Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as

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Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as Nucleocapsid Protein 7 (NCp7) Inhibitors with anti-HIV Activity Luca Sancineto, Alice Mariotti, Luana Bagnoli, Francesca Marini, Jenny Desantis, Nunzio Iraci, Claudio Santi, Christophe Pannecouque, and Oriana Tabarrini J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01183 • Publication Date (Web): 27 Nov 2015 Downloaded from http://pubs.acs.org on November 29, 2015

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Journal of Medicinal Chemistry

Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as Nucleocapsid Protein 7 (NCp7) Inhibitors with anti-HIV Activity #

Luca Sancineto, § Alice Mariotti, § Luana Bagnoli, § Francesca Marini, §Jenny Desantis, Nunzio Iraci, #

§

Claudio Santi, §* Christophe Pannecouque,¥* Oriana Tabarrini#*

Department of Pharmaceutical Sciences, Group of Catalysis and Organic Green Chemistry,

University of Perugia, Via del Liceo 1, Perugia 06100, Italy. #

Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, Perugia

06100, Italy. ¥

KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and

Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium.

TITLE RUNNING HEAD: Selenium-containing NCp7 inhibitors

ABSTRACT The interest in the synthesis of Se-containing compounds is growing with the discovery of derivatives exhibiting various biological activities. In this manuscript, we have identified a series of 2,2’-diselenobisbenzamides (DISeBAs) as novel HIV retroviral nucleocapsid protein 7 (NCp7) inhibitors. Due to its pleiotropic functions in the whole viral life cycle and its mutation intolerant nature, NCp7 represents a target of great interest which is not reached by any anti-HIV agent in clinical use. Using the diselenobisbenzoic scaffold, aminoacid and benzenesulfonamide derivatives were prepared and biologically profiled against different models of HIV infection. The

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incorporation of amino acids such as glycine and glutamate into DISeBAs 7 and 8 resulted into a selective anti-HIV activity against both acutely and chronically infected cells, as well as into an interesting virucidal effect. DISeBAs demonstrated broad antiretroviral activity, encompassing HIV-1 drug-resistant strains including clinical isolates, as well as simian immunodeficiency virus (SIV). Time of addition experiments, along with the observed dose dependent inhibition of the Gag precursor proper processing, confirmed that their mechanism of action is based on NCp7 inhibition.

INTRODUCTION For a long time selenium has been mainly considered a poison, probably as a consequence of studies that correlated its bioaccumulation to important disorders in both animals1 and humans,2 including a hypothetic correlation with cancerogenesis.3 In the second half of the 20th century, selenium became

instead of interest for its biochemical properties, being

recognized as an essential nutritional trace element, with its deficiency correlated to various human diseases.4 Nowadays it is confirmed that selenium plays a key role when incorporated as selenocysteine (Sec), into biologically relevant selenoproteins5 such as the glutathione peroxidase (GPx). GPx was the first discovered selenoprotein in mammals, and has been extensively studied because of its central role in the defense against the oxidative stress and of other diverse functions.6 Although many aspects of selenium physiological and pathological roles need to be clarified, literature reports agree on its protective role during HIV infection.7,8 Being selenium a key nutrient in antioxidant defense, its deficiency facilitates the oxidative stress onset, thus contributing to immune deregulation and HIV replication.9 Daily selenium supplementation to AIDS patients has shown to suppress the progression of HIV-1 viral load and to increase


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CD4+ lymphocytes counts with a general feeling of well-being when compared to patients receiving a placebo.7,10,11 The toxicity of selenium is an old and debated issue that for a long time has been hampering the idea of Se-containing compounds as potential therapeutics,12 On the contrary, over the last years there has been a growing interest in the synthesis of such compounds, that yielded promising antioxidants, enzyme mimics and inhibitors, immunomodulators, cytoprotectors, antitumoral, anti-inflammatory, antihypertensive, and anti-infective agents.13-16 The introduction of ebselen and ethaselen into clinical trials for the treatment of diabetes complications and non-small cell lung cancer respectively,17,18 recently paved the way for an improved exploitation of selenium in medicinal chemistry. A few examples of selenorganic compounds with anti-HIV activity are known. The first was ebselen,19 identified about 20 years ago through the knowledge that HIV-1 gene expression and viral replication can be induced, in addition to the cytokine stimulation, by oxidative stress. More recently, other Se-containing compounds were synthesized to test their anti-HIV activity. In particular, selenium replaced oxygen in a series of 2’,3’-dideoxynucleosides,14 while 1,2,3-selenadiazole thioacetanilide derivatives were designed as non-nucleoside reverse transcriptase inhibitors by replacing sulfur with selenium.20 While the 4’-seleno-2’,3’dideoxynucleosides did not show any anti-HIV activity, some selenodiazoles derivatives displayed anti-HIV-1 activities comparable to those of their sulfanyldiazole analogues. HIV-1 infection can be effectively controlled by the combination antiretroviral therapy (cART) which improves the life quality in infected individuals, but unfortunately fails to eradicate the virus even after decades of treatment.21 This limitation together with the emergence of multidrug-resistant HIV strains demands for new highly potent drugs capable of interfering with viral processes that are not targeted by the currently approved anti-HIV drugs. Given our interest in selenium chemistry 22-26 and in the identification of anti-HIV agents,27-34


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we have planned the synthesis of a series of organoselenium compounds with the aim to identify novel anti-HIV agents. Our attention was caught by the 2,2’-dithiobisbenzamides (DIBAs, Figure 1)35,36 and their peculiar anti-HIV mechanism of action. Indeed, these compounds inhibit the retroviral nucleocapsid protein 7 (NCp7 - the mature form of NCp), which is not reached by any antiHIV agent in clinical use.37 NCp7, is a small basic protein containing two copies of the strictly conserved zinc finger (ZF) motif C(X)2C(X)4H(X)4C (also termed CCHC; X= any aa). These motifs are known to chelate Zn2+ ions with subpicomolar affinities through their cysteine and histidine residues. In HIV-1, NCp7 contains 55 aa and is released by proteasedirected cleavage of the Gag polyprotein precursor. Thanks to its nucleic acids binding and chaperoning properties, NCp7 is essential in several phases of the HIV replicative cycle, including reverse transcription, integration, and virus assembly. In the latter step, the NCp7 domain of the Gag precursor directs genomic vRNA selection, RNA packaging and dimerization. Considering the essential and multiple functions in both early and late stages of HIV-1 replication, as well as the conserved chelating motif, NCp7 represents an attractive target to complement the cART in the hope to overcome the growing concern on drug resistance selection. With the general principle of bioisosterism in mind,38 and considering the ability of selenium to coordinate bivalent zinc cations,39-40 we decided to synthesize and evaluate the anti-HIV activity of a series of 2,2’-diselenobisbenzamides (DISeBAs).

DESIGN OF DISeBAs DIBA compounds (Figure 1), discovered by a random screening at the National Cancer Institute, are among the best characterized NCp7 inhibitors.35 They are electrophilic compounds that are nucleophilically attacked by a key cysteine residue of the ZF domain of


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NCp7, leading to covalent modification of the protein and subsequent zinc ejection with intramolecular disulfide bond formation. This “zinc-ejector” property translates into a broad spectrum anti-HIV activity, also encompassing clinical isolates, with no tendency to select for resistant strains.35,36 Starting from DIBAs, we decided to synthesize a series of variously functionalized diselenides replacing the sulfur atom with the selenium one (compounds 1-10, Figure 1). This simple modification permits to obtain diselenides that are strong antioxidants that have been poorly explored in medicinal chemistry so far.41 DISeBAs should exert anti-HIV activity by both NCp7 inhibition and antioxidant cell protection. The sulfonamide derivative 1 was prepared as strict analogue of DIBA-1, the hit compound of the DIBA series. Based on the putative mechanism of action, one of the selenium atoms should act as electrophile once nucleophilically attacked by the cysteine residue of NCp7. Selenium electrophilicity is considerably affected by non-bonding interactions established with neighboring heteroatoms.42 In order to explore the effect of oxygen instead of nitrogen in this intramolecular interaction, the ester derivative 2 was prepared. Benzene ring was then replaced by a pyridine moiety in compound 3. Concerning the amino acids derivatives, the design first entailed the use of the isoleucine residue (compound 4) as strict analogue of DIBA-4. The selection of the other amino acid substituents on the diselenobisbenzoic core was made mainly applying the Mekler-Idlis (MI) amino acids pairing theory,43 that investigates how amino acids are able to select residues they preferentially interact with. It is well known that C49 is most reactive cysteine residue of NCp7,37 that is surrounded by D48 (N-terminal side) and T50 (C-terminal side), as shown in Figure 2. Based on the MI theory, the introduction of valine (compound 5) as well as isoleucine (compound 4), alanine (compound 6) or glycine (compound 7), would allow a better interaction with D48, C49 or


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T50 respectively, thus increasing the likelihood for the diselenide moiety to properly react with the cysteine sulphydryl group. Taking in consideration the strong overall basicity of the target protein,37 acidic side chainfeatured amino acids were also introduced synthesizing compounds 8 and 9. Finally, proline derivative 10 was prepared as disubstituted amide sample. All the intermediate ester derivatives of compounds 4-10 synthesis, were also tested to collect additional SAR clues.

