Synthesis, Antiprotozoal Activity, and Cheminformatic Analysis of 2-Phenyl-2H-Indazole Derivatives

Indazole is an important scaffold in medicinal chemistry. At present, the progress on synthetic methodologies has allowed the preparation of several new indazole derivatives with interesting pharmacological properties. Particularly, the antiprotozoal activity of indazole derivatives have been recently reported. Herein, a series of 22 indazole derivatives was synthesized and studied as antiprotozoals. The 2-phenyl-2H-indazole scaffold was accessed by a one-pot procedure, which includes a combination of ultrasound synthesis under neat conditions as well as Cadogan’s cyclization. Moreover, some compounds were derivatized to have an appropriate set to provide structure-activity relationships (SAR) information. Whereas the antiprotozoal activity of six of these compounds against E. histolytica, G. intestinalis, and T. vaginalis had been previously reported, the activity of the additional 16 compounds was evaluated against these same protozoa. The biological assays revealed structural features that favor the antiprotozoal activity against the three protozoans tested, e.g., electron withdrawing groups at the 2-phenyl ring. It is important to mention that the indazole derivatives possess strong antiprotozoal activity and are also characterized by a continuous SAR.


Introduction
Indazole is an important scaffold in medicinal chemistry. So far, a few drugs containing the indazole moiety are currently available for clinical use; for instance, the antiemetic granisetron, the nonsteroidal anti-inflammatory drugs benzydamine and bendazac, the anticancer agents pazopanib, axitinib, niraparib and entrectinib. From these, niraparib and entrectinib were recently approved in 2017 and 2019 respectively [1,2]. At present, the progress on synthetic methodologies has allowed the preparation of several new indazole derivatives with interesting pharmacological properties [3][4][5][6]. Particularly, the antiprotozoal activity of indazole derivatives against Entamoeba histolytica [7], Giardia intestinalis and Trichomonas vaginalis have been recently reported [8,9]. E. histolytica and G. intestinalis are intestinal protozoa, which causes amebiasis and giardiasis, respectively. Worldwide 50 million amebiasis cases and 100,000 deaths annually are estimated [10], whereas 280 million people are affected by G. intestinalis [11]. Both diseases have high impact in the public health, causing diarrhea, which is a major factor in morbidity and mortality, affecting mainly infant population [12]. On the other hand, trichomoniasis, caused by T. vaginalis is the most common non-viral sexually transmitted disease [13]. According to the World Health Organization, more than 156 million new cases are estimated annually worldwide [14]. Trichomoniasis can be the cause of cervicitis, urethritis, vaginitis, and genital ulceration; additionally, it has been associated with preterm labor, low-birth weight, sterility, cervical cancer, and a predisposition to HIV infection [14][15][16]. Although some chemotherapeutic agents are available to treat amebiasis, giardiasis and trichomoniasis, e.g., nitroderivatives such as metronidazole [13,16,17], the increased drug resistance [13,[16][17][18][19], and absence of new approved drugs with alternative mechanism of action, lead us to search new antiprotozoal compounds, particularly based on the indazole scaffold. Herein, 22 indazole derivatives were synthesized employing a practical one-pot process. Whereas six of these compounds have been previously reported to have activity against E. histolytica, G. intestinalis, and T. vaginalis, in this study, the activity of the additional 16 compounds against these three parasites was evaluated. Moreover, a SAR analysis and activity landscape studies were performed to highlight relevant structural features for the biological activity and to identify the SAR nature of the compounds studied.

