Novel anti-adenovirus agents based on aminoglycerol and piperazine scaffolds: design, synthesis and in vitro biological evaluation

Department of Organic and Medicinal Chemistry Department of Pharmacy, Health and Nutritional Sciences NOVEL ANTI-ADENOVIRUS AGENTS BASED ON AMINOGLYCEROL AND PIPERAZINE SCAFFOLDS: DESIGN, SYNTHESIS AND IN VITRO BIOLOGICAL EVALUATION PhD Thesis in joint supervision Sarah Mazzotta Seville, September 2020 Department of Organic and Medicinal Chemistry Department of Pharmacy, Health and Nutritional Sciences NOVEL ANTI-ADENOVIRUS AGENTS BASED ON AMINOGLYCEROL AND PIPERAZINE SCAFFOLDS: DESIGN, SYNTHESIS AND IN VITRO BIOLOGICAL EVALUATION PhD Thesis in joint supervision Sarah Mazzotta Seville, September 2020 Supervisors (University of Seville) Supervisor (University of Calabria) Margarita Vega Holm Francesca Aiello Fernando Iglesias Guerra José Manuel Vega Pérez Departamento de Química Orgánica y Farmacéutica Dipartimento di Farmacia e Scienze della Salute e della Nutrizione NOVEL ANTI-ADENOVIRUS AGENTS BASED ON AMINOGLYCEROL AND PIPERAZINE SCAFFOLDS: DESIGN, SYNTHESIS AND IN VITRO BIOLOGICAL EVALUATION Tesis Doctoral en Cotutela Sarah Mazzotta Sevilla, septiembre 2020 Directores (Universidad de Sevilla) Directora (Università della Calabria) Margarita Vega Holm (Tutora) Francesca Aiello Fernando Iglesias Guerra José Manuel Vega Pérez Margarita Vega Holm, Fernando Iglesias Guerra, José Manuel Vega Pérez and Francesca Aiello. Hereby certify: That Sarah Mazzota has carried out under our direction, and under joint superv1s1on, in the Department of Organic and Medicinal Chemistry of the Faculty of Pharmacy of the University of Seville, and in the Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, the research leading to the Doctoral Thesis entitled: NOVEL ANTI-ADENOVIRUS AGENTS BASED ON AMINOGL YCEROL AND PIPERAZINE SCAFFOLDS: DESIGN, SYNTHESIS AND IN VITRO BIOLOGICAL EV ALUATION. Once this manuscript has been drafted, it has been supervised by us and we find it compliant with the requirements to be presented as a thesis to aspire to the degree of Doctor by the University of Seville and the University of Calabria, before the committee that is duly appointed in its day. And for the record, in compliance with current provisions, we issue this in Seville on July 16, 2020. Signed: Margarita Vega Holm Signed: Fernando Iglesias Guerra Signed: Francesca Aiello When someone you love becomes a memory, the memory becomes a priceless treasure. 4 6.2.2 Cytotoxicity assay 158 6.2.3 Plaque assay 158 6.2.4 Nuclear-associated HAdV genomes 159 6.2.5 HAdV yield reduction 159 6.2.6 DNA and mRNA quantification by real-time PCR 159 6.2.7 Antiviral activity of compound combinations 160 6.2.8 Phi29 DNA polymerase amplification efficiency assay 161 6.2.9 Hamster serum stability assay 161 6.2.10 Statistical Analyses 162 Chapter 7. Conclusions 163 Chapter 8. Homodrimane scaffold for the development of selective TRPV4 antagonists 165 8.1 Insights into TRPV4 channel and its functions 165 8.1.1 Structure and localization 165 8.1.2 Therapeutic opportunities of TRPV4 modulators 167 8.2 Design of new homodrimane-based compounds 169 8.3 Chemical modification of (+)-sclareolide -Pathway A: Semi-synthesis of homodrymanyl amides (265-280) -Pathway B: semi-synthesis of homodrymanyl acid esters (282-284) -Pathway C: semi-synthesis of homodrymanyl diol esters and ether (286-289) 172 172 174 175 8.4 In vitro pharmacological characterization 177 8.5 Conclusion 180 8.6 Experimental part 8.6.1 General chemical methods -General procedure 21. Synthesis of homodrimanyl amides through lacton ring opening reaction of (+)-sclareolide (265-267) -General procedure 22. Synthesis of homodrimanyl acid ester through lacton ring opening reaction of (+)-sclareolide (282, 283) -General procedure 23. Synthesis of homodrimanyl methyl ester derivative through lacton ring opening reaction of (+)-sclareolide (284) - Procedure 24. Lactone ring reduction reaction of (+)-sclareolide (285) - General procedure 25. Synthesis of homodrimanyl diol esters (286-289) - Procedure 26. Synthesis of homodrimanyl diol ether (289) 181 181 182 188 189 190 190 191 8.6.2 Biological methods 192 5 -TRPV1 and TRPV4 channel assays 192 References 195 List of Figures 207 List of Schemes 209 List of Tables 210 6 7 LIST OF ABBREVIATIONS A - AA: arachidonic acid - ADME: absorption, distribution, metabolism, and excretion - ADP: adenovirus death proteins - AEA: arachidonoyl ethanolamide - AGE: acute gastroenteritis - ANK: ankyrin - ANPs: acyclic nucleoside phosphonates - ATCC: american type culture collection - AVP: adenovirus protease