RESULTS AND DISCUSSION CHEMISTRY As shown in Scheme 1, the synthetic route to the title Se-organic compounds first entailed the preparation of the target acid 11, which was obtained in good yield following a previously reported procedure44 in which the diazonium salt prepared starting from anthranilic acid undergoes nucleophilic displacement using a freshly prepared aqueous solution of Na2Se2. The diselenide salt was in turn obtained reacting elemental selenium with a basic aqueous solution of NaBH4 at room temperature. Treatment of 11 with a stoichiometric amount of SOCl2 afforded the acid chloride, which was directly coupled with sulphanilamide in dry THF or 4-hydroxybenzenesulfonamide in dry DMF using Et3N as base to give 1 and 2, respectively. The nicotinoyl derivative 3 was prepared from 12,45 in turn obtained from 2-chloronicotinic acid by reaction with a freshly prepared ethanolic solution of Na2Se2. The target diselenide was obtained in high yield without any further purification. Nicotinic derivative 12 was converted to the corresponding acid chloride that was then coupled with sulphanilamide to give compound 3. A one-pot domino reaction permitted all the amino acid-functionalized derivatives to be obtained. In particular, the key synthone 11 was coupled with the selected amino esters in moderate to good yields using dicyclohexylcarbodiimide (DCC) as dehydrating and N-


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hydroxybenzotriazole (HOBt) as activating agents in dry THF at room temperature. Basic hydrolysis under mild conditions of the ester intermediates 13-19 afforded the target acids 410. 77

Se NMR analysis performed on all the synthesized compounds, confirmed the presence of

the diselenide moiety. With the exception of the nicotinoyl derivatives, the chemical shift of all the compounds was around 430-450 ppm, which is the typical δ value of the diselenides. In compounds 12 and 3, 77Se chemical shift resulted to be strongly deshielded (530 ppm) by the presence of the aromatic nitrogen.

BIOLOGICAL ACTIVITY AND COMPUTATIONAL STUDIES

The newly synthesized compounds were initially evaluated for their anti-HIV-1 (IIIB) and anti-HIV-2 (ROD) activity in acutely infected MT-4 cells and the cytotoxicity of the compounds was assessed in parallel. As shown in Table 1, some compounds endowed with an anti-HIV activity at subtoxic, micromolar concentrations were obtained. The best profile was shown by amino acid derivatives 4-8, which showed good antiviral activity against both HIV1 and HIV-2 with EC50s ranging from 3.15 to 13.91 µM that combined with very low cytotoxicity levels led to respectable SI values. A comparison of these data with those reported for the corresponding DIBAs in CEM cells,36 clearly reveals how favorable the sulfur/selenium switch appears. Indeed, while the antiviral profile of isoleucine derivative 4 is superimposable to that of its sulfur analogue DIBA-4 (EC50 = 9 µM; CC50 > 120 µM; SI > 13.3),36 the other DISeBAs show an improved ability to reduce viral replication when compared with the corresponding DIBAs, for which no appreciable anti-HIV activities was measured in CEM cells.36 To make a direct comparison possible, we re-synthesized and tested in MT-4 cells the glycine DIBA derivative 20, the analogue of our best selenorganic compound 7. A behavior similar to that seen in CEM cells


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was observed, with compound 20 lacking of anti-HIV activity at concentrations lower than those cytotoxic (CC50 of 58.07 µM), and compound 7 showing a good potency (EC50 of 3.1 µM and 3.5 µM against HIV-1 and HIV-2, respectively) coupled with the absence of cytotoxicity (CC50) up to 117.0 µM. The presence of amino acid moieties such as aspartic acid (compound 9) and proline (compound 10), resulted in diselenide derivatives devoid of cytotoxicity but likewise lacking of antiviral activity. All of the ester precursors (13-19) showed a common behavior being always more potent than the acid counterparts, with EC50 values in the low micromolar range, as in compound 16 which is the most potent with EC50 = 0.91 µM and 0.74 µM on HIV-1 and HIV-2, respectively. The higher activity could be explained by an improved cellular permeability that in part could also explain the concomitant more pronounced cytotoxicity. An additional cause for the esters toxicity could be their minor acidity; indeed, due to the strong basicity of the protein, it is known that neutral or basic derivatives are generally less NCp7-specific.46 The sulfonamidic derivatives 1 and 3 showed good antiviral activity albeit coupled with an increased toxicity if compared to amino acid derivatives. Both the antiretroviral activity and cytotoxicity of 1 are superimposable to those measured in MT-4 cells for the re-synthesized DIBA-1. The ester derivative 2 is devoid of any antiviral activity at concentrations lower than those cytotoxic, plausibly indicating that the nonbonded interaction between chalcogens is detrimental for target recognition. A further explanation for this unexpected result could be the inability of compound 2 to cyclize leading to the benzisoselenazolone form. DIBAs are indeed known for their propensity to cyclize under physiological conditions and both forms are endowed with superimposable biological activities.47 Once verified that the replacement of sulfur with selenium permits a selective antiviral activity to be retained or even improved, some efforts were devoted to the identification of the


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mechanism of action responsible for the antiviral activity of DISeBAs. To pinpoint the step of the viral replicative cycle inhibited by these compounds, time-ofaddition (TOA) experiments were carried out on three of the best derivatives, i.e. 4, 7, and 8, and the results were compared to reference compounds whose mode of action is known. This experiment determines how long the addition of a compound can be postponed before its antiviral activity is lost. Indeed, when an inhibitor that interferes with the binding of the virus to the host cell is present at the time of virus addition, it will inhibit virus replication. However, when this binding inhibitor is added after binding to the host cell has occurred, it will no longer be able to interfere with the replication at that point. In this assay, cells were infected at a high multiplicity of infection, and each compound to test was added at 1, 2, 3, 4, 5, 6, 7, and 8 h after infection, as indicated in Figure 3. Virus replication was monitored by measuring p24 capsid protein concentrations in the supernatant, 31 h after infection. Depending on the drug target, the addition of the compound could be delayed for hours, specific for each compound, without losing its antiviral activity. For the selected reference inhibitors, dextran sulfate 8000 (DS8000), the bicyclam CXCR4-antagonist AMD3100, the nucleoside reverse transcriptase inhibitor AZT and the protease inhibitor ritonavir, addition can be delayed until about 0, 0, 4, and 17 hours post-infection, respectively. In the TOA experiments, the addition of the three Se-compounds affected the amount of produced virus, as assessed by quantification of p24 in the supernatant, similarly to the protease inhibitor ritonavir. However, besides the fact that the detected p24 concentrations for all the compounds studied and ritonavir are approximately half of the amount detected for the control condition, the lapse of time the addition of the compound can be delayed is different for the three studied DISeBAs. Indeed, as shown in Figure 3 they appear to interfere with distinct steps in HIV replicative cycle: early entry for glutamic derivative 8, early functions of NCp7 for glycine derivative 7, late functions of NCp7 for isoleucin derivative 4.


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As it is known that certain NCp7 inhibitors prevent Gag precursor processing, resulting in accumulation of aggregated and unprocessed Gag polyprotein in native virions, we investigated whether treatment with DISeBAs would elicit a similar effect. For this experiment, chronically HIV-1(IIIB) infected HuT-78 cells were treated with different concentration of Se-compounds. The western blot analysis of the progeny virus in the supernatant demonstrated that treatment with either of the three compounds did result in an effective accumulation of unprocessed Gag polyprotein, analogously to what observed for the protease inhibitor ritonavir and the zinc ejector SRR-SB3,48 used as controls (Figure 4). All of the tested compounds dose dependently inhibited the Gag processing, with glutamic derivative 8 exerting the best activity, being the p24 production hampered even at the lower concentration (30.4 µM) used. Thus, although from the TOA experiments some differences emerged among the three studied DISeBAs, all of them have in common the ability to interfere with Gag processing, as confirmed by the western blot experiments. Compounds 4, 7 and 8 were also investigated in silico for their interaction with NCp7 and mode of inhibition. Starting from the assumption that, analogously to DIBAs,35 the diselenide moiety is nucleophilically attacked by the most reactive cysteine of NCp7, i.e. C49, Glide covalent docking49 was used to generate models of the three compounds bound to NCp7 before and after the nucleophilic attack. In a simplistic manner, we can consider covalent binding as a two steps process. In the first one the ligand binds non covalently to the protein in a conformation suitable for the reaction that takes place in the second step. Glide covalent docking uses molecular mechanic methods to model the covalent docking, and calculates affinity scores as averages of the pre- and post- reacted GlideScores. Compounds 4, 7 and 8 docked non covalently to NCp7 in a similar fashion, with one of their selenium atoms interacting with residues 46-50 and engaging several electrostatic interactions with polar residues such as K38, K47 and R52 (Figure 5, Table S1).