Synthesis of 2-Phenyl-2H-Indazole Derivatives
The synthetic route for the synthesis of 2-phenyl-2H-indazole derivatives is shown in Scheme 1. For this purpose, a new practical modification of the Cadogan's method using an ultrasound assisted one-pot synthesis was employed. This method uses a combination of ultrasound synthesis as previously described by Crawford under neat conditions as well as the Cadogan's cyclization by refluxing the Schiff base with triethyl phosphite [20,21]. A comparison of the employed one-pot procedure and a previously reported methodology for representative examples is displayed in Table 1. Noteworthy, the one-pot method leads to similar or better yields than our previous report [8]. Moreover, it is important to mention that our method avoids one purification step, save solvents, and reduce the work up time. To expand the scope of the method, the number of compounds available for biological assays, and the diversity of substituents for SAR analysis, derivatives with chlorine, methoxy, methoxycarbonyl, fluorine and trifluoromethyl groups at the 2-or 3positions of the phenyl substituent were also synthesized. It is to be noted that compound 18, having a 2-(methoxycarbonyl)phenyl substituent, was not obtained by employing the described method, instead, 18a having a 2-cyanophenyl was synthesized as alternative intermediary to obtain 18; however a slight method modification was also needed to achieve the compound 18a (vide infra). Since compounds substituted with OH and COOH are important from the medicinal chemistry point of view due to the fact that it forms key protein-ligand interactions (e.g., hydrogen bonds and salt bridges) [22], derivatives having hydroxyl (7, 14 and 21) and carboxyl (8, 15 and 22) groups were synthesized by hydrolysis of precursors 3, 10, 17, 4, 11 and 18a. All synthesized compounds were characterized by 1 H NMR and 13 C NMR spectra and the new structures were also characterized by mass spectrometry. The nuclear magnetic resonance spectra of all compounds can be found in Figures S1-S23 in the Supplementary Materials.

Antiprotozoal Activity
Compounds 5, 6, 9-22 were tested in vitro against E. histolytica, G. intestinalis, and T. vaginalis. It is important to mention that the activity of compounds 1-4, 7 and 8 against these three protozoa were previously reported by our group [8], however, the values were included in Table 2 in order to support the SAR analysis. Against E. histolytica, the first interesting finding was that all tested compounds were more potent than 1H-indazole reported by López-Vallejo et al. (IC 50 = 0.740 µM) [7]. It is important to note that biological assays were performed using the same strain, under the same conditions as well as the same laboratory. The results suggest that 2-phenyl substitution is important for the antiprotozoal activity, since this modification increased 9-fold the activity. The best potency was found for 2-phenyl-2H-indazole derivatives substituted with methoxycarbonyl (4, 11 and 18), 4-chlorophenyl (2) and 2-(trifluoromethyl)phenyl (20) (IC 50 < 0.050 µM). On the other hand, the results for the assays performed against G. intestinalis showed that the derivatives substituted with 2-chlorophenyl (16), 2-(methoxycarbonyl)phenyl (18), 2-(trifluoromethyl)phenyl (20) and 2-carboxyphenyl (22) displayed the best activity (IC 50 < 0.050 µM). Moreover, 4-and 3-(trifluoromethyl)phenyl derivatives (6 and 13 respectively) showed favorable effect on the giardicidal activity. It is worth to emphasize that compounds were slightly more potent against E. histolytica and G. intestinalis than T. vaginalis. Hence, against T. vaginalis, the best activity (IC 50 < 0.070 µM) was found for the derivatives substituted with 3-(methoxycarbonyl)phenyl (11), 3-(trifluoromethyl)phenyl (13) and 3-carboxyphenyl (15), as well as the 2-chlorophenyl (16) and 2-carboxyphenyl (22). The IC 50 values and SAR data displayed in Figure 1, indicate that electron-withdrawing substituents attached to the 2-phenyl ring are favorable for the antiprotozoal activity against the three evaluated protozoa.

