Synthesis and Anti-Inflammatory Activity of New Alkyl-Substituted Phthalimide 1H-1,2,3-Triazole Derivatives

Four new 1,2,3-triazole phthalimide derivatives with a potent anti-inflammatory activity have been synthesized in the good yields by the 1,3-dipolar cycloaddition reaction from N-(azido-alkyl)phthalimides and terminal alkynes. The anti-inflammatory activity was determined by injecting carrageenan through the plantar tissue of the right hind paw of Swiss white mice to produce inflammation. All the compounds 3a–c and 5a–c exhibited an important anti-inflammatory activity; the best activity was found for the compounds 3b and 5c, which showed to be able to decrease by 69% and 56.2% carrageenan-induced edema in mice. These compounds may also offer a future promise as a new anti-inflammatory agent.


Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for reducing pain and swellings associated with inflammation and represent an area in continuous and evergrowing development. Phthalimide derivatives are an interesting class of compounds because they possess important biological activities [1], such as anti-inflammatory [2] and hypolipidemic [3,4] ones. Glycoconjugates play an important role in many biological processes, including cellular recognition, particularly in cases of inflammation, tumor metastasis, and immune response in bacterial and viral infections [5].
The literature describes a series of N-phthalimidomethyl 2,3-dideoxy-and 2,3-unsaturated sugar derivatives that showed a potent anti-inflammatory activity, by exhibiting reduction of the edema induced by carrageenan [2]. The anti-inflammatory activity has also been recently related to 1,2,3-triazole derivatives [6]. The 1,2,3-triazole is a fivemembered ring structure containing three nitrogen atoms and is a ring bioisosterism case of the 1,2,4-triazole. Triazoles and derivatives display a diversity of biological activities [7], such as anti-inflammatory [6,8], hypolipidemic [4], antimicrobial [9], anticonvulsant, [10] and anti-nociceptive [11] ones. Most of these compounds and their related activity are associated with an anti-inflammatory profile [8,9]. 1,2,3-Triazole represents a class of compounds that has awakened an interest in our research group by the synthesis of new heterocyclic derivatives and their biological evaluation [12][13][14]. In this work the synthesis and antiinflammatory activity of six compounds based on 1,2,3triazoles it were performed. One group of compounds was 1,2,3-triazoles linked to unsaturated carbohydrate and phthalimides (CTP), and another one was 1,2,3-triazoles linked to phthalimides (PTP), as shown in Figure 1. These compounds were obtained through the reaction between N-(azido-alkyl) phthalimides and terminal alkynes employing Cu-AAC (Cu-Catalyzed Azide Alkyne Cycloadditon) reaction [15].

Acute
Anti-Inflammatory Activity. The drugs used for comparison purposes were 3a-c, 5a-c, ibuprofen, and ASA. All compounds were suspended in 1% carboxymethylcellulose (CMC) and single dose of 250 mg/Kg was administered intraperitoneally, in the morning [18]. Other animal group received 1% CMC. Two positive and one negative antiinflammatory control tests were done in three animal groups by intraperitoneal administration of 250 mg/Kg of acetylsalicylic acid (ASA), a standard dose for pharmacological comparative tests, 250 mg/Kg of ibuprofen (Laboratory Teuto Brazilian Ltd., Brazil), and 0.9% of aqueous saline solution, respectively. The anti-inflammatory activity was determined by Levy's method [19]. Carrageenan (Sigma, St. Louis, USA), 0.1 mL of a 1% solution in 0.9% NaCl, was injected through the plantar tissue of the right hind paw of each mouse to produce inflammation. After four hours, the animals were sacrificed under anesthesia and their paws were cut and weighed. The results were analyzed according to the percentage of inflammation reduction as described earlier [19].

Calculation of Octanol-Water Partition Coefficient
− log P [20]. The structures of compounds 3a-c and 5a-c were analyzed in ACD/Labs that contains a database available for this procedure. The values of octanol-water partition coefficient (log P) for these compounds were predicted using Advanced Chemistry Development Inc. (ACD/Labs, algorithm version: v5.0.0.184).

Statistics.
All results are expressed as mean ± SEM for experiments. Statistical evaluation was undertaken by analysis of variance (ANOVA) followed by Turkey test for multiple comparisons. P < 0.05 was used as the criterion of statistical significance.

Results and Discussion
3.1. Chemistry. The synthetic strategy to obtain the compounds 3a-c consists of two convergent steps. First, the alkyne carbohydrate 1 was prepared in 82% of yield according to Ferrier's protocol [16]. In parallel, the synthesis of N-(azido-alkyl)phthalimides 2a-c was achieved from the corresponding N-(bromoalkyl)phthalimide in good yields (70%-75%). Finally, the glycoconjugate 1,2,3-triazoles 3ac were initially prepared using the protocol described in a previous work [12] to afford the compound 3a in moderate yield (48%). Thus, when 1 drop of triethylamine was added the desired compounds were obtained in good yield (90%). Under this optimized conditions the compounds 3a-c were synthesized in good yields of 68%-90% after column chromatography on silica gel (Scheme 1). The N-propargyl phthalimide 4 was prepared from potassium phthalimide and propargyl bromide after being stirred at room temperature for 24 h. The reaction between alkyne 4 and N-(azido-alkyl)-phthalimides 2a-c was carried out at the same conditions described above. This facile protocol provided the bis-phthalimides 1,2,3-triazole 5a-c in good yields of 62%-88%, as shown in Scheme 2.