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Despite the encouraging results obtained with the MI-based design, docking results highlight how the design of next-generation DISeBAs will have to account not only for the interaction with C49 bound residues. The optimization of nonbonded interaction with further residues, such as K38, H44, M46, K47 and R52, might in fact facilitate the molecular recognition between NCp7 and DISeBAs, leading to a higher drug efficacy. Once the nucleophilic attack takes place and the leaving group gets away, the covalently attached part of DISeBAs interacts with residues K38, M46, K47, E51 and R52 as shown in Figure 6. For its RNA-binding properties, NCp7 requires intact ZFs,37 we then used the predicted conformations of the post-reacted complexes (Figure 6) as inputs for molecular dynamics (MD) simulation to quantify ZF architecture alterations (Figure 7). The root mean square deviation (RMSD) of the zinc atom and of the atoms it is chelated by, i.e. the γ-sulphurs of C36, C39 and C49 and the ε-nitrogen of H44, show values (averaged during 10 ns long MD simulations ± SD) of 1.21 ± 0.22 Å, 2.29 ± 0.26 Å and 3.96 ± 0.48 Å for NCp7 covalently bound to compounds 4, 7, and 8, respectively. On the contrary, simulation of unbound NCp7 shows a very stable architecture, with an average RMSD value of 0.42 ± 0.04 Å. It is evident that, once the studied compounds covalently bind to C49, the original architecture of the ZF gets substantially modified, plausibly preluding to zinc ejection. As mentioned before, cART is effective in hindering HIV-1 replication but it fails to eradicate the virus even after decades of treatment. The major barrier to eradication is due to the existence of latent reservoirs that hide copies of the virus, which can trigger a new systemic infection upon discontinuation of therapy.50 Being NCp7 involved in post integrative events in the HIV-1 life cycle, its inhibition results in the interference of late phases of viral replication, as confirmed by the activity in latently infected cells of certain NCp7 inhibitors.35 Thus, DISeBAs 4, 7, 8, including the benzensulfonamide derivative 1, were evaluated on


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persistently infected HuT-78(IIIB) cells. DIBA-1 and SRR-SB, were also included as reference compounds, together with the positive and negative controls, ritonavir and nevirapine, respectively. HuT-78 cells continuously produce HIV-1(IIIB) from the integrated proviral DNA. After removal of the already-produced viruses, the cells were exposed to serial dilutions of the compounds and the quantity of newly-produced virus was compared with a control condition. As shown in Table 2, all the compounds inhibited the release of infectious virus in persistently infected cells at micromolar concentrations and positive SI values, with a behavior similar to that observed in acutely infected MT-4 cells. With the exception of the isoleucine derivative 4, which showed an EC50 of 73 µM, all of the compounds exerted a low micromolar activity, ranging from 8 to 15 µM, coupled with the absence of cytotoxicity. This activity is better than that showed by the reference compound SRR-SB-003 albeit worse than that of the resynthesized DIBA-1, which is however endowed with a pronounced toxicity. DIBAs and several others NCp7 inhibitors act by chemically attacking the ZF domain of NCp7 and offer a unique approach to inhibit and inactivate cell-free viruses, with a potential utility as topical microbicides. The mechanism of HIV sexual contagion is still not perfectly known, but could indeed involve a cell-free virus transmission.51 Thus, to preliminarily investigate their potential application as topical microbicides, derivatives 4, 7, and 8 were assayed for their ability to inactivate cell-free HIV-1(IIIB). Cellfree virus stock was treated with the compounds for 1 hour and this stock was diluted prior to titration on target cells to demonstrate reduction in virus infectivity, as compared to untreated controls. As shown in Figure 8, compounds 7 and 8, the most potent in both MT-4 and HuT78 (IIIB) cells, showed a clear virucidal effect, completely reducing the virus infectivity at 125 µg/mL. Finally, to assess the antiviral spectrum of action of DISeBAs, compounds 4, 7, and 8 were evaluated against HIV-1 strains that are resistant to NNRTIs (Res056), ritonavir (IIIB/RIT),


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and AZT (ADP/141), including clinical isolates L6 and B12, which are resistant to RTIs (both NRTIs and NNRTIs) and ritonavir, respectively, along with simian immunodeficiency virus (SIV). As shown in Table 3, the compounds displayed, against all the drug resistant strains assayed, a profile perfectly superimposable to that obtained using wild type strains, whereas nevirapine, ritonavir, and AZT, markedly lost inhibitory potential against their respective resistant HIV-1 mutants. The inhibitory activity against SIV, at least comparable to the ones observed against HIV-1 and HIV-2, proves that the activity is not limited to human retroviruses.

CONCLUSIONS Isosteric substitution of sulfur or oxygen with selenium in biologically active compounds is recently producing interesting new interesting chemotypes. We applied this approach to the anti-HIV research field, where rare examples of Se-containing derivatives have been reported so far. To this purpose, DIBA compounds appeared particularly suitable due to their structure and, most importantly, peculiar anti-HIV mechanism of action. Indeed, they inhibit NCp7, a conserved retroviral protein that exerts essential and multiple functions in both early and late stages of viral replication. Based on DIBAs, a series of diselenides named DISeBAs were efficiently synthesized and profiled for their anti-HIV properties. The most interesting results were achieved with glycine and glutamate derivatives 7 and 8, followed by isoleucine derivative 4, that showed a good and broad anti-HIV activity with EC50 values in the low micromolar range and respectable SIs in both acutely (MT-4) and persistently (HuT-78(IIIB)) infected cells. Noteworthy, DISeBAs retained full anti-HIV-1 activity against several drug resistant strains, including clinical isolates. Amino acidic derivatives 7 and 8 showed virucidal effects, suggesting their potential use as topical anti-HIV agents. Analogously to known NCp7 inhibitors, DISeBAs are able to prevent the Gag precursor proper processing indicating that they are able to recognize NCp7 even when it is still part of


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the polyprotein. However, in TOA experiments they did not display a unique behavior, interfering with different steps in the HIV replicative cycle. The interaction between NCp7 and DISeBAs was studied in silico, prompting an optimization on nonbonded protein/inhibitor contacts regarding not only C49 bound residues, but even residues such as K38, H44, M46, K47 and R52, which resulted to be involved in molecular recognition of DISeBAs. The direct comparison of DISeBAs with their sulfur analogues, purposely re-synthesized, led to surely conclude that bioisosteric substitution of sulfur with selenium is feasible and might pave the way for a new class of anti-HIV compounds acting through NCp7 inhibition.

EXPERIMENTAL SECTION Chemistry All reactions were routinely checked by TLC on silica gel 60F254 (Merck) and visualized by using UV or iodine. Flash column chromatography separations were carried out on Merck silica gel 60 (mesh 230-400). Melting points were determined in capillary tubes (Büchi Electrothermal Mod. 9100) and are uncorrected. HRMS spectra were registered on Agilent Technologies 6540 UHD Accurate Mass Q-TOF LC/MS, HPLC 1290 Infinity. Purities of the compounds were determined by UHPLC and were ≥98% pure or by elemental analyses and the data for C, H, and N are within ± 0.4% of the theoretical values (purity of ≥95%). UHPLC conditions were as follows: column, Phenomenex AERIS Peptide 1.2 mm × 1000 mm (1.7 µm); flow rate, 0.8 mL/min; acquisition time, 20 min; DAD 190−650 nm; oven temperature, 45 °C; gradient of acetonitrile in water containing 0.1% of formic acid (0−100% in 20 min). MS: Bruker MicroOTOF QII with an electrospray ionization (ESI) source. Elemental analyses were performed on a Fisons elemental analyzer, Model EA1108CHN. NMR experiments were obtained at 25 °C on a Bruker DPX 200 spectrometer operating at 200 MHz for 1H, and 50.31 MHz for

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C experiments or in a Bruker DRX spectrometer

operating at 400 MHz for 1H 100.62 MHz for 13C and 76.0 MHz for 77SeNMR experiments.


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H and 13C and 77Se chemical shifts (δ) are reported in parts per million (ppm) and they are

relative to TMS 0.0 ppm and PhSeSePh 364.0 ppm, respectively, and the residual solvent peak of chloroform (δ = 7.26) or dimethylsulfoxide (δ = 2.48) were used as an internal standard. Data are reported as: chemical shift (multiplicity, coupling constants where applicable, number of hydrogen atoms). Abbreviations are: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), dt (double of triplet), tt (triplet of triplet), m (multiplet), br (broad signal). Coupling constant (J) quoted in Hertz (Hz) to the nearest 0.1 Hz. Reagents and solvents were purchased from common commercial suppliers and were used without further purifications. After extraction, organic solutions were dried over anhydrous Na2SO4, filtered, and concentrated with a Büchi rotary evaporator under reduced pressure. Yields are of purified products and were not optimized. All starting materials were commercially available unless otherwise indicated.

2,2'-Diselanediyldinicotinic acid (12). Sodium (0.19 g, 8.25 mmol) was added to absolute ethanol (5 ml). After the initial vigorous reaction had subsided, NaBH4 (0.031 g, 0.83 mmol) was added in an ice bath. The above mixture was dropped slowly into a solution of powdered selenium (0.45 g, 5.78 mmol) dispersed in 3 ml of absolute ethanol. After stirring for 1h at room temperature, the mixture was stirred for another 0.5 h at 70 °C to finish the reaction. The reaction mixture was slowly warmed to RT and 2-chloronicotinic acid (0.91 g, 5.78 mmol) was added portion wise. After the addition the temperature was raised to 70 °C. After 3 hours the reaction mixture was evaporated in vacuo giving an orange solid which was treated with 10 % HCl giving 500 mg of the title compound 12 a yellow solid which was then crystallized with DMF (60 % yield), mp 224-227 °C (litt mp 218-220 °C).57 1H NMR 400 MHz (DMSO-d6) δ = 7.30 (dd, J= 4.8 and 7.8 Hz, 1H, H-5), 8.20 (dd, J= 1.7 and 7.8 Hz, 1H, H-4), 8.55 (dd, J= 1.7 and 4.3 Hz, 1H, H-6), 14.10 (bs, 1H, OH);

13

C NMR 100 MHz

(DMSO-d6) δ 120.7, 125.9, 139.1, 153.3, 161.9, 168.1 ppm; 77Se NMR (76 MHz, DMSO-d6)


15


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Page 16 of 50

δ = 531.12 ppm. HRMS m/z [M + H+] calcd for C12H9N2O4Se2 404.8893 found 404.8885.