Cheminformatic Analysis
Compounds 1-22 were also analyzed employing cheminformatic tools and compared against antiprotozoal databases of benzimidazole derivatives [7,[23][24][25][26][27][28][29][30][31][32][33][34][35][36] and ChEMBL compounds [37], which have also reported activities against E. histolytica, G. intestinalis and T. vaginalis. It is worthwhile to mention that benzimidazole derivatives compose an important reference database since a high number of compounds were tested under the same method and conditions. These benzimidazole derivatives have been the reference database for several cheminformatic and QSAR studies as antiprotozoals [31,34,[38][39][40]; and the data available has been expanded during the last years. Figure 2 shows a representation of the property space and the activity distribution of the 22 synthesized indazole derivatives as compared to benzimidazole derivatives and ChEMBL collection for each analyzed parasite [41]. Although, 2-phenyl-2H-indazole derivatives are localized in a very focalized space as compared to benzimidazole and ChEMBL compounds, their activity values are similar to the majority of benzimidazoles, but higher than ChEMBL compounds in most cases, as is visually depicted as violin plots in Figure 2. Although the number and diversity of indazole derivatives synthesized and tested herein are still low, it is of our interest to expand the database of indazole derivatives as well as their activity landscape to search for more potent compounds. compounds in most cases, as is visually depicted as violin plots in Figure 2. Although the number and diversity of indazole derivatives synthesized and tested herein are still low, it is of our interest to expand the database of indazole derivatives as well as their activity landscape to search for more potent compounds. To visualize the activity landscape of the synthesized indazole derivatives as compared to the benzimidazole reference database, chemotype-based Structure-Activity Similarity (SAS) maps were generated ( Figure 3) [42]. A general form of the SAS map is presented in Figure 4. SAS maps are divided in four regions: region I is associated with scaffold hopping (or side chain hopping), region II denotes smooth SAR and region IV indicates discontinuous SAR and activity cliffs. The structure similarity threshold was established by the median similarity of the pairwise comparisons plotted in each map and depends on the database and the fingerprint employed, whereas the activity similarity threshold was established on two units in activity difference (100-fold difference in potency). Since Similarity-Property principle establish that similar compounds have similar properties (e.g., biological activity), all indazole derivatives fall in such definition [43]. Different SAR classifications have been previously defined as continuous (smooth), heterogeneous and discontinuous (rough) SAR [43,44]. Moreover, the implications in medicinal chemistry, disadvantages or opportunities implied on different SAR classification has been widely discussed [44,45]. Therefore, the 22 indazole derivatives studied are characterized by a smooth or continuous SAR (high similarity and low activity differences; region II) as compared to the benzimidazole derivatives database where a heterogeneous SAR can be observed [38,39]. Therefore, the antiprotozoal indazole database should be expanded by including more diverse substituted derivatives at different positions to carry out QSAR studies and lead optimization. To visualize the activity landscape of the synthesized indazole derivatives as compared to the benzimidazole reference database, chemotype-based Structure-Activity Similarity (SAS) maps were generated ( Figure 3) [42]. A general form of the SAS map is presented in Figure 4. SAS maps are divided in four regions: region I is associated with scaffold hopping (or side chain hopping), region II denotes smooth SAR and region IV indicates discontinuous SAR and activity cliffs. The structure similarity threshold was established by the median similarity of the pairwise comparisons plotted in each map and depends on the database and the fingerprint employed, whereas the activity similarity threshold was established on two units in activity difference (100-fold difference in potency). Since Similarity-Property principle establish that similar compounds have similar properties (e.g., biological activity), all indazole derivatives fall in such definition [43]. Different SAR classifications have been previously defined as continuous (smooth), heterogeneous and discontinuous (rough) SAR [43,44]. Moreover, the implications in medicinal chemistry, disadvantages or opportunities implied on different SAR classification has been widely discussed [44,45]. Therefore, the 22 indazole derivatives studied are characterized by a smooth or continuous SAR (high similarity and low activity differences; region II) as compared to the benzimidazole derivatives database where a heterogeneous SAR can be observed [38,39]. Therefore, the antiprotozoal indazole database should be expanded by including more diverse substituted derivatives at different positions to carry out QSAR studies and lead optimization.

Chemicals and Instruments
All chemicals and starting materials were obtained from Sigma-Aldrich (Sigma-Aldrich, Toluca, MEX, Mexico). Reactions were monitored by TLC on 0.

Chemicals and Instruments
All chemicals and starting materials were obtained from Sigma-Aldrich (Sigma-Aldrich, Toluca, MEX, Mexico). Reactions were monitored by TLC on 0.

