Probing Adamantane Arylhydroxamic Acids against Trypanosoma brucei and Trypanosoma cruzi

In this work, we present the synthesis and the anti-trypanosomal activity of the 2-(4(adamant-1-yl)phenyl)-N-hydroxyarylamides, 1a,b and the 2-(4-(adamant-1-yl)phenoxy)-N-hydroxyacetamide, 1c. The 4-(adamant-1-yl)phenyland 4-(adamant-1-yl)phenoxymoieties, which are endowed with promising drug-like properties, are functionalized at the side chain termini as hydroxamic acids. The phenoxy acetohydroxamic derivative, 1c, shows the most interesting profile in terms of activity and toxicity against trypanosomes and merits further investigation.


Introduction
One-sixth of the global population, more than 1 billion, is infected with one or more neglected tropical diseases (NTDs) and an additional 2 billion are at risk [1]. Among the "most neglected" NTDs are the two types of trypanosomiasis: human African trypanosomiasis (HAT) and Chagas disease (CD) [2]. Public-private consortia bridge the gap between research and the current therapeutic regimens against these diseases [3,4]. Largescale phenotypic screening has identified many drug candidates that are active against the kinetoplastid protozoa. However, validated targets against the parasitic diseases are fewer because of the complexities of pathogen biology and, in some cases, insufficiently robust genetic approaches [5]. The challenge for new classes of effective therapeutic agents involves investigation of target-based approaches of drug design [6].
The current drugs used against HAT and CD are suboptimal. The approved regimen presents many restrictions, such as serious adverse side effects, excessive toxicity, limited efficacy and increasing resistance [3,4]. The older therapeutical arsenal consists of pentamidine, eflornithine, suramin, melarsoprol and nifurtimox, while newer agents are benznidazole and the latter fexinidazole and acoziborole [5]. Over the last decade, the scientific community has made tremendous efforts toward NTD control and many more late-stage candidates will be generated in the pipeline, in due course [7][8][9].
ank 2022, 2022, x FOR PEER REVIEW 2 o acids exhibit metal-chelating properties and act as inhibitors of metalloproteins with wide spectrum of therapeutic roles [19]. Acetohydroxamic adamantane derivatives ha shown significant activity against T. brucei [20] (Figure 1). We now explore the role of t phenyl or phenoxy substitution at the C1 position of the adamantane cage regarding t functional hydroxamic end of the side chain in the 1a,b and c derivatives and their activ against trypanosomes.

Chemistry
The synthesis of the hydroxamic acids 1a-c was accomplished by the reactions show in Scheme 1. Carboxylic acids 3-5 were coupled with O-benzylhydroxylamine in the pr ence of 1,1′-carbonyldiimidazole (CDI) in THF to give the corresponding O-benzyl h droxamates 2a-c, in yields of 68-74%. Subsequent O-benzyl deprotection by hydrogen ysis in the presence of Pd/C in ethanol afforded the target hydroxamic acids, 1a-c, in hi yield (90-95%). The synthesis of the carboxylic acids 3 and 4 (Scheme 1) has been describ in our previously published protocols [18,21], while the preparation of the 2-(4-(adama tan-1-yl)phenoxy)acetic acid 5 was reported by Lee et al. [22].

Biological Evaluation
The new hydroxamic acid analogues 1a-c were tested for their activity against t bloodstream form Trypanosoma brucei and Trypanosoma cruzi epimastigotes and the resu are presented in Tables 1 and 2.

Chemistry
The synthesis of the hydroxamic acids 1a-c was accomplished by the reactions shown in Scheme 1. Carboxylic acids 3-5 were coupled with O-benzylhydroxylamine in the presence of 1,1 -carbonyldiimidazole (CDI) in THF to give the corresponding O-benzyl hydroxamates 2a-c, in yields of 68-74%. Subsequent O-benzyl deprotection by hydrogenolysis in the presence of Pd/C in ethanol afforded the target hydroxamic acids, 1a-c, in high yield (90-95%). The synthesis of the carboxylic acids 3 and 4 (Scheme 1) has been described in our previously published protocols [18,21], while the preparation of the 2-(4-(adamantan-1-yl)phenoxy)acetic acid 5 was reported by Lee et al. [22].
Molbank 2022, 2022, x FOR PEER REVIEW 2 of 8 acids exhibit metal-chelating properties and act as inhibitors of metalloproteins with a wide spectrum of therapeutic roles [19]. Acetohydroxamic adamantane derivatives have shown significant activity against T. brucei [20] (Figure 1). We now explore the role of the phenyl or phenoxy substitution at the C1 position of the adamantane cage regarding the functional hydroxamic end of the side chain in the 1a,b and c derivatives and their activity against trypanosomes.