2,2'-Diselanediylbis(N-(4-sulfamoylphenyl)benzamide)

(1).

To

a

refluxed

and

magnetically stirred suspension of 1144 (200 mg, 0.5 mmol) in dry benzene (3 ml) thionyl chloride (0.1 ml, 1.25 mmol) was added and the reaction was continued until the diselenide had dissolved (ca. 4 h). After this period, benzene and thionyl chloride were evaporated in vacuo washing the residue three times with dry benzene. The crude diselenide chloride was used without further purification. To a solution of sulfanilamide (0,19 g, 1.15 mmol) and triethylamine (0.15 ml, 2.3 mmol) in dry THF, the solution of freshly prepared acid chloride was added once diluted in THF. After 5 hours the reaction mixture was evaporated in vacuo and the residue was washed with 10 % HCl and then with water. The red-orange precipitate was filtered off and purified by flash chromatography eluting with DCM/MeOH 5%. An orange solid was obtained and crystallized from EtOH/DMF giving the title compound 1 as a yellowish solid (yield 45 %), mp 310- 315 °C. 1H NMR 400 MHz (DMSO-d6) δ = 7.35 (bs, 3H, NH2), 7.45 (t, J= 7.3 Hz, 1H, H-5), 7.65 (t, J= 7.6 Hz, 1H, H-4), 7.80 (d, J= 8.7 Hz, 2H, H-3’ and H-5’), 7.85 (d, J= 8.5 Hz, 2H, H-2’ and H-6’), 7.90 (d, J= 7.6 Hz, 1H, H-3), 8.00 (d, J= 8.1 Hz, 1H, H-6); 13C NMR 50.31 MHz (DMSO-d6) δ =124.5; 126.4; 126.9; 127.3; 128.6; 128.9; 133.2; 139.2; 140.9; 143.3; 165.8 ppm;

77

Se NMR (76 MHz, DMSO-d6) δ = ppm

444.4.

2,2'-Diselanediylbis(N-(4-sulfamoylphenyl)benzoate) (2). To a refluxed and magnetically stirred suspension of 1144 (300 mg, 0.75 mmol) in dry benzene (4.5 ml) thionyl chloride (0.13 ml, 1.875 mmol) was added and the reaction was continued until the diselenide had dissolved (ca. 4 h). After this period, benzene and thionyl chloride were evaporated in vacuo washing the residue three times with dry benzene. The crude diselenide chloride was used without further purification. To a solution of 4-hydroxybenzenesulfonamide (0.3 g, 1.172 mmol) and


16

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triethylamine (0.24 ml, 1.72 mmol) in dry DMF, the solution of freshly prepare acid chloride was added once diluted in dry DMF. The reaction mixture was then slowly warmed to 80 °C. After 5 hours the reaction mixture was poured in ice water and the pH was adjusted to 7 using 10 % HCl, the resulting precipitate was filtered off and was purified by flash chromatography eluting with DCM 1% MeOH. 30 mg (yield 35 %) of the title compound 2 were obtained, mp 275 - 277 °C. 1H NMR 400 MHz (DMSO-d6) δ = 7.45 (bs, 2H, NH2), 7.50 (t, J= 7.4 Hz, 1H, H-5), 7.55 (d, J= 6.75 Hz, 2H, H-3’ and H-5’), 7.60 (t, J= 7.3 Hz, 1H, H-4), 7.75 (d, J= 8.2 Hz, 1H, H-3), 7.90 (d, J= 8.7 Hz, 2H, H-2’ and H-6’), 8.30 (d, J= 7.8 Hz, 1H, H-6). 13C NMR 100 MHz (DMSO-d6) δ = 123.1, 127.1, 127.6, 127.9, 130.3, 133.9, 134.7, 135.4, 142.5, 153.1, 165.8 ppm. 77Se NMR (76 MHz, DMSO-d6) δ = 445.54 ppm. 2,2'-Diselanediylbis(N-(4-sulfamoylphenyl)nicotinamide)

(3).

To

a

refluxed

and

magnetically stirred suspension of 12 (200 mg, 0.5 mmol) in dry benzene (3 ml) thionyl chloride (0.1 ml, 1.25 mmol) was added and the reaction was continued until gas evolution was observed (ca. 6 h). After this period, benzene and thionyl chloride were evaporated in vacuo washing the residue three times with dry benzene. The crude diselenide chloride was used without further purification. To a solution of sulfanilamide (0.19 g, 1.15 mmol) and triethylamine (0.15 ml, 2.3 mmol) in dry THF, the solution of freshly prepared acid chloride was added once diluted in THF. After 5 hours the reaction mixture was evaporated in vacuo and the concentrate was treated with ice-water. A yellow residue was formed and collected by filtration. The solid was then suspended in 10 % NaHCO3 and filtered giving the title 3 compound as a white solid (100 mg, 50 % yield), mp 274-277 °C. 1H NMR 400 MHz (DMSO-d6) δ = 7.30 (bs, 2H, NH2), 7.50 (dd, J= 4.7 and 7.8 Hz, 1H, H-5), 7.80 (s, 4H, H-2’, H-3’, H-5’ and H6’), 8.20 (dd, J= 1.7 and 7.8 Hz, 1H, H-4), 8.75 (dd, J= 1.7 and 4.3 Hz, 1H, H-6);

13

C NMR 100 MHz (DMSO-d6) δ 120.6, 127.0, 127.8, 128.9, 136.4, 139.4, 141.6,

159.5, 165.9 ppm; 77Se NMR (76 MHz, DMSO-d6): δ = 523.64 ppm.


17


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Page 18 of 50

General Procedure for Coupling Reaction : Method A. (2S,3S)-methyl 2-(2-((2-(((2S,3S)1-methoxy-3-methyl-1-oxopentan-2-yl) carbamoyl) phenyl) diselanyl)benzamido)-3methylpentanoate (13). To a solution 1144 (300 mg, 0.75 mmol) in dry THF (8 ml), DCC (390 mg, 1.875 mmol) was added portion wise under argon atmosphere maintaining the temperature below 0 °C. After 20 min, a solution of HOBt (255 mg, 1.875 mmol) in dry THF (4 ml) was added, and the resulting light yellow solution was allowed to stir for additional 20 min. Meanwhile a suspension of isoleucine methyl ester hydrochloride (290 mg, 1.6 mmol) and Et3N (0.22 ml, 1.6 mmol) in dry THF was prepared and added by dropping. After 8 hours the reaction mixture was filtrated in vacuo and the filtrate is diluted with EtOAc and washed with 1N HCl, 5 % NaHCO3, water, and finally with brine. The organic layers were dried with anhydrous sodium sulfate and evaporated in vacuo giving a yellow oil which was purified by flash chromatography eluting with DCM to 4 % MeOH. The residue so obtained was crystallized with DCM/Petroleum Ether 2:1, giving 250 mg (60 % yield) of title compound as a white solid, mp 149 - 152 °C. 1H NMR 400 MHz (DMSO-d6) δ = 0.85 (t, J= 7.4 Hz, 3H CH2CH3), 0.90 (d, J= 6.8 Hz, 3H, CHCH3), 1.25-1.35, 1.50-1.55 and 1.95-2.00 (m, each 1H, aliphatic CH), 3.70 (s, 3H, OCH3), 4.40 (t, J= 7.6 Hz, 1H, asymmetric CH), 7.35 (t, J= 7.4 Hz, 1H, H-5), 7.40 (t, J= 6.5 Hz, 1H, H-4), 7.70 (d, J= 7.9 Hz, 1H, H-3), 7.90 (d, J= 7.5 Hz, 1H, H-6), 8.90 (bd, J= 7.7 Hz, NH);

13

C NMR 100 MHz (DMSO-d6) δ = 11.3, 15.9, 25.6,

36.0, 52.1, 57.9, 126.5, 129.0, 130.3, 132.2, 132.4, 133.0, 168.3, 172.3 ppm;

77

Se NMR (76

MHz, DMSO-d6): δ = 441.3 ppm. HRMS m/z [M + H+] calcd for C28H37N2O6Se2 657.0977 found 657.0982.

