Synthesis of 4,7,9-Trihydroxy[1]benzofuro[3,2-d] pyrimidine-6-carboxamide: Evaluation of Cytotoxicity and Inhibition of Protein Kinase C ( Ca Pkc1)

The protein kinase Pkc1 of Candida albicans ( Ca Pkc1), one of the key proteins involved in MAPK pathway, is described as a regulator of cell wall integrity during growth, morphogenesis, and response to cell wall stress. The (–)-cercosporamide is an antifungal natural product isolated from the phytopathogen fungus Cercosporidium henningsii . This phytoxin was found to inhibit selectively Ca Pkc1 and constitutes an interesting model for the design of novel antifungal molecules. In this research, 4,7,9-trihydroxy[1]benzofuro[3,2-d]pyrimidine-6-carboxamide ( 13 ) derived from (–)-cercosporamide was synthesized via a seven-step procedure by well-known reactions and evaluation of cytotoxicity and inhibition of Ca Pkc1. The bioassay showed CaPkc1 inhibitory activity 87% higher and cytotoxicity 100 times less than the reference, (–)-cercosporamide.


Introduction
Candidiasis exists in all regions of the world; however, there are significant variations between regions and it is difficult to give an average incidence of candidiasis. Although many studies have been carried out annually, the type of population recruited is variable and the results rendered are difficult to compare. In addition, it is absolutely necessary to know the profile of the local epidemiology in order to best adapt the antifungal treatment. American studies report that the annual incidence of Candida infections is 8 per 100,000 inhabitants [1,2]. In Europe, Candida spp. are responsible for 2-3% of septicemia ranking between 6th and 10th among pathogens most frequent [3]. e distribution of Candida species in patients with candidiasis has changed over the past 20 years. e incidence of Candida albicans decreased (from 70% to 50%), while the incidence of non-albicans Candida (NAC) species, including C. glabrata, C. krusei, C. tropicalis, C. auris, and C. parapsilosis has increased [4]. Until recently, C. albicans remains mainly responsible for these invasive infections, but the distribution of NAC species evolves and differs according to the regions of the world [5]. In C. albicans, protein kinase C (CaPkc1) is one of the proteins involved in the MAPK pathway [6]. It is described as a regulator of the integrity of the cell wall during growth, morphogenesis, and the response to cell wall stress [7]. Treatment of fungal infections is very difficult because of limited therapeutic arsenal and emergence of resistance to the two antifungal major classes: azoles and echinocandins. Consequently, the search for new targets and new therapeutic strategies is a priority. In this context, targeting PKCmediated signal transduction pathway represents a new attractive strategy for antifungal therapy. e (-)-cercosporamide is an antifungal natural product isolated from the phytopathogen fungus Cercosporidium henningsii [8]. is phytoxin was found to inhibit selectively Pkc1 kinase of C. albicans (IC 50 � 44 nM) [9] and constitutes an interesting model for the design of novel antifungal molecules. In addition, research results suggest dihydroxybenzofurancarboxamide ring as the pharmacophore of the molecule, justifying the strategy for the design of new tricyclic compounds [10]. Regarding this, our expertise in the synthesis of pyrimidine-fused heterocycles as biological agents targeting kinases was used for the design of the new compounds, constituting the third ring of the benzofuro[3,2-d]pyrimidine derivatives described [11]. In this study, we designed and synthesized a new compound 13, which remained the dihydroxybenzofuran-carboxamide ring (part A, excluding asymmetric carbon), replacing part B with pyrimidine ring (Figure 1) targeting CaPkc1. e introduction of a pyrimidine ring into the structure is capable of providing donor or acceptor groups for hydrogen bonds and can cause additional interactions with amino acids at the active site for better kinase inhibitory activity and improved selectivity. Figure 2, analogues of (-)-cercosporamide were prepared from 3,5-dimethoxyphenol (1). Diiodination of compound 1 was performed very quickly at low temperature in the presence of NIS, followed by O-alkylation with a base and ethyl bromoacetate [12,13] to afford 3 in 72%. Subsequent cyanation of this intermediate in the presence of cuprous cyanide in DMF gave ester 4 in 75% [14]. Cyclization using sodium hydride furnished benzofuran ring bearing the amino group at position 3 and ester function at position 2 [15]. Tricyclic pyrimidine derivative 6 was synthesized from benzofuran precursor 5 [16]. From compound 6, attempts for the conversion of nitrile to carboxamide remained unsuccessful [13,[17][18][19].