B - BAA: bisandrographolide - BCV: brincidofovir - BOILED-egg: brain or intestinal estimated permeation method C - CaM: calmodulin - CAR: coxsackievirus and adenovirus receptor - CC50: cytotoxic concentration 50% - CDV: cidofovir - CI: chemical Ionization - CMV: cytomegalovirus - COSY: correlation spectroscopy - CPE: cytopathic effect - CuAAC: copper(I)-catalyzed azide alkyne cycloaddition D - DBP: DNA-binding protein - DCM: dichloromethane - DEPT: distortionless enhancement by polarization transfer 8 - DIBAL-H: diisobutylaluminium hydride - DMAP: 4-dimethylaminopyridine - DMEM: dulbecco/vogt modified eagle's minimal essential - DMF: dimethylformamide - DMSO: dimetilsulfoxide - DSG-2: desmoglein-2 E - ECGC: epigallocatechin gallate - EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - EMEM: minimum essential medium eagle - ESI: electrospray ionization F - FAB: fast atom bombardment - FBS: fetal bovine serum - FDA: food and drug administration G - GAPDH: glyceraldehyde 3-phosphate dehydrogenase - GCV: ganciclovir - GI: gastrointestinal H - HadV: human Adenoviruses - HATs: histone acetyltransferases - HBV: hepatitis B virus - HCV: hepatitis C virus - HDACs: histone deacetylases - HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - HIV: human immunodeficiency virus - HMBC: heteronuclear multiple bond correlation - HRMS: high resolution mass spectrometry - HSCT: hematopoietic stem cell transplantation 9 - HSPGs: heparan sulfate proteoglycans - HSQC: heteronuclear single quantum correlation I - IAV: influenza A virus - IC50: half maximal inhibitory concentration - IP3: inositolo trifosfato - ITRs: inverted terminal repeats - IVIg: intravenous immunoglobulin L - LC-MS: liquid chromatography–mass spectrometry M - mCPBA: meta-chloroperoxybenzoic acid - MLTU: major late transcriptional unit - MOI: multiplicity of infection - MRM: multiple reaction monitoring N - NA: not active - NE: nuclear envelope - NMR: nuclear magnetic resonance - NO: nitric oxide - NOESY: nuclear overhauser effect spectroscopy O - ODE: octadecyloxyethyl P - PCR: polymerase chain reaction - PKC: protein kinase C 10 - PRD: `proline-rich domains - pTP, preterminal protein R - RAR: retinoic acid receptor - RT: renal transplantation - rt: room temperature - RT-PCR: reverse transcription polymerase chain reaction S - SAHA: suberoylanilide hydroxamic acid - SAR: structure–activity relationship - SD: standard deviation - SI: selectivity index - SOT: solid organ transplantation T - TCID50: 50% tissue culture infective dose - THF: tetrahydrofuran - TLC: thin-layer chromatography - TLR: toll-like receptor - TMD: transmembrane domain - TMS: tetramethylsilane - TP: terminal protein - TRP: transient receptor potential cation channels U - UV: ultraviolet V - VPA: valproic acid - VSLD: voltage sensor-like domai 11 CHAPTER 1: AN OVERVIEW OF ADENOVIRUS BIOLOGY AND DISEASES CHAPTER 1 AN OVERVIEW OF ADENOVIRUS BIOLOGY AND DISEASES Human adenovirus (HAdV) is a DNA virus that causes severe diseases in immunocompromised hosts [1]. It was isolated from human adenoids in the 1953 and was associated with some respiratory infections [2]. HAdVs belong to the Mastadenovirus genus of the family Adenoviridae and include more than 80 serotypes classified into 7 species (HAdV A-G). Among these, 2 and 5 (species C) are the most common studied, though many types belong to the species D [3]. Species designation depends on several features such as genome organization of E3 region, phylogenetic distance, nucleotide composition, oncogenicity in rodents, host range, crossneutralization, recombine capacity [4]. The diversity of species is the result of the recombination between capsid protein genes and this factor improves the pathogenicity and virulence of the new viruses [3,5]. Figure 1. Adenovirus diversity [5]. 12 CHAPTER 1: AN OVERVIEW OF ADENOVIRUS BIOLOGY AND DISEASES 1.1 Viral genome HAdVs present an icosahedral capsid including a linear and double-stranded DNA genome of 26-45 kb depending on the serotype. In particular, HAdV type 5 has a ~36 kb genome which encodes more than 40 proteins in its transcription units [5]. During Adenovirus lytic infections, HAdV genome encodes at least 25 early gene products and 15 late gene products before and after the viral DNA replication (B, Figure 1) [6]. The proteins from the early regions E1, E2, E3, E4 (Figure 2) are involved in the beginning of viral replication; in particular, immediate-early E1A proteins are the first to be transcribed and activate the transcription of the delayed-early genes and re-programme cellular gene expression in inflected cells, facilitating the viral