(2S,2'S)-Diethyl

2,2'-((2,2'-diselanediylbis(benzoyl))

bis

(azanediyl))bis(3-

methylbutanoate) (14). The compound was obtained following the general procedure A. Yellow solid, yield 64 %, mp 158-160 °C. 1H NMR 400 MHz (DMSO-d6) δ = 0.95 (d, J= 7.0


18

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Hz, 3H, CHCH3), 1.00 (d, J= 7.0 Hz, 3H, CHCH3), 2.20 (sept, J= 7.0 Hz, 1H, CH(CH3)2), 4.10-4.20 (m, 2H, CH2CH3), 4.30 (t, J= 7.0 Hz, asymmetric CH), 7.35-7.40 (m, 2H, H-4 and H-5), 7.70 (dd, J= 7.0 and 1.2 Hz, 1H, H-3), 7.90 (dd, J= 7.0 and 1.4 Hz, 1H, H-6), 8.90 (bd, J= 8.0 Hz, 1H, NH);

13

C NMR 100 MHz (DMSO-d6) δ = 14.5, 19.3, 19.5, 29.9, 59.2, 60.8,

126.5, 129.1, 130.3, 132.2, 132.3, 168.4, 171.7 ppm;

77

Se NMR (76 MHz, DMSO-d6): δ =

439.8 ppm. HRMS m/z [M + H+] calcd for C28H37N2O6Se2 657.0977 found 657.0985.

(2S,2'S)-Dimethyl 2,2' - ( (2,2' – diselanediylbis (benzoyl)) bis(azanediyl)) dipropanoate (15). The compound was obtained following the general procedure A. White solid, yield 42 %, mp 168-171°C 1H NMR 400 MHz (DMSO-d6) δ = 1.45 (d, J= 7.1 Hz, 3H, CH3), 3.65 (s, 3H, OCH3), 4.55-4.60 (m, 1H, asymmetric CH), 7.35 (td, J= 7.7 Hz and 1.2 Hz, 1H, H-5), 7.45 (td, J= 7.4 Hz and 1.3 Hz, 1H, H-4), 7.70 (dd, J= 8.1 and 1.2 Hz, 1H, H-3), 7.90 (dd, J= 7.6 and 1.4 Hz, 1H, H-6), 9.00 (bd, J= 7.0 Hz, NH);

13

C NMR 100 MHz (DMSO-d6) δ =

39.2.7, 48.3, 52.4, 126.5, 132.4, 132.5, 132.6, 167.7, 173.2 ppm;

77

Se NMR (76 MHz,

DMSO-d6): δ = 443.9 ppm.

Diethyl 2,2'-((2,2'-diselanediylbis(benzoyl)) bis(azanediyl)) diacetate (16) The compound was obtained following the general procedure A. White solid, 67 % yield, mp 130-134 °C. 1H NMR 400 MHz (DMSO-d6) δ = 1.20 (t, J= 7.11 Hz, 3H, CH3), 3.95 (d, J= 5.83 Hz, 2H, NHCH2), 4.15 (q, J= 7.09 Hz, 2H, CH2CH3), 7.35 (t, J= 6.82 Hz, 1H, H-5), 7.40 (t, J= 6.59 Hz, 1H, H-4), 7.70 (d, J= 7.89 Hz, 1H, H-3), 7.85 (d, J= 7.58 Hz, 1H, H-6), 9.20 (bt, J= 5.74 Hz, NH);

13

C NMR 100 MHz (DMSO-d6) δ = 14.5, 41.7, 61.1, 126.7, 128.6, 130.4, 132.3,

132.5, 132.6, 170.1 ppm; 77Se NMR (76 MHz, DMSO-d6): δ = 443.60 ppm. HRMS m/z [M + H+] calcd for C22H25N2O6Se2 573.0038 found 573.0039.

(2S,2'S)-Tetraethyl 2,2'- ((2,2'-diselanediylbis(benzoyl)) bis(azanediyl))dipentanedioate


19


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Page 20 of 50

(17). The compound was obtained following the general procedure A. Light yellow solid, 75 % yield, mp 134-137 °C. 1H NMR 400 MHz (DMSO-d6) δ = 1.15 (t, J= 7.1 Hz, 3H CH3), 1.20 (t, J= 7.1 Hz, 3H, CH3), 1.95-2.10 (m, 2H, diasterotopic CH2), 2.45 (t, J= 7.9 Hz, 2H CH2CO), 4.00 (q, J= 7.1 Hz, 2H, CH2), 4.10 (q, J= 7.1 Hz, 2H, CH2), 4.45-4.50 (m, 1H, CH), 7.30 (td, J= 1.3 and 7.5 Hz, 1H, H-5), 7.35 (td, J= 1.5 and 7.4 Hz, 1H, H-4), 7.65 (dd, J= 1.1 and 7.9 Hz, 1H, H-3), 7.85 (dd, J= 1.5 and 7.5 Hz, 1H, H-6), 9.00 (bd, J= 7.4 Hz, 1H, NH); 13

C NMR 100 MHz (DMSO-d6) δ = 14.5, 26.0, 30.5, 52.5, 60.4, 61.2, 126.7, 128.9, 130.4,

132.4, 132.5, 132.6, 166.2, 171.9, 172.6 ppm.

77

Se NMR (76 MHz, DMSO-d6): δ = 441.7

ppm. HRMS m/z [M + H+] calcd for C32H41N2O6Se2 773.1086 found 773.1093.

(2S,2'S)-Tetramethyl 2,2'-((2,2'-diselanediylbis(benzoyl)) bis(azanediyl))disuccinate (18). The compound was obtained following the general procedure A. Light yellow solid, yield 63 %, mp 168-170°C. 1H NMR 200 MHz (DMSO-d6) δ = 2.90-3.00 (m, 2H, CH2), 3.65 and 3.70 (s, each 3H, OCH3), 4.95-5.00 (m, 1H, asymmetric CH), 7.35-7.40 (m, 2H, H-5 and H-4), 7.74 (d, J= 7.9 Hz, 1H, H-3), 7.85 (dd, J= 7.5 and 2.0 Hz, 1H, H-6), 9.25 (bd, J= 8.3 Hz, NH); 13

C NMR 100 MHz (DMSO-d6) δ = 50.7, 64.8, 67.1, 67.8, 141.7, 143.7, 145.5, 147.4, 147.5,

147.6, 182.7, 185.9, 186.4 ppm; 77Se NMR (76 MHz, DMSO-d6): δ = 444.2 ppm. HRMS m/z [M + Na+] calcd for C26H28N2NaO10Se2 710.9967 found 710.9975.

(2S,2'S)-Dimethyl 1,1'-(2,2'-diselanediylbis(benzoyl)) bis (pyrrolidine-2-carboxylate) (19). The compound was obtained following the general procedure A. Light yellow solid, yield 55 %, mp 104-106 °C. 1H NMR 400 MHz (DMSO-d6) δ =1.90-2.00 (m, 3H, pro CH2 and pro CH), 2.20-2.25 (m, 1H, pro CH), 3.45-3.50 (m, 2H, pro CH), 3.65 (s, 3H, OCH3), 4.50 (dd, J= 4.4 and 8.0 Hz, 1H, asymmetric CH), 7.35 (t, J= 7.3 Hz, 1H, H-5), 7.40 (t, J= 7.1 Hz, 1H, H-4), 7.45 (d, J= 7.1 Hz, 1H, H-3), 7.75 (d, J= 7.3 Hz, 1H, H-6); 13C NMR 100 MHz (DMSO-d6) δ = 25.3, 29.3, 49.8, 52.3, 59.3, 127.4, 127.6, 130.1, 131.5, 131.8, 136.2; 167.7;


20

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172.4 ppm; 77Se NMR (76 MHz, DMSO-d6): δ = 433.08 ppm. HRMS m/z [M + H+] calcd for C26H28N2O6Se2 625.0351 found 625.0357.

General Procedure for Hydrolysis Reaction: Method B. (2S,3S)-2-(2-((2-(((1S,2S)-1Carboxy-2-methylbutyl)carbamoyl) phenyl) diselanyl) benzamido)-3-methylpentanoic acid (4). A suspension of ester (100 mg, 0.15 mmol) in 10 % NaOH (2 ml) and water 1 ml was allowed to stir at room temperature for 10 h. the reaction was then filtered and the filtrate was acidified with 10 % HCl until a white precipitate was formed. The solid was collected by filtration and then treated with diethyl ether giving the title compound as a white solid (57 mg, 59 % yield), mp 195 - 197 °C. 1H NMR 200 MHz (DMSO-d6) δ = 0.80 (t, J= 7.4 Hz, 3H CH2CH3), 0.90 (d, J= 6.7 Hz, 3H CHCH3), 1.10-1.30, 1.45-1.55 and 1.95-2.00 (m, each 1H, aliphatic CH), 4.30 (t, J= 7.5 Hz, 1H, NHCH), 7.25-7.35 (m, 2H, H-5 and H-4), 7.60 (d, J= 7.5 Hz, 1H, H-3), 7.85 (d, J= 6.8 Hz, 1H, H-6), 8.75 (bd, J= 7.9 Hz, 1H, NH); 13C NMR 100 MHz (DMSO-d6) δ = 11.4, 16.0, 25.5, 57.8, 126.5, 129.0, 130.3, 132.2, 132.4, 133.2, 168.1, 173.3 ppm; 77Se NMR (76 MHz, DMSO-d6): δ = 440.8 ppm. HRMS m/z [M + H+] calcd for C26H33N2O6Se2 629.0664 found 629.0666.

