory effect. It is worth to mentioning that nitro and trifluoromethyl groups exert its electron-withdrawing effect through different mechanisms; nitro group by resonance effect, but trifluoromethyl group by inductive effect. Additionally, the introduction of trifluoromethyl substituent has proven to improve absorption and metabolic stability of many bioactive compounds [⁴⁵-⁴⁶].

Afterwards, encouraging results were observed when synthesized compounds were tested on TPA acute inflammation model. This time, all compounds were anti-inflammatory in a range of 31.68-52.02 % edema inhibition. Once again trifluoromethyl derivative 6 was the more potent hit, almost comparable to indomethacin (60.32%) (Table 3). Identical edema inhibition was observed for derivative 2 (52.02%), being both the more lipophilic compounds of the series (logP = 3.82, 2.94, respectively) (Table 3), and thereby having a better absorption through mice skin. Afterwards, it was observed that the more methoxy groups were present in the aromatic ring, the more potency was achieved. Thus, edema inhibition of piperlotine C > 1 > piperlotine A (42.24%, 41.76% and 32.21%, respectively) (Table 3). Finally, the nitro derivative 5 was the less active hit of the series (31.68% edema inhibition). Nonetheless, it is noticeable than 5 exhibited anti-inflammatory activity by TPA topical model, but pro-inflammatory activity was observed when carrageenan model was carried out. Additional experiments are needed to clarify this dual behavior previously observed in our group [⁴⁷].

In conclusion, the topical anti-inflammatory activity of 2 and 6 was remarkably in comparison to positive control -indomethacin-. More interesting, compound 6, the more active hit trough oral and topical administration has been previously described as an anticonvulsant agent on mice [³-⁴] and it could serve as a useful scaffold in the search of potent central nervous system anti-inflammatory agents.

On the other hand, α,β-unsaturated amides have been probed as antimycobacterial agents in several works [⁹-¹², ⁴⁹], especially piperine amide analogs which had more potent antimycobacterial activity than first line drugs as ethambutol and isoniazid on Mycobacterium tuberculosis H37Rv strain [50]. Based on this, we tested synthesized piperlotines against MTb, where only piperlotine A and compounds 2 and 6 showed slight activity (MIC = 50 µg/mL) (Table 4) compared to isoniazid and rifampicin (MIC < 1 µg/mL) [¹¹-¹²], but comparable to other structurally related Piper α,β-unsaturated amides as: sarmentine, brachyamide B and guineensine (MIC = 50 µg/mL) [⁵¹] and higher than cinnamic acid itself (MIC = 100 µg/mL) [¹¹].

Furthermore, several authors have reported synergistic activity of cinnamic acid amide derivatives [¹², ⁵²-⁵³] in combination with isoniazid or rifampicin. Then, we suggest piperlotines synthesized in this work could be used in combination with classical treatments on MTb strains.

Conclusion

Two piperlotine-like compounds 5-6 were easily synthesized from β-amido phosphonate 4 and an aromatic aldehyde in good yields. No solvent or additional heating was needed and only (E)-diastereomer was obtained. Next, synthesized and previously obtained piperlotines were tested as in vivo anti-inflammatory agents for the first time. In this respect, trifluoromethyl derivative 6 had remarkably anti-inflammatory activity in two acute inflammation models on mice (comparable or higher than indomethacin, the commercial anti-inflammatory drug reference), being active through oral and topical administration. Finally, piperlotine A, and derivatives 2, 6 exhibited slight antibacterial activity on MTb H37Rv strain, and could be used in combination with first line antituberculosis drugs in future investigations, pointing the potential of piperlotines and its derivatives to further medicinal chemistry research.