replication [7]. The splicing process affords five several transcripts of the primary E1A. The most important forms (289R, 243R) interacting with cellular proteins is implicated in cell cycle and epigenetic regulation, transcription factors, thus increase the viral gene expression and promote the infection. E1A proteins can be identified through in vitro studies during the late infection [8]. Proteins from E1B regions in cooperation with E4 are involved in ubiquitination of cellular proteins, inactivation of cellular DNA damage response and viral mRNAs transport [9]. The early region E2 consists of two transcriptional units, E2A and E2B, which different polyadenylation sites. They code for the three proteins required for viral DNA replication: E2A codes for the DNA-binding protein (DBP), while E2B codes for the precursor terminal protein (pTP) and the viral DNA polymerase [10,11]. E3 proteins are implicated in immunomodulatory functions in infected host cells. Furthermore, E3 region encodes the HAdV death protein (ADP); it was expressed from late promoter to improve the cell lysis and the virus release after complete replication [12]. E3 region was generally delated in order to generate viral vectors used in gene therapy [13]. E4 region encode proteins that regulate the transition to late phase of infection. They are involved in the regulation of viral transcription and RNA splicing; moreover, proteins from this region interfere with cell signaling and DNA repair, contributing to cell transformation and oncogenicity [14]. The adenovirus major late transcription unit (MLTU) encodes multiple proteins from L1 to L5 regions by an alternative splicing and polyadenylation (B, Figure 1) [15]. A L1 product (52/55K) is expressed prior to the replication and promote the expression of L1 IIIa and L2-L5, that code for structural components of the capsid. L4 promoter is important for the late gene expression [16,17]. 13 CHAPTER 1: AN OVERVIEW OF ADENOVIRUS BIOLOGY AND DISEASES Figure 2. Adenovirus virion (A) and genome (B) [18]. 1.2 Virion and life cycle Structurally, HAdV consists of two main elements, an external capsid and an inner core which includes viral DNA genome and histone-like proteins. The HAdV icosahedral capsid with a diameter of ~70–100 nm exists in four forms (different in DNA quantity) between which only one are fully infectious. Capsid is mostly composed of three major proteins: 240 hexon trimers (protein II) that contribute to the capsid mass, 12 penton base pentamers (protein III) and 12 fiber trimers (protein IV) [19], that act in the cell internalization; and four minor proteins (IIIa, VI, VIII, IX) that preserve the capsid and connect it to a nucleoprotein core [20]. Other four proteins (VII, V, Mu, terminal protein TP) are connected with viral DNA inside de virion. (A, Figure 2); In particular, core protein VII promotes the DNA packaging and stabilize chromatin structure [21], while V represent a linker between DNA and internal capsid [22,23]. All viruses need to bind to specific receptors on cellular membranes in order to infect the host. In particular, HAdV life cycle begin with the cellular entry through two receptor interactions. Primarily, the terminal knob domain of viral fibers binds the coxsackie and adenovirus receptor (CAR). Next, an Arg-Gly-Asp (RGD) motif of penton base engages cellular integrins (αvβ3 and αvβ5), promoting the virus internalization by clathrin-mediated endocytosis [24]. In this process, fibers are dissociated and penton bases changes its conformation, weakening interactions with the capsid [25]. The uncoating proceeds with the vertex and V proteins release from early endosome. Protein VI liberation plays a key role in the subsequent viral particles secretion into the cytosol [26]. HAdV causes a progressive disruption of cell endosome in a mild acid pH condition in order to release the virion, 20 CHAPTER 1: AN OVERVIEW OF ADENOVIRUS BIOLOGY AND DISEASES rate from HAdV infections (less than 20%) [54,55]. Levels Clinical studies displayed the efficacy of CDV in the treatment of acute adenoviral keratoconjunctivitis in immunocompromised patients, especially when its administration was associated with immunotherapy [49]. Unfortunately, low oral bioavailability, nephrotoxicity and myelosuppression are limiting factors for CDV clinical use. Of an intravenous dose of CDV only 10% of drug is absorbed, while the remaining part (90%) is expelled in the urine by filtration and tubular secretion processes. Despite its rapid excretion, CDV is absorbed by proximal tubular cells through organic anion transporters and secreted into the