(2S,2'S)-2,2'-((2,2'-diselanediylbis(benzoyl))bis(azanediyl))bis(3-methylbutanoic

acid)

(5). The title compound was obtained following the general procedure B. White solid, 84 % yield, mp 138°-141 °C. 1H NMR 400 MHz (DMSO-d6) δ = 1.00 (m, 6H, CH(CH3)2), 2.20 (sept, J= 7.0 Hz, 1H, CH(CH3)2), 4.30 (t, J= 7.0 Hz, asymmetric CH), 7.30 (t, J= 7.3 Hz, 1H, H-4), 7.40 (t, J= 6.9 Hz, 1H, H-5), 7.70 (d, J= 8.0 Hz, 1H, H-3), 7.90 (d, J= 7.5 Hz, 1H, H-6), 8.80 (bd, J= 8.1 Hz, 1H, NH);

13

C NMR 100 MHz (DMSO-d6) δ = 19.2, 19.8, 29.9, 58.9,

126.6, 129.1, 130.3, 132.4, 133.3, 168.3, 173.3 ppm;

77


440.6 ppm. HRMS m/z [M + H+] calcd for C24H29N2O6Se2 601.0351 found 601.0356.


21


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Page 22 of 50

(2S,2'S)-2,2'-((2,2'-Diselanediylbis(benzoyl)) bis(azanediyl)) dipropanoic acid (6). The title compound was obtained following the general procedure B. White solid, 73 % yield, mp 204 - 207 °C. 1H NMR 400 MHz (DMSO-d6) δ = 1.45 (d, J= 7.3 Hz, 3H, CH3), 4.45-4.50 (m, 1H, asymmetric CH), 7.35 (t, J= 7.3 Hz, 1H, H-5), 7.40 (t, J= 7.3 Hz, 1H, H-4), 7.80 (d, J= 7.8 Hz, 1H, H-3), 7.90 (d, J= 7.1 Hz, 1H, H-6), 8.95 (bd, J= 7.0 Hz, NH), 12.65 (bs, 1H, OH); 13

C NMR 100 MHz (DMSO-d6) δ = 17.3, 48.8, 126.5, 128.8, 130.4, 132.5, 132.6, 167.7,

173.4 ppm; 77Se NMR (76 MHz, DMSO-d6): δ = 443.6 ppm. HRMS m/z [M + Na+] calcd for C20H20N2NaO6Se2 566.9544 found 566.9550.

2,2'-((2,2'-Diselanediylbis(benzoyl)) bis (azanediyl)) diacetic acid (7). The title compound was obtained following the general procedure B. White solid, 70 % yield, mp 220-223 °C. 1H NMR 400 MHz (DMSO-d6) δ = 3.90 (d, J= 5.7 Hz, 2H, CH2), 7.35 (t, J= 7.5 Hz, 1H, H-5), 7.40 (t, J= 6.1 Hz, 1H, H-4), 7.70 (d, J= 7.9 Hz, 1H, H-3), 7.85 (dd, J= 1.27 and 7.58 Hz, 1H, H-6), 9.10 (bt, J= 6.0 Hz, NH); 13C NMR 100 MHz (DMSO-d6) δ = 41.6, 126.1, 126.7, 128.5, 130.4, 132.3, 132.4, 168.0, 171.3 ppm; 77Se NMR (76 MHz, DMSO-d6): δ = 443.72 ppm. (2S,2'S)-2,2'-((2,2'-Diselanediylbis(benzoyl)) bis (azanediyl))dipentanedioic acid (8). The title compound was obtained following the general procedure B. White solid, 83 % yield, mp 181-183 °C. 1H NMR 400 MHz (DMSO-d6) δ = 2.00-2.15 (m, 2H, CHCH2), 2.40 (t, J= 7.2 Hz, 2H CH2CO), 4.40-4.45 (m, 1H, CH), 7.35 (t, J= 7.3 Hz, 1H, H-5), 7.40 (t, J= 7.8 Hz, 1H, H-4), 7.70 (d, J= 8.0 Hz, 1H, H-3), 7.90 (d, J= 7.5 Hz, 1H, H-6), 8.90 (bd, J= 7.4 Hz, NH), 12.45 (bs, 1H, CO2H);

13

C NMR 100 MHz (DMSO-d6) δ = 26.2, 30.7, 52.5, 126.5, 128.8,

130.3, 132.3, 132.5, 132.6, 168.0, 173.5, 174.2; 77Se NMR (76 MHz, DMSO-d6): δ = 443.00 ppm. HRMS m/z [M + H+] calcd for C24H25N2O10Se2 660.9834 found 660.9840. (2S,2'S)-2,2'-((2,2'-Diselanediylbis(benzoyl)) bis (azanediyl)) disuccinic acid (9). The title


22

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compound was obtained following the general procedure B. Orange solid, 65 % yield, mp 202 – 205 °C. 1H NMR 200 MHz (DMSO-d6) δ = 2.75 (dd, J= 8.0 and 16.3 Hz, 1H, diasterotiopic CH), 2.85 (dd, J= 5.9 and 16.4 Hz, 1H, diasterotiopic CH), 4.70-4.80 (m, 1H, asymmetric CH), 7.30 - 7.45 (m, 2H, H-5 and H-4), 7.70 (d, J= 7.6 Hz, 1H, H-3), 7.85 (d, J= 6.0 Hz, 1H, H-6), 9.10 (bd, J= 7.7 Hz, 1H, NH);

13

C NMR 100 MHz (DMSO-d6) δ = 36.0, 49.9, 126.5,

128.7, 130.4, 132.4, 132.6, 167.6, 172.0, 172.6 ppm;

77


444.7 ppm. HRMS m/z [M + Na+] calcd for C22H20N2NaO10Se2 654.9341 found 654.9346. (2S,2'S)-1,1'-(2,2'-Diselanediylbis (benzoyl))bis(pyrrolidine-2-carboxylic acid) (10). The title compound was obtained following the general procedure B. Yellow solid, quantitative yield, mp 164 -166°C. 1H NMR 400 MHz (DMSO-d6) δ =1.80-2.00 (m, 3H, aliphatic CH2 and CH), 2.20-2.25 (m, 1H, aliphatic CH), 3.45-3.50 (m, 2H, aliphatic CH), 4.40 (dd, J= 4.4 and 8.0 Hz, 1H, asymmetric CH), 7.30-7.40 (m, 2H, H-4 and H-5), 7.45 (d, J= 7.5 Hz, 1H, H3), 7.75 (d, J= 7.7 Hz, 1H, H-6), 12.75 (bs, 1H, COOH) ppm; 13C NMR 100 MHz (DMSO-d6) δ = 25.3, 29.3, 49.8, 59.3, 127.3, 127.7, 130.2, 131.2, 131.5, 136.4, 167.6, 173.3 ppm;

77

Se

NMR (76 MHz, DMSO-d6): δ = 432.5 ppm. HRMS m/z [M + H+] calcd for C24H25N2O6Se2 597.0043 found 597.0044.

Molecular Modeling Protein and ligands preparation. NCp7 coordinates were downloaded from the RCSB Protein Data Bank, PDB ID: 1MFS.52 The Schrodinger Protein Preparation Wizard53 was used on the first of 30 NMR conformations to obtain a satisfactory starting structure for the following studies. The orientation of hydroxyl groups on Ser, Thr and Tyr, the side chains of Asn and Gln residues, and the protonation state of His residues were optimized. N- and C- terminal residues were capped with ACE and NMA residues, respectively. The ionization and tautomeric states of His,

Asp, Glu, Arg and Lys were adjusted to match a pH of 7.4. The structure was finally


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submitted to a restrained minimization (OPLS2005 force field)54 that was stopped when RMSD of heavy atoms reached 0.30 Å. Ligands were sketched using the Maestro55 interface and 3D coordinates were generated using LigPrep.71 Ionization/tautomeric states were predicted for a pH range of 7±1 using Epik.57 The most populated ionization state for each ligand was retained. Docking studies. Glide covalent docking49,58 was used to predict the covalently bound conformations of compounds 4, 7 and 8 to NCp7. Docking space was defined as a 30 Å cubic box centered on C49, which was even set as reactive residue. Two docking grids were computed, in one of them the Zn atom coordinated by C49 was retained, while it was removed in the other one, to test whether the presence of the metal ion would have influenced docking results, that were instead almost identical for the two target structures (reported docking scores refer to the structure in which the Zn atom was retained). Nucleophilic attack to selenium by sulphur was not provided in the default reaction types, a custom chemistry file accounting for these reaction and reactants was then written. No docking constrain to reference position was applied, even though a distance constrain between the reactive atoms is applied by the glide covalent docking protocol. Docking mode was set to “thorough” with a minimization radius of 6 Å from the ligand. Binding affinities were estimated using Glide, and only the top scoring poses were retained. Molecular dynamics simulations. MD simulations of NCp7 covalently bound to compounds 4, 7 and 8 were set and run using Desmond MD system.59 The simulated environment was built using the system builder utility, with the structures being neutralized by Na+ and Clions, which were added until a concentration of 0.15 M was reached. Simulations were run in explicit solvent, using the TIP4P water model60 in a Periodic Boundary Conditions orthorhombic box. A series of minimizations and short MD simulations were carried out to relax the model system, by means of a relaxation protocol consisting of six stages: (i)


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minimization with the solute restrained; (ii) minimization without restraints; (iii) simulation (12 ps) in the NVT ensemble using a Berendsen thermostat (10 K) with non-hydrogen solute atoms restrained; (iv) simulation (12 ps) in the NPT ensemble using a Berendsen thermostat (10 K) and a Berendsen barostat (1 atm) with non-hydrogen solute atoms restrained; (v) simulation (24 ps) in the NPT ensemble using a Berendsen thermostat (300 K) and a Berendsen barostat (1 atm) with non-hydrogen solute atoms restrained; (vi) unrestrained simulation (24 ps) in the NPT ensemble using a Berendsen thermostat (300 K) and a Berendsen barostat (1 atm). At this point, 10 ns long MD simulations were carried out at a temperature of 300° K in the NPT ensemble using a Nose-Hoover chain thermostat and a Martyna-Tobias-Klein barostat (1.01325 bar). Trajectory analyses were performed using the Desmond simulation event analysis tool for the RMSD calculations.

Biology Cells and Viruses. MT-4 and HuT-78 cells were grown and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 0.1% sodium bicarbonate and 20 µg gentamicin per ml. The HIV-1(IIIB) strain was provided by R.C. Gallo and M. Popovic.61 HIV-2 (ROD) was obtained from L. Montagnier.62 HIV-1 (ADP/141) is a recombinant AZT-resistant HIV-1 strain (RT mutations D67N, K70R, T215F and K219Q) obtained through the Medical Research Council’s AIDS Reagent Project, National Institute for Biological Standards and Control, London, United Kingdom and was contributed by B Larder and S Kemp 3.63 RES056 is a double RT (K103N;Y181C) NNRTI-resistant mutant selected from IIIB.64 Simian immunodeficiency virus (SIV MAC251) was originally isolated by Daniel et al65 and was obtained from C. Bruck (Smith Kline –RIT, Rixensart, Belgium); SIV stocks were prepared from the supernatants of SIV-infected MT-4 cells. HIV-1 (B12), a ritonavir resistant HIV-1 strain (mutations L10I, I15V; I54V; L63P, V82A and I85V) was isolated from an HIV-1-infected patient after one year of ritonavir monotherapy (Courtesy of


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B. Clotet and L. Ruiz, Barcelona, Spain).66 HIV-1 IIIB/ritonavir is a ritonavir resistant strain selected from HIV-1 (IIIB) in vitro. HIV-1 L6 is a clinical isolate from a seropositive patient after sequential treatment with the dideoxynucleoside analogues AZT, ddI, ddC, d4T, and 3TC and the HIV-1-specific NNRTI loviride (R89439) (RT mutations: V75I, F77L, K103N, F116Y, Q151M, and M184V).67 In Vitro Antiviral Assays. Evaluation of the antiviral activity of the compounds against HIV-1 strain IIIB in MT-4 cells was performed using the MTT assay as previously described.68,69 Stock solutions (10 x final concentration) of test compounds were added in 25 µl volumes to two series of triplicate wells so as to allow simultaneous evaluation of their effects on mockand HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman instruments, Fullerton, CA). Untreated HIV- and mock-infected cell samples were included as controls. HIV-1(IIIB) stock (50 µl) at 100-300 CCID50 (50 % cell culture infectious doses) or culture medium was added to either the infected or mock-infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effects of test compound on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells were centrifuged for 5 minutes at 220 g and the supernatant was discarded. The MT-4 cells were resuspended at 6 x 105 cells/ml and 50 µl volumes were transferred to the microtiter tray wells. Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically using the MTT assay. The MTT assay is based on the reduction of yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (Acros Organics) by mitochondrial dehydrogenase activity in metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Infinite M1000, Tecan), at two wavelengths (540 and 690 nm). All data were


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calculated using the median absorbance value of three wells. The 50% cytotoxic concentration (CC50) was defined as the concentration of the test compound that reduced the absorbance (OD540) of the mock-infected control sample by 50%. The concentration achieving 50% protection against the cytopathic effect of the virus in infected cells was defined as the 50% effective concentration (EC50). Treatment of HuT-78(IIIB) Persistently Infected Cells, Virion Analysis and Western Blot. Chronically-infected HIV-1 IIIB HuT-78 cells (HuT-78(IIIB) were washed four times with PBS to remove all free virions before treatment and 2x105 cells were resuspended in 1 ml compound-containing medium for 43 hours at 37°C. Then, virions were prepared from clarified supernatants (10 min at 300 g) by centrifugation at 36,670 g for 2 hours at 4°C. Protein from lysed virions were separated by SDS-PAGE on a NuPage Novex 4-12% BisTris gel (Invitrogen) and transferred on a hydrophobic polyvinylidene difluoride (PVDF) membrane (Amersham Hybond-P). The blot was blocked overnight at 4°C by 5% dry milk powder in western blot wash solution (WBWS; PBS + 0.5% Tween 20), washed three times for 5 minutes with WBWS and incubated with a mouse anti-HIV-1 p24 antibody (1:5000) from Abcam. The blot was then washed three times for 5 minutes with WBWS and incubated for 1 h with a goat anti-mouse IgG-HRP secondary antibody (1:2500) from Santa Cruz Biotechnology. The blot was washed three times for 5 minutes with WBWS and after 5 minutes of incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) it was developed. Antiviral Effect of Test Compounds in Persistently HIV-1 Infected Cells. The antiviral activities of test compounds against persistent HIV-1 infection were based on the inhibition of p24 antigen production in chronically-infected HuT-78(IIIB) cells. Test compounds (500 µl/well of 2X final concentration) were added in a 48-well plate and 7 serial 5 fold


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dilutions were made for each compound. Untreated samples were also included as controls. HuT-78 cells persistently-infected with HIV-1 IIIB (HuT-78(IIIB)) were washed four times with PBS 1X to remove all free virions before treatment and seeded 2 × 105 cells/well in 500µl/well. After 43 h at 37°C, HIV-1 production was determined by measuring p24 antigen in the supernatant (p24 antigen enzyme-linked immunosorbent assay; Perkin-Elmer,Brussels, Belgium). Cytotoxicity of test compounds on HuT-78(IIIB) cells was tested in parallel using an MTT assay and evaluating the cell culture microscopically. Time-of-Addition Experiments. Time-of-addition experiments were adapted from ref.70,71 Briefly, MT-4 cells were infected with HIV-1(IIIB) at an m.o.i. of 0.5. Following a 1 hour adsorption period cells were distributed in a 96-well tray at 45,000 cells/well and incubated at 37°C. Test compounds were added at different times (0, 1, 2, 3, 4, 5, 6, 7, 8, 24, and 25h) after infection. HIV-1 production was determined at 31 hr postinfection via a p24 enzymelinked immunosorbent assay (Perkin Elmer, Brussels, Belgium). Dextran sulfate was used at 12.5 µM, AMD3100 at 9.9 µM, AZT at 1.9 µM, , Ritonavir at 2.8 µM, SeGly at 97.2 µM, SeGluT at 91.1 µM and SeIle at 143.2 µM. Virucidal Effect. Aliquots of a HIV stock (HIV-1 strain IIIB) were incubated with various concentrations of compound in a final volume of 100 µl RPMI-1640 culture medium with 10% FCS for 1 hour at 37°C. Subsequently, the samples were diluted 4000 times with complete medium so that the residual concentration of compound present was far below its IC50. The drug-treated and diluted virus suspension was then used to infect susceptible MT-4 T-cells to quantify the viral infectivity by titration and CCID50 calculation.68 Control experiments with AZT indicated that this procedure effectively diluted the compound to concentrations well below its effective antiviral concentration.


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ASSOCIATED CONTENT Supporting information Pre- and post-reacted, and average docking scores for compounds 4, 7 and 8 (Table S1) and the Table S2 containing Elemental Analysis data for target compounds 1-10 . This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *OT: phone, + 39 075 585 5139; fax, +39 075 585 5115; e-mail, [email protected]. *CP: phone, +3216332171; fax, +3216332131; e-mail, [email protected] *CS: phone, + 39 075 5855102; fax, +39 0755855116 e-mail, [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This

work

was

supported

by

CINMPIS

project,

“RATIONAL

DESIGN

OF

ORGANOSELENIUM AND ORGANOSULFUR COMPOUNDS AS INNOVATIVE ANTIHIV AGENTS”. Fondazione Cassa di Risparmio di Perugia is also gratefully acknowledged. This research was undertaken as part of the scientific activity of the international multidisciplinary network “SeS Redox and Catalysis”. LS thanks Angela Mencaglia for the fruitful scientific discussions.


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ABBREVIATIONS USED cART, Combined Antiretroviral Therapy; DCC, Dicyclohexylcarbodiimide; DIBAs, Dithiobisbenzamides; DISeBAs, 2,2’-Diselenobisbenzamides; GPx, Glutathione Peroxidase; HOBt, N-hydroxybenzotriazole; MI, Mekler-Idlis; NCp7, Nucleocapsid Protein 7; Sec, Selenocysteine; TOA, Time of Addition; ZF, Zinc Finger.

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52. Lee, B. M.; De Guzman, R. N.; Turner, B. G.; Tjandra, N., Summers, M. F. Dynamical behavior of the HIV-1 nucleocapsid protein. J. Mol. Biol. 1998, 279, 633– 649. 53. Schrödinger Suite Protein Preparation Wizard, Schrödinger LLC, New York, NY, 2015. 54. Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J. Am. Chem. Soc. 1996, 118, 11225–11236. 55. Maestro, Schrödinger LLC, New York, NY, 2015. 56. LigPrep, Schrödinger LLC, New York, NY, 2015. 57. Epik, Schrödinger LLC. New York, NY. 58. Prime, Schrödinger LLC, New York, NY, 2015. 59. Desmond Molecular Dynamics System, D. E. Shaw Research. New York, NY, 2015. 60. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; & Klein, M. L. (). Comparison of simple potential functions for simulating liquid water. Journal of Chemical Physics, 1983, 79, 926-935. 61. Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science. 1984, 224, 497-500. 62. Barré-Sinoussi, F.; Chermann, J. C.; Rey, F.; Nugeyre, M. T.; Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blin, C.; Vézinet-Brun, F.; Rouzioux, C., Rozenbaum, W.; Montagnier, L. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science. 1983, 220, 868-871. 63. Larder, B. A.; Kemp, S. D.Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989, 246, 1155-1158.


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Table 1. Anti-HIV-1 and -HIV-2 activity and Cytotoxicity of DISeBAs in MT-4 cells HIV-1(IIIB)

HIV-2(ROD)

SId b,c

compd

CC50 (µM) EC50 (µM)

a,c

EC50 (µM)

d

SI (IIIB) (ROD)

a,c

1

2.82 ± 0.38

3.03 ± 0.22

17.43 ± 0.84

6

6

2

> 18.62

> 18.62

18.62 ± 1.55

150.95

150.95 ± 11.10

151.89

151.89 ± 23.76

7.48

> 748

> 935

0.28 ± 0.04

> 15

> 15

> 60

1

DIBA-1 Ritonavir AZT Nevirapine a

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EC50: concentration of compound required to achieve 50% protection of MT-4 cells from HIV

induced cytopathogenicity, as determined by the MTT method. b CC50: concentration of compound that reduces the viability of mock-infected cells by 50%, as determined by the MTT method. represent mean values ± standard deviations for at least two separate experiments.

d

c

All data

SI: ratio of

CC50/EC50.


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Table 2. Antiviral Activity and Cytotoxicity of selected DISeBAs in HIV-1 Chronically Infected (Hut-78 (IIIB) Cells. EC50 (µM) a,c

CC50 (µM)b,c

SId

1

8.25 ± 3.99

> 35

>4

4

73.2 ± 21.7

>162

> 2.2

7

14.1 ± 8.94

108 ± 13

7.8

8

15.5 ± 4.86

100 ± 27

6.5

DIBA-1

2.66 ± 0.09

18.4 ± 10.26

6.9

SRR-SB-003

29 ± 17

>145

>5.00

Ritonavir

0.26 ± 0.08

>14

>54

Nevirapine

>7.5 ±

>7.5

-

compd

a

EC50: concentration of compound required to achieve 50% reduction

of p24 production in HIV-1 infected cells.

b

CC50: concentration of

compound that reduces the viability of cells by 50%, as determined by the MTT method. c All data represent mean values ± standard deviations for at least two separate experiments. dSI: ratio of CC50/EC50.


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Table 3. Antiviral profile of DISeBAs.a Strain HIV-1RES056

b

HIV-1IIIB/RITc

HIV-1L6

d

HIV-1B12

e

HIV-1ADP/141f

SIVMac251 a

g

4

7

8

EC50

28.01 ± 4.15

3.51 ± 0.14

3.61 ± 0.28

CC50

164.37 ± 13.41

117.02 ± 15.40

107.28 ± 3.86

SI

6

33

29

EC50

25.81 ± 1.91

3.30 ± 0.12

5.42 ± 0.04

CC50

164.37 ± 13.41

117.02 ± 15.40

107.28 ± 3.86

SI

6

36

20

EC50

25.92 ± 0.44

3.44 ± 0.01

6.59 ± 0.17

CC50

164.37 ± 13.41

117.02 ± 15.40

107.28 ± 3.86

SI

6

34

16

EC50

27.71 ± 3.15

6.43 ± 0.85

14.5 ± 5.06

CC50

164.37 ± 13.41

117.02 ± 15.40

107.28 ± 3.86

SI

6

18

8

EC50

14.71 ± 1.94

4.51 ± 1.32

2.8 ± 0.52

CC50

164.37 ± 13.41

117.02 ± 15.40

107.28 ± 3.86

SI

11

26

38

EC50

17.42 ± 0.33

2.21 ± 0.06

1.7 ± 0.12

CC50

164.37 ± 13.41

117.02 ± 15.40

107.28 ± 3.86

SI

9

52

63

All of the experiments were carried out in MT-4 cells. EC50 and CC50 are expressed in

µM, SI=CC50/EC50,; bNNRTIs resistant (Nevirapine EC50 > 15 µM; CC50 > 15); c

ritonavir resistant (Ritonavir EC50 = 2.77 ± 0.94 µM; CC50 > 5.55); dclinical isolate

resistant to both NRTIs and NNRTIs (Nevirapine EC50 > 15 µM; CC50 > 15; AZT EC50 > 7.48 µM; CC50 > 7.48 µM); eclinical isolate resistant to ritonavir (Ritonavir EC50 =


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1.28 ± 0.08 µM; CC50 > 5.55); fAZT-resistant (AZT EC50 = 0.03 ± 0.003 µM; CC50 > 7.48 µM); gNevirapine EC50 > 15 µM; CC50 > 15; Ritonavir EC50 = 0.14 ± 0.05 µM; CC50 > 5.55.

Figure legends Figure 1. Structure of DISeBAs synthesized in this study along with sulfur analogues DIBA1 and DIBA-4.

Figure 2. Section of the distal zinc-finger domain. Zinc coordinating cysteine (C) 49 is the most nucleophilic residue among NCp7 structure, it is flanked by aspartic acid (D) 48 and threonine (T) 50. The complementary amino acids suggested by Mekler-Idlis theory are given.

Figure 3. Time-of-addition experiment. MT-4 cells were infected with HIV-1, and the test compounds were added at different time points after infection. Virus production was determined by p24 Ag production in the supernatant at 31 h post infection. Pink squares, control; triangles, AMD3100, CXCR4 inhibitor; magenta crosses, dextran sulfate (DS8000); pink crosses, AZT, non nucleoside reverse transcriptase inhibitor; red circles, ritonavir,


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Page 44 of 50

protease inhibitor; magenta line, 4; green line, 7; blue line, 8.

Figure 4. Inhibition of Gag processing. HIV-1 IIIB virions from persistently infected HuT-78 cells treated with different concentrations of compound were analyzed by gel electrophoresis and immunoblotted with antibody against capsid. 1:untreated control; 2: AZT (3.8 µM); 3: ritonavir (2.8 µM); 4: SRR-SB3 (14.5 µM); 5: 8 (91.1 µM); 6: 8 (30.4 µM); 7: 4 (95.5 µM); 8: 4 (31.8 µM); 9: 7 (116.7 µM); 10: 7 (38.9 µM).

Figure 5. Schematic ligand interaction diagrams of pre-reacted bound conformations of compounds 4 (panel A), 7 (panel B) and 8 (panel C).

Figure 6. NCp7/4, NCp7/7 and NCp7/8 covalent complexes. NCp7 is depicted as grey surface, cartoons and sticks. Zinc ions are represented by magenta spheres, while compounds 4, 7, 8 are depicted as magenta, green and cyan sticks, respectively.

Figure 7. title. RMSD values of the γ-carbons of C36, C39 and C49 and the ε-nitrogen of H44 as a function of MD simulation time.

Figure 8. Virucidal Effect on HIV-1 IIIB. Inactivation of isolated HIV-1 IIIB particles by DISeBAs. Virus stock was exposed to different concentrations of compound and the 50% cell culture infectious dose (CCID50) of treated and untreated virus stock was determined by the Reed and Muench method.

Scheme Footnotes Scheme 1a


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a

Reagents and conditions: i) NaNO2, HCl, H2O, rt; ii) Na2Se2, H2O, 0 to 110 °C; iii) SOCl2,

reflux then benzensulphonamide derivative, Et3N, dry THF or DMF, rt; iv) amino acid ester hydrochloride, DCC, HOBt, Et3N, dry THF, rt; v) NaOH 5%, rt; vi) Na2Se2 ethanolic solution, 80°C; vii) SOCl2, reflux; viii) sulphanilamide, Et3N, dry THF, rt.

Figure 1


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Page 46 of 50

Figure 2

Figure 3

Figure 4.


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Figure 5

Figure 6


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Figure 7

Figure 8


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Scheme 1.

“Table of Contents Graphic”


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Page 50 of 50

Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as Nucleocapsid Protein 7 (NCp7) Inhibitors with anti-HIV Activity Luca Sancineto, Alice Mariotti, Luana Bagnoli, Francesca Marini, Jenny Desantis, Nunzio Iraci, Caudio Santi, Christophe Pannecouque, Oriana Tabarrini


50

Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as

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