Synthesis and Biological Evaluation of Substituted Desloratadines as Potent Arginine Vasopressin V2 Receptor Antagonists

Twenty-one non-peptide substituted desloratadine class compounds were synthesized as novel arginine vasopressin receptor antagonists from desloratadine via successive acylation, reduction and acylation reactions. Their structures were characterized by 1H-NMR and HRMS, their biological activity was evaluated by in vitro and in vivo studies. The in vitro binding assay and cAMP accumulation assay indicated that these compounds are potent selective V2 receptor antagonists. Among them compounds 1n, 1t and 1v exhibited both high affinity and promising selectivity for V2 receptors. The in vivo diuretic assay demonstrated that 1t presented remarkable diuretic activity. In conclusion, 1t is a potent novel AVP V2 receptor antagonist candidate.

Numerous AVP receptor antagonists were developed and evaluated in recent decades [24][25][26][27][28][29][30]. A few of them have undergone sufficient clinical trials to be on the market, such as the dual V1a/V2 receptor antagonist conivaptan and the selective V2 receptor antagonist tolvaptan approved for the treatment of hyponatremia in the USA. Another promising selective AVP V2 receptor antagonist, lixivaptan, is still undergoing phase 3 clinical trials at this moment. The structures of most extant AVP receptor antagonists include a benzene-fused seven membered ring system (ring A) and two aromatic rings (ring B and ring C) linked through amide bonds. Recently, we reported some amide and sulfamide derivatives of desloratadine, which are potent AVP V2 receptor antagonists [31]. Desloratadine is a selective, H 1 -receptor antagonist, and also has anti-inflammatory activity [32]. In the previous study, ring A and ring B of classic V2 receptor antagonists were replaced by desloratadine (Figure 1). In a continuous study, we synthesized several compounds centered on a desloratadine scaffold as ring A (compounds 1a, 1b, 1h and 1i, see Table 1) and found that they exhibited potent diuretic activity [33]. Therefore, additional compounds with a desloratadine scaffold as ring A were designed, synthesized and evaluated. Herein, we report the synthesis and biological evaluation of this series of substituted desloratadine designed as potent AVP V2 receptor agonists.

Results and Discussion
The synthetic routes used in this study are illustrated in Schemes 1 and 2, respectively. As shown in Scheme 1, the acylation of 2 with a p-nitrobenzoyl chloride, p-nitrobenzene sulfonyl chloride or m-nitrobenzene sulfonyl chloride provided 4a-c, which were subsequently reduced with SnCl 2 to provide the corresponding anilines 5a-c in satisfied yields. Acylation of 5a-c with alkyl chloride or alkylsulfonyl chloride yielded the target compounds 1a-f. Similarly, as shown in Scheme 2, acylation of 5a-c with substituted benzoyl chlorides or benzenesulfonyl chlorides gave target product 1g-y. Scheme 1. Synthetic route to 1a-f. Twenty-one compounds 1c-g, 1j-y were synthesized and characterized by 1 H-NMR and HRMS. In order to provide a comprehensive understanding of the structure-activity relationships, compounds 1a, 1b, 1h and 1i were included in this research as well. The structures of the target compounds 1a-y and evaluation of the biological features were summarized in Table 1. The binding affinity was determined by a radioligand binding assay and cAMP assay on V1a and V2 over-expressing cells. These compounds had specific affinity to human AVP receptors. Furthermore, they showed high selectivity to V2 receptors. When ring C was replaced by an alkyl group with straight chain, their binding constants to V2 receptor were reduced significantly along the length increase of the carbon chain. Halogen-substituted alkyl group slightly increased their binding affinity to V2 receptors. When ring C was a substituted benzene ring, the different substituted positions of methyl or halogen did not significantly affect the binding affinity to V2 receptors or V1a/V2 selectivity of the compounds.

Reagents and Conditions
Moreover, meta-substituted compounds had a relatively higher binding affinity to V2 receptors than ortho-substituted or para-substituted compounds. Compounds 1x and 1y showed poor binding activity to both V1a and V2 receptors. Compounds 1a, 1n, 1t and 1v presented encouraging binding affinity, with both remarkable binding affinity and selectivity for the V2 receptor.
Several compounds with satisfactory binding affinity were selected to conduct the in vivo diuretic assay, with 1a as the reference compound [33]. As shown in Table 1, it is evident that the selected compounds have significant diuretic activity, as they strongly increased urine volume compared with the control group. Compound 1t exhibited an excellent diuretic activity which was equivalent to tolvaptan. It is very difficult to declare the relationship between diuretic activity and binding affinity. Because of specific differences in the vasopressin receptors, it may be difficult to draw a direct comparison between the diuretic assay in rats and the binding assay in cells expressing the human receptor.

Biological Evaluation
The in vitro evaluation was done by a slightly modified method we reported previously [31]. An in vitro radioligand binding assay was performed to determine the binding affinity of the candidates to human V2 and V1a receptors. The functional activity was then subsequently determined by measuring the activation or inhibition of vasopressin induced cAMP accumulation in V2 receptor expressing cells. We investigated some potent derivatives for in vivo diuretic activity in conscious hydrated male Sprague-Dawley rats at 8 weeks of age (body weight: (260 ± 20) g). Urine volume was measured 20 h after oral administration of the test compounds.

Conclusions
Twenty-one derivatives of desloratadine designed as AVP V2 receptor antagonists were synthesized and characterized by 1 H-NMR, HRMS and HPLC. Their biological activity was evaluated by in vitro radioligand binding assay, cAMP assay and in vivo diuretic assay. Compounds 1n, 1t and 1v exhibited both high affinity and promising selectivity for V2 receptors. The selected compounds showed promising diuretic results in rats, especially compound 1t, which produced a total urine volume equivalent to tolvaptan during the experimental period. Through the present studies, compound 1t, which has good efficacy both in vitro and in vivo, could be a novel AVP V2 receptor antagonist candidate. Further preclinical studies are however still required.