Chemistry. As shown in
Another strategy was to introduce the carboxamide group before the cyclization step. As shown in Figure 3, a direct aminocarbonylation of compound 1 use chlorosulfonylisocyanate (CSI) as electrophilic reagent afforded benzamide derivative 8 after acidic hydrolysis of the corresponding N-chlorosulfonyl carboxamide intermediate [20]. Next, the iodination of compound 8 was performed very quickly at 0°C in the presence of NIS, followed by cyanation in the presence of cuprous cyanide in DMF, and then, O-alkylation with a base and ethyl bromoacetate gave ester 11 in 90%. Afterwards, the same reaction sequence as previously described in Scheme 1 was applied to obtain derivative 7 bearing the carbamoyl appendage at C-6 position of the azaheterocycle. Finally, demethylation of the methoxy groups was carried out by heating in neat pyridine hydrochloride at 200°C under microwave irradiation to provide the desired 4,7,9-trihydroxy [1]benzofuro [3, 2-d] pyrimidine-6-carboxamide (13) in 43% [21]. e structures of all compounds synthesized were confirmed using MS, 1 H, and 13 C NMR spectrum (Supplementary Materials).

Biological Activity.
e inhibition of PKC activity (CaPkc1) was initially investigated in order to check the involvement of this protein as the putative target of 4, 7, 9trihydroxy [1]benzofuro [3,2-d]pyrimidine-6-carboxamide (13). Pkc activity of C. albicans (CAAL93 sensitive to fluconazole); total protein extracts was measured using ELISA-based PKC Kinase Activity Assay (Enzo Life Sciences, Inc.) [22]. PKC inhibition activity was expressed as the ratio between the absorbance measured for protein extract treated with compounds 13 (100 µM) and untreated protein extract. e reference natural (-)-cercosporamide was provided by extraction and purification from a Mycosphaerella henningsii culture using a modified procedure of the literature [8,23].
Mean percentage of inhibition of PKC activity was obtained for each compound (100 µM) and compared with mean results obtained for (-)-cercosporamide (100 µM) applying a one-way ANOVA followed by multiple comparisons with an uncorrected Fisher's LSD test. P values were expressed for the uncorrected Fisher's LSD test (p < 0.00001). To investigate in vitro cytotoxic activity of 4,7,9-trihydroxy [1]benzofuro [3,2-d]pyrimidine-6-carboxamide (13), assay on HeLa cells was performed. As given in Table 1, compound 13 proved to be at least 100 times less cytotoxic than (-)-cercosporamide. is shows that compound 13 has the potential to become a new selective effect on fungal cells, not toxic to other cells. As depicted in Table 1, when used at 100 µM, (-)-cercosporamide displayed a moderate Pkc inhibitory activity of 53 ± 3% in our conditions. Interestingly, compound 13 showed statistically (p < 0.05) significant higher inhibition with values of 87 ± 2% at 100 µM. It is possible that the pyrimidine ring in part B in the structure has an additional -OH group resulting in stronger binding capacity of compound 13 to the center associated with CaPkc1, increasing the ability to inhibit CaPkc1 of compound 13.

Conclusions
An efficient and practically feasible procedure for the synthesis of 4,7,9-trihydroxy [1]benzofuro[3,2-d]pyrimidine-6carboxamide via a seven-step procedure using common chemical reactions such as direct aminocarbonylation, iodonation, cyanation, O-alkylation, cyclizaction, and demethylation has been described. In this research, the initial biological assay of compound 13 suggests further chemical modifications to evaluate the structure-activity relationship of this naturally derived potential compound, (-)-cercosporamide.

Experimental
All reactions were monitored by TLC analysis using Merck silicagel 60F-254 thin-layer plates. Column chromatography was carried out on silicagel Merck 60 (70-230 mesh ASTM).    Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane as internal standard, and coupling constants (J) are given in Hertz (Hz). e following abbreviations are used to describe peak patterns when appropriate: s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Mass spectra were recorded using an electrospray ionization method with the Waters ZQ 2000 spectrometer. Microwave-promoted reactions were performed on a CEM Discover SP monomode apparatus. Elemental analyses were performed on a ermo Scientific Elemental Analyser Flash EA 1112 and were found within ±0.4% of the theoretical values. (2). A solution of 3,5dimethoxyphenol (1) (1.00 g, 6.50 mmol) in dichloromethane (20 mL) was cooled to −78°C. N-Iodosuccinimide (3.22 g, 14.3 mmol) was added, and the reaction was maintained at −78°C for 2 min. e reaction was quenched at −78°C by the addition of 10% aqueous potassium carbonate solution (20 mL) and warmed to room temperature. e reaction was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). e combined organic extracts were dried over sodium sulfate and filtered. e solvent was evaporated in vacuo, and the residue was purified by silica gel column chromatography using cyclohexane/ethyl acetate (9/1) as an eluent system to give compound 2 as a beige solid (1.87 g, 71%).

Data Availability
e data used to support the findings of this study are included within the article and in Supplementary Materials.

Conflicts of Interest
e authors declare that they have no conflicts of interest.