Pharmacology.
All the compounds exhibited antiinflammatory activity when compared with ASA as well as ibuprofen. The compounds 3a, 3b, and 3c reduced carrageenan-induced edema in Swiss white mice by 33.7%, 69%, and 44%, respectively. This result is significant (P < 0.001) when compared with the control group treated with the saline solution ( Table 1).
The compounds 5a, 5b, and 5c were able to reduce the edema by 17%, 25%, and 56.2%, respectively, as shown in Table 1. Therefore, substances 3b and 5c showed the best anti-inflammatory activity in terms of edema inhibition.
The 3b and 5c compounds, which showed the greatest anti-inflammatory effect, were selected for the experiment of determining the effective dose. The study of effective dose has shown that closely at concentration of 225 mg/Kg the compounds 3b and 5c have 50% of anti-inflammatory activity ( Figure 2). The results of anti-inflammatory dose-response Ibuprofen ASA Anti-inflammatory activity (%) Figure 2: Anti-inflammatory dose-response curves for compounds 3b, 5c, ASA, and ibuprofen using carrageenan to produce inflammation in the paw of mice. curves for the compounds 3b and 5c showed similar results to the ASA at all concentrations tested. On the other hand, the anti-inflammatory effect of compounds 3b and 5c was similar to ibuprofen only when administered at high dose (350 mg/Kg), as shown in Figure 2.
According to Table 1 the acute anti-inflammatory activity of considering the compounds 3a-c containing phthalimide linked to carbohydrate (CTP) the best anti-inflammatory Table 1: Acute anti-inflammatory activity of 1,2,3-triazole phthalimides 3a-c and 5a-c after carrageenin-induced edema at dose 250 mg· kg −1 .
activity was observed for the compound 3b that contains three methylene groups. However, for compounds 5a-c that have two moieties of phthalimide connected to a triazole ring (PTP), when increasing the size of aliphatic chain, the antiinflammatory activity is also increased from 17% (n = 2) to 56.2% (n = 4) ( Table 1 and Figure 3). Hydrophobic interactions play a key role in the folding and maintenance of the three-dimensional structure of proteins, as well as in the binding of ligands (e.g., drugs) to protein targets [21]. Protein-ligand binding is partially driven by lipophilic interaction, and the log P for a class of ligand compounds will depend on the nature of protein.
In fact, it is known in the literature that chain branching or homologation can cause the molecule to bind more or less well to the receptors responsible for specific biological activity [22].
In order to evaluate the chain effect on the antiinflammatory activity we have calculated the partition coefficient (log P) using the ADC/Labs PhysChem Predictor. The log P values are important data to be considered for drug design and their pharmacokinetics properties; they indicate the lipophilic tendency or the ability to penetrate lipid barriers (lipophilicity) [23].
The chain effect for the biological activity in both classes of the compounds 3 and 5 is shown in Table 2, and the log P ranged from 1.53 to 2.04 and from 2.37 to 2.89, respectively.
The compounds 3a-c have a portion carbohydrate, and for this class we can note that a peak anti-inflammatory activity occurred with the 3-phthalimide propyl-1,2,3-triazole 3b (Figure 3). On the other hand, the class of compounds 5a-c is more lipophilic and showed to be sensitive to antiinflammatory activity when the methylene groups were introduced.
In recent works, a new series of 1-[2-(1H-tetrazol-5yl)ethyl]-1H-benzo[d] [1,2,3]triazoles have shown anti-inflammatory activity (11-47%) against carrageenan-induced paw edema, whereas the standard drug diclofenac sodium showed 61% [24]. Other novel series of azoles, such as pyrazole, were obtained by reacting chalcones and hydrazine hydrate, and they exhibited low inhibition of paw edema ranging 1.08-31.05%, and these results corresponded to a percentage of the indomethacin inhibition [25]. However, in the present work, the compounds 3b and 5c exhibited better anti-inflammatory activity ( Figure 2) than that reported by Khalil [25], when considering the anti-inflammatory effect of 3b and 5c observed with 50 mg/Kg (lower dose) in relation to the same concentration of ibuprofen or ASA instead of that of the saline.
Recently, a series of new 1,2,4-triazoles obtained by reacting acyl 1,2,4-triazoles with various secondary amines, exhibited appreciable inhibition [8]; when, administered at a dose of 100 mg/Kg, the percentage inhibition reached 55.6%, the comparison was performed with the reference drug indomethacin (62.5% at 100 mg/Kg). These results are similar to the compounds 3b and 5c, which were administered by us at a dose of 250 mg/Kg.
There are scientific evidences that the bis-heterocyclic compounds encompassing 2-mercapto benzothiazole and 1,2,3-triazole showed significant binding potential towards COX enzyme thus lowering the paw edema induced by carrageenan [6]. The class of monoacylated 5-amino-1,2,4-triazole derivatives exhibited potent anti-inflammatory activity (at 5 mg/kg, oral dose level) in carrageenan-induced rat paw edema test [26]. These results also contributed to show that many structure containing the heterocyclic triazole possessed high anti-inflammatory activity. 6 The Scientific World Journal New 1H-(1,2,3-triazole)phthalimide derivatives are interesting drugs due to their potential anti-inflammatory activity, thus deserving further studies in order to understand the mechanism of action. In conclusion, the results suggest that these compounds may also offer a future promise as a new anti-inflammatory agent.