lumen. The resulting improvement of intracellular levels of CDV causes tubular necrosis. For this reason, the hydratation before and after the CDV therapy promotes the drug elimination and prevents the nephrotoxicity [56]. -Brincidofovir (BCV). BCV is a lipid ester conjugate of CDV, with improved oral bioavailability and reduced toxicity (4, Figure 4) compared to CDV. The presence of a lipid moiety allows an efficient absorption through lipid uptake pathways of enterocytes simulating the endogenous lipid lysophosphatidylcholine [57]. Thus a high intracellular concentration of BCV achieves suitable antiviral activity by its conversion in CDV diphosphate. In particular, it demonstrated in vitro inhibitory activity against double-strand DNA viruses such as herpes simplex virus, polyomaviruses, papillomaviruses, poxviruses, cytomegalovirus (CMV) and adenovirus (serotypes 3, 5, 7, 8, 31) [49,58]. BCV results to be effective in immunosuppressed animal models with HAdV infections. Administration of BCV in HAdV-infected patients, for which the CDV therapy failed, provided better results in terms of efficacy and toxicity. Nephrotoxicity are not observed during BCV therapy (Phase II clinical trial-NCT01231344) because of its low plasma levels and weak propensity for renal storage [58]. At present, it is subjected to Phase III of clinical trials (NCT02087306) but significant gastrointestinal (GI) disorders including diarrhea, nausea, vomiting and pain have been observed in HSCT patients, limiting its use in therapy [59,60]. -Anti-HAdV vaccines. A live oral vaccine against HAdV type 4 and 7 was approved from 1971 to 1997 for the use in US military units. This vaccine resulted to be safe and effective in several clinical trials, reaching a 100-fold reduction of HAdV diseases, mainly respiratory ones. Nevertheless, this vaccine never was available to the general public and there are no vaccines approved today for adenoviral infections [61]. The unsatisfactory results of current antiviral drugs highlight the need of new effective anti-HAdV agents for clinical use in association to immune system reconstitution therapy. 21 CHAPTER 2: NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY CHAPTER 2 NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY 2.1 Potential targets useful in HAdV drug discovery Over the years, many researchers discovered novel compounds with inhibitory activity against HAdV serotypes in cell culture and animal model. Different targets could be considered useful in the design of specific anti-HAdV agents, especially proteins involved in critical roles of viral entry, replication or maturation processes [62]. HAdV cell entry in host cells implicates the attachment of virions to several cellular receptors. The most common receptors used by HAdV serotypes are CAR, CD46 and desmoglein 2 (DSG-2), although serotype D typically use sialic acid as entry receptor and heparan sulfate proteoglycans (HSPGs) mediate HAdV-C (2 and 5) cell entry [63]. Other potential targets are integrins, whose inhibition provide a block of viral internalization by clathrin-mediated endocytosis, or virus microtubule transport to the nucleus. The Ad replication proteins are the major explored targets for the development of new antiviral drugs. Among them are included E1A protein, an important regulator of other early genes expression [64], and proteins implicated in viral replication: DNA polymerase, pTP and DBP. HAdV proteases such as adenain (AVP) constitute additional potential targets due to their role in viral maturation process [62]. Many current antiviral therapies are directed to viral proteins or enzymes, although host targets have been identified in order to avoid the development of resistance mechanisms. Since HAdV associates with nucleosomes in the host nucleus, the viral gene expression is also affected by cellular epigenetic regulator proteins such as histone acetyltransferases (HATs) and histone deacetylases (HDACs), suggesting that these proteins could be represent interesting host targets for the development of new anti-HAdV candidates [65]. Protein p21 have an important role in the termination of cell cycle; it also promotes cells resistance to Adenovirus infection. Indeed, the upregulation of p21 expression may lead to viral replication inhibition. Toll-like receptors (TLR) pathways are activated by viruses and promote an increase of pro-inflammatory cytokines, in the case of HAdV B and C. The use of TLR pathways ligands could be an efficient approach to control HAdV infections [66]. Also retinoic acid receptor (RAR) represents a potential host target for anti-HAdV agents. A downregulation of RARβ mRNA was observed during Ad infections; on the contrary, the RARβ overexpression was associated with a decreased of virus spread. For this reason, RAR agonist could be effective in the treatment of HAdV infections [67]. 