Chemistry
The synthesis of the hydroxamic acids 1a-c was accomplished by the reactions shown in Scheme 1. Carboxylic acids 3-5 were coupled with O-benzylhydroxylamine in the presence of 1,1′-carbonyldiimidazole (CDI) in THF to give the corresponding O-benzyl hydroxamates 2a-c, in yields of 68-74%. Subsequent O-benzyl deprotection by hydrogenolysis in the presence of Pd/C in ethanol afforded the target hydroxamic acids, 1a-c, in high yield (90-95%). The synthesis of the carboxylic acids 3 and 4 (Scheme 1) has been described in our previously published protocols [18,21], while the preparation of the 2-(4-(adamantan-1-yl)phenoxy)acetic acid 5 was reported by Lee et al. [22]. Scheme 1. Synthesis of the hydroxamic acid analogues 1a-c.

Biological Evaluation
The new hydroxamic acid analogues 1a-c were tested for their activity against the bloodstream form Trypanosoma brucei and Trypanosoma cruzi epimastigotes and the results are presented in Tables 1 and 2.

Biological Evaluation
The new hydroxamic acid analogues 1a-c were tested for their activity against the bloodstream form Trypanosoma brucei and Trypanosoma cruzi epimastigotes and the results are presented in Tables 1 and 2.  We found that the acetohydroxamide 1b was more potent than the benzohydroxamide 1a, which had little activity against trypanosomes. Of the three derivatives tested, the phenoxy acetamide 1c was the most active. Insertion of the oxygen atom of the phenoxy group enhanced activity against T. brucei more than ninefold compared with 1b (IC 50 of 1c versus IC 50 of 1b, Table 1). For comparison purposes, the phenoxy acetamide 1c was 12-fold more potent against T. cruzi epimastigotes and more than eight times more potent against the T. brucei bloodstream forms than benznidazole and fexinidazole, two heterocyclic nitro-drugs that are used to treat T. cruzi and T. brucei, respectively. The activity of both compounds against T. cruzi followed the same pattern. These results have prompted us to further investigate these structure-activity relationships and pursue in the future the synthesis of related structures.

Chemistry
All chemicals and solvents were obtained from commercial suppliers and used without further purification. Concentrated refers to the removal of solvents with a rotary evaporator at normal water aspirator pressure, followed by further evacuation on a high-vacuum line. Reactions were monitored by thin-layer chromatography. Thin-layer chromatography was performed using E. Merck precoated silica gel 60 F 254 plates. Developed TLC plates were visualized with UV light (254 nm) and iodine. The chromatographic purification of the products was carried out using Silica gel 60 (40-63 µm, 230-400 mesh ASTM, Silica flash). Melting points were determined on a Büchi 530 apparatus and are uncorrected. Microanalyses were carried out by the NCSR Demokritos, Greece, and the results obtained had a maximum deviation of ±0.4% from the theoretical value.
The 1 H and 13 C NMR spectra for the hydroxamic acid derivatives described in this report (compounds 1a-c), are consistent with a Z/E conformation of these molecules in solution. The detected double set of characteristic peaks in the 1 H and 13 C NMR spectra confirm the presence of the two carbonyl rotamers. The assignment of the Z or E rotamers was based on literature data concerning Z/E geometry studies in simple hydroxamate structures (Figure 2) [23][24][25][26][27].

Cytotoxic Activity against Rat Skeletal Myoblast L6 Cells
Cytotoxicity against mammalian cells was assessed using microtitre plates. Briefly, L6 cells (a rat skeletal muscle line) were seeded at 1 × 10 4 mL −1 in 200 µL of RPMI-1640 growth medium containing seven different compound concentrations in a range previously established to encompass both the IC 50 and IC 90 values. The plates were incubated for 6 days at 37 • C and 20 µL Alamar Blue (Biosource Ltd, Wilton, North Yorkshire England.) was then added to each well. After an additional 8 h incubation, the fluorescence was determined using a Gemini fluorescent plate reader (Molecular Devices). Inhibition of growth was calculated by comparison with control values and IC 50 and IC 90 values were determined in triplicate using linear regression analysis.

Trypanosoma brucei Culturing and Drug Testing
Bloodstream form T. brucei (strain 427) were cultured at 37 • C in modified Iscove's medium. Trypanocidal activity was assessed by growing parasites in microtiter plates in the presence of various drug concentrations. Parasites were seeded at 0.25 × 10 −5 mL −1 in 200 µL of growth medium containing seven different compound concentrations in a range previously established to encompass both the IC 50 and IC 90 values. The plates were incubated for 48 h at 37 • C and 20 µL Alamar Blue was then added to each well. After an additional overnight incubation, the fluorescence was determined using a Gemini fluorescent plate reader (Molecular Devices).

Trypanosoma cruzi Culturing and Drug Testing
T. cruzi epimastigotes (strain CL Brener) were cultured at 28 • C in supplemented RPMI-1640 medium. Trypanocidal activity was assessed by growing parasites in microtiter plates in the presence of various drug concentrations. Parasites were seeded at 2.5 × 10 −5 mL −1 in 200 µL of growth medium containing 7 different compound concentrations in a range previously established to encompass both the IC 50 and IC 90 values. The plates were incubated for 4 days at 28 • C and 20 µL Alamar Blue was then added to each well. After an additional 3 days incubation, the fluorescence was determined using a Gemini fluorescent plate reader (Molecular Devices).