Diketopiperazine-Based, Flexible Tadalafil Analogues: Synthesis, Crystal Structures and Biological Activity Profile.

Phosphodiesterase 5 (PDE5) is one of the most extensively studied phosphodiesterases that is highly specific for cyclic-GMP hydrolysis. PDE5 became a target for drug development based on its efficacy for treatment of erectile dysfunction. In the present study, we synthesized four novel analogues of the phosphodiesterase type 5 (PDE5) inhibitor-tadalafil, which differs in (i) ligand flexibility (rigid structure of tadalafil vs. conformational flexibility of newly synthesized compounds), (ii) stereochemistry associated with applied amino acid building blocks, and (iii) substitution with bromine atom in the piperonyl moiety. For both the intermediate and final compounds as well as for the parent molecule, we have established the crystal structures and performed a detailed analysis of their structural features. The initial screening of the cytotoxic effect on 16 different human cancer and non-cancer derived cell lines revealed that in most cases, the parent compound exhibited a stronger cytotoxic effect than new derivatives, except for two cell lines: HEK 293T (derived from a normal embryonic kidney, that expresses a mutant version of SV40 large T antigen) and MCF7 (breast adenocarcinoma). Two independent studies on the inhibition of PDE5 activity, based on both pure enzyme assay and modulation of the release of nitric oxide from platelets under the influence of tadalafil and its analogues revealed that, unlike a reference compound that showed strong PDE5 inhibitory activity, the newly obtained compounds did not have a noticeable effect on PDE5 activity in the range of concentrations tested. Finally, we performed an investigation of the toxicological effect of synthesized compounds on Caenorhabditis elegans in the highest applied concentration of 6a,b and 7a,b (160 µM) and did not find any effect that would suggest disturbance to the life cycle of Caenorhabditis elegans. The lack of toxicity observed in Caenorhabditis elegans and enhanced, strengthened selectivity and activity toward the MCF7 cell line made 7a,b good leading structures for further structure activity optimization and makes 7a,b a reasonable starting point for the search of new, selective cytotoxic agents.

Extending the use of tadalafil scaffold: Development of novel selective phosphodiesterase 5 inhibitors and histone deacetylase inhibitors.

Herein we present the synthesis and characterization of a novel chemical series of tadalafil analogues that display different pharmacological profiles. Compounds that have the 6R, 12aR configuration and terminal carboxylic acid group at the side chain arising from the piperazinedione nitrogen were potent PDE5 inhibitors, with compound 11 having almost equal potency to tadalafil and superior selectivity over PDE11, the most common off-target for tadalafil. Modifying the stereochemistry into 6S, 12aS configuration and adopting the hydroxamic acid moiety as a terminal group gave rise to compounds that only inhibited HDAC. Dual PDE5/HDAC inhibition could be achieved with compounds having 6R, 12aR configuration and hydroxamic acid moiety as a terminal group. The anticancer activity of the synthesized compounds was evaluated against a diverse number of cell lines of different origin. The compounds elicited anticancer activity against cell lines belonging to lymphoproliferative cancer as well as solid tumors. Despite the previous reports suggesting anticancer activity of PDE5 inhibitors, the growth inhibitory activity of the compounds seemed to be solely dependent on HDAC inhibition. Compound 26 (pan HDAC IC50 = 14 nM, PDE5 IC50 = 46 nM) displayed the most potent anticancer activity in the present series and was shown to induce apoptosis in Molt-4 cells. HDAC isoform selectivity testing for compound 26 showed that it is more selective for HDAC6 and 8 over HDAC1 by more than 20-fold.

Characterization and Stability of Amorphous Tadalafil and Four Crystalline Polymorphs.

Tadalafil (TD), a phosphodiesterase-5 (PDE-5) inhibitor with poor oral bioavailability. The aim of the study was to prepare and characterize three crystalline polymorphs of TD (II, III, and IV) and the tadalafil amorphous form (TD-AM). TD polymorphs and TD-AM were prepared and characterized by polarized light microscope (PLM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), X-ray powder diffractometry (XRPD), and Fourier-transform (FT)IR, followed by the dissolution testing, physical stabilities and polymorphic transformation studies. TD-I and TD-II were found to be enantiotropically related, while TD-III was monotiotropically related to TD-I with heat release. Among all studied polymorphs, TD-AM demonstrated an extremely high intrinsic dissolution rate with most prolonged higher saturated concentration during dissolution, while TD-II, TD-III, and TD-IV converted to TD-I easily by supersaturation-mediated phase transformation. Upon heating under 60°C for 3 h and storing at long-term stability condition for 3 months, no phase transformation was detected for TD-I, TD-III, and TD-AM, while TD-II and TD-IV easily transformed to TD-I and TD-III, respectively. The higher intrinsic dissolution rate, prolonged supersaturated state during dissolution and favorable physical stability of TD-AM made it to be a very promising candidate for further product development.

ATR-FTIR characterization of generic brand-named and counterfeit sildenafil- and tadalafil-based tablets found on the Brazilian market.