General Procedure for O-Demethylation
The methoxy-2-phenylindazole compounds (3, 10 or 17) (4 mmol) were dissolved in dichloromethane (12 mL) and cooled to 0 • C under N 2 atmosphere. Afterwards, boron tribromide (12 mmol, 12 mL of 1 M solution in dichloromethane) was added and the reaction mixture was stirred at room temperature overnight. The solvent was removed by evaporation and a saturated sodium bicarbonate solution was added. The resulting solid was filtered and dried under vacuum. The crude product was purified using a short column packed with silica gel and ethyl acetate-hexanes (6:4) as a mobile phase.

General Procedure for the Hydrolysis of Ester Derivatives
The methyl ester compounds (4 or 11, 1.2 mmol) were dissolved in methanol (7.5 mL) and an aqueous solution of NaOH (3.6 mmol in 3 mL of water) was added. The reaction mixture was refluxed until the starting material was totally consumed. The mixture was cooled on ice and acidified to pH 1 with HCl to induce precipitation. The solid was filtered under vacuum and dried.

Antiprotozoal Activity Assays
Trichomonas vaginalis strain GT3, Giardia intestinalis isolate IMSS:0981:1 and Entamoeba histolytica strain HM1-IMSS were used in all the experiments. Trophozoites of G. intestinalis were maintained in a TYI-S-33 medium supplemented with 10% calf serum and bovine bile. E. histolytica and T. vaginalis trophozoites were maintained in TYI-S-33 medium supplemented with 10% bovine serum. Briefly, 5 × 10 4 trophozoites of G. intestinalis or T. vaginalis, or 6 × 10 3 trophozoites of E. histolytica were incubated for 48 h at 37 • C with different concentrations of the compound to be tested, each added as solutions in DMSO. As a negative control, parasites received an equivalent amount of DMSO only, while albendazole (ABZ) and metronidazole (MTZ) were included as positive controls. At the end of the treatment period, the cells were washed and subcultured for another 48 h in fresh medium to which no drug was added. The trophozoites were then counted with a hemocytometer and the 50% inhibitory concentration (IC 50 ), together with the respective 95% confidence limit was calculated by Probit analysis. Experiments were carried out in triplicate and repeated at least twice.

Structure-Activity Similarity (SAS) Maps
SAS maps were calculated by employing the Activity Landscape Plotter V1 [53]. Molecular ACCess System (MACCS) keys fingerprint (166 bits) and Extended Connectivity Fingerprint (ECFP) with a diameter four were used [54,55]. Each map was generated by plotting molecular similarity in the X-axis and the absolute value of the activity differences in the Y-axis. The Tanimoto coefficient was used to calculate N(N − 1)/2 pairwise structural similarities (SS i,j ) for each pair of molecules i and j included in each scaffold class (benzimidazole or indazole). Moreover, N(N − 1)/2 pairwise activity differences, determined by the equation |∆A(T) i,j | = |A(T) i − A(T) j |, were calculated for indazole and benzimidazole datasets; where A(T) i and A(T) j are the activities of the ith and jth molecules (j > i), in pIC 50 , tested against the target parasite T [42]. SAS maps were divided in four regions employing a threshold of two units on activity difference (100-fold in potency) and the median similarity of the total datapoints plotted in each map (including indazole and benzimidazole derivatives). Briefly, Regions I and II are associated with scaffold hopping (or side chain hopping) and smooth SAR, respectively, whereas Region IV indicates discontinuous SAR (Figure 4).

Conclusions
A one-pot procedure including a combination of ultrasound synthesis under neat conditions and a Cadogan's cyclization was implemented for the synthesis of 2-phenyl-2Hindazole derivatives. The one-pot method leads to similar or better yields as compared to our previous reports. The biological assays revealed that electron withdrawing groups at the 2-phenyl ring are favorable for antiprotozoal activity. Furthermore, although the SAR information obtained from indazole derivatives as antiprotozoals is still limited, cheminformatic analysis for the 22 compounds studied, highlight their potent values and their smooth SAR nature as compared to reference databases. More studies are needed to expand the activity landscape of indazole derivatives; however, these results represent a valuable start-point toward the optimization of indazole derivatives as antiprotozoals.