22 CHAPTER 2: NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY 2.2 Drug repositioning Drug repurposing is an effective strategy to detect new uses of existing drugs that are approved for other pharmacological indication, allowing low risks about safety and reduced development costs. Several drugs have been identified as inhibitors of HAdV infections. Valproic acid (VPA) is a medication employed in the treatment of epilepsy or bipolar disorder and the first reported HDAC inhibitor with demonstrated anti-HAdV activity (5, Figure 5). In a reported study, typical cytopathic effects (CPE) of infected cells were observed in absence of VPA, while no CPE occurred in the presence of this drug. It significantly affected viral replication and spread in cell culture. The majority of its effects seems to be related to VPA-induced upregulation of p21 expression with subsequent cell-cycle arrest, but also to the dysfunctional upregulation of viral E1A expression, that resulted in a block of late promoter induction. The role of HDACs in HAdV infection still must be clarify [68]. Also the suberoylanilide hydroxamic acid vorinostat (SAHA) is a member of HDAC inhibitors with antiviral properties (6, Figure 5). It is approved for the use in refractory or relapsed cutaneous T cell lymphoma, but showed also a broad spectrum of epigenetic activities. Since HDACs are associated with reduction of gene expression and cell cycle, an HDAC inhibitor should provide reverse effects due to an increase of histone acetylation. Unexpectedly, 6 achieved a significant decrease of HAdV5 gene expression and of E1A protein levels in cell cultures, influencing negatively several steps of virus cycle and virus yield at nanomolar concentration. Other HDAC inhibitors such as apicidin and panobinostat demonstrated antiviral activity, besides 6 [65]. Modulators of RNA splicing, such as cardiotonic steroids digoxin (7) and digitoxin (8) used for the heart failure, have been described as anti-HAdV agents (Figure 5). Both drugs inhibited several steps of viral replication in serotype 5, 31, 35 by alteration of E1A RNA splicing and resulted impairment of major late genes expression [69]. Figure 5. HDAC inhibitors and cardiotonic steroids with HAdV activity. 23 CHAPTER 2: NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY Mifepristone is an another commercially available steroid drug. It is approved by FDA for the medical termination of intrauterine pregnancy but it should be formally assessed as repurposed drug for the treatment of HAdV-induced diseases (9, Figure 5), due to its reported inhibition of HAdV infections in cells and mice. It showed in vitro activity against HAdV-5 at low micromolar concentration (EC50 =1.9 μM) with low cytotoxicity, interfering with virus translocation into de nucleus and with the replication of viral genome [70]. Among the RAR agonists potentially useful in antiviral therapy, tazarotene is a retinoid approved for the topic treatment of psoriasis, that was able to inhibit HAdV infection in vitro (10, Figure 6), with IC50 values of 8.34 μM for Ad5, 13.75 μM for Ad7 and 11.36 μM for Ad55. This effect was mediated by the binding of tazarotene to RARβ, with subsequent reduction of viral DNA replication and late hexon protein expression in a dose-dependent manner [67]. Polyphenolic compounds, especially from green tea, have been found to have antiviral activity. Among catechins, epigallocatechin gallate (EGCG) is the best broad-spectrum compound (11, Figure 6), showing activity against several viruses such as human immunodeficiency virus (HIV), adenovirus (AdV), influenza A virus (IAV), hepatitis B (HBV) and hepatitis C (HCV) virus. EGCG interacted with viral glycoproteins, interfering with the attachment of virus on host cell membrane. In particular, the competition between EGCG and virions for the binding to heparan sulfate or sialic acid glycoproteins have been observed, displayed IC50 values of 3.7 µM for HAdV binding inhibition [63]. Figure 6. Diversified potential repurposed drugs for the treatment of HAdV infections. The salicylanilide antielminting drugs niclosanide (12), oxyclozanide (13), and rafoxanide (14) displayed significant anti-adenovirus activity by suggested different ways (Figure 7). Niclosamide and rafoxanide interfered with the virus transport to the nucleus, whereas oxyclozanide inhibited HAdV early gene E