Viagra and Cialis are among the most counterfeited medicines in many parts of the world, including Brazil. Despite the many studies that have been made regarding discrimination between genuine and counterfeit samples, most published works do not contemplate generic and similar versions of these medicines and also do not explore excipients/adjuvants contributions when characterizing genuine and suspected samples. In this study, we present our findings in exploring ATR-FTIR spectral profiles for characterizing both genuine and questioned samples of several generic and brand-name sildenafil- and tadalafil-based tablets available on the Brazilian market, including Viagra and Cialis. Multi-component spectral matching (deconvolution), objective visual comparison and correlation tests were used during analysis. Besides from allowing simple and quick identification of counterfeits, results obtained evidenced the strong spectral similarities between generic and brand-named tablets employing the same active ingredient and the indistinguishability between samples produced by the same manufacturer, generic or not. For all sildenafil-based and some tadalafil-based tablets tested, differentiation between samples from different manufacturers, attributed to slight variations in excipients/adjuvants proportions, was achieved, thus allowing the possibility of tracing an unknown/unidentified tablet back to a specific manufacturer.

Synthesis and Structure Revision of Dimeric Tadalafil Analogue Adulterants in Dietary Supplements.

A number of phosphodiesterase 5 (PDE5) inhibitors approved by authorities have been used successfully in the treatment of erectile dysfunction. These medicines must be prescribed carefully due to their adverse effects, but they and their analogues are being illegally added to dietary supplements. These illegal dietary supplements pose a significant risk to public health. Several dimeric tadalafil analogues have been synthesized for use as reference standards in the inspection of functional foods that are mainly advertised as sexual enhancement products. During the course of this synthesis, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) was proven to be the reagent of choice for amide coupling to produce these dimeric tadalafil analogues. Moreover, the trans-isomer structures tentatively assigned for the isolated dimeric tadalafil analogues (bisprehomotadalafil and bisprecyclopentyltadalafil) found in dietary supplements are now revised to cis-isomer structures.

Design of PVP/VA S-630 based tadalafil solid dispersion to enhance the dissolution rate.

Tadalafil (TDF) is a Biopharmaceutics Classification System (BCS) class II drug; the efficacy thereof is critically limited by inherent poor water solubility. Solid dispersion (SD) techniques are widely used to improve the bioavailability of drugs that are poorly water-soluble. Herein, we used an SD technique to improve the solubility and in vitro dissolution rate of TDF; a solvent evaporation method was applied involving the use of hydrophilic carriers (PVP/VA S-630) and assistants (malic acid or meglumine). The TDF-SD formulations were evaluated in terms of the solubility, in vitro dissolution, and stability. Physical properties were confirmed by field-emission scanning electron microscopy, differential scanning calorimetry, powder X-ray diffraction, and Fourier transform infrared spectroscopy (FT-IR). TDF-SD formulations containing assistants (malic acid or meglumine) and various solubilizers exhibited significantly enhanced solubility in distilled water (DW) (up to 27.3-fold; 18.5±0.16µg/mL with PVP/VA S-630) compared with TDF alone (0.73±0.08µg/mL). However, the dissolution rate of malic acid based formulation was decreased as the PVP/VA S-630 content increased compared to meglumine based formulation. Thus, the optimal TDF-SD formulation (TDF/meglumine/PVP/VA S-630/Aerosil 200: 1/3/5/3) exhibited a greater dissolution rate (89.1±3.9%) than TDF alone (6.2±2.5%) and Cialis® powder (16.0±1.9%) in DW. The final TDF-SD formulation was amorphous in nature and exhibited good stability. In conclusion, TDF-SD was successfully improved in vitro dissolution rate of TDF compared to commercial products (Cialis®) in the dissolution media without sodium lauryl sulfate (SLS).

High energy ball milling and supercritical carbon dioxide impregnation as co-processing methods to improve dissolution of tadalafil.

Tadalafil (TD) is a crystalline drug of a high melting point (Tm=299°C) and limited solubility in water (<5µg/mL). These properties may result in reduced and variable bioavailability after oral administration. Since the melting of TD is followed by its decomposition, the drug processing at high temperatures is limited. The aim of the research is, therefore, to improve the dissolution of TD by its co-processing with the hydrophilic polymer Soluplus® (SL) at temperatures below 40°C. In this study, two methods, i.e. high energy ball-milling and supercritical carbon dioxide impregnation (scCO2) are compared, with the aim to predict their suitability for the vitrification of TD. The influence of the amount of SL and the kind of co-processing method on TD thermal properties is analyzed. The results show that only the high energy ball milling process makes it possible to obtain a completely amorphous form of TD, with the characteristic X-ray 'halo' pattern. The intensity of the Bragg peaks diminishes for all the formulations treated with scCO2, but these samples remain crystalline. The MDSC results show that high energy ball milling is capable of forcing the mixing of TD and SL at a molecular level, providing a homogeneous amorphous solid solution. The glass transition temperatures (Tg), determined for the co-milled formulations, range from 79°C to 139°C and they are higher than Tg of pure SL (ca. 70°C) and lower than Tg of pure TD (ca. 149°C). In contrast to the co-milled formulations which are in the form of powder, all the formulations after scCO2 impregnation form a hard residue, sticking to the reaction vessel, which needs to be ground before analysis or further processing. Finally, the dissolution studies show that not only has SL a beneficial effect on the amount of TD dissolved, but also both co-processing methods make the dissolution enhancement of TD possible. After co-processing by scCO2, the amount of TD dissolved increases with the decreasing amount of SL, whereas in the case of the co-milled formulations, the higher the amount of SL in the glassy solution is, the higher the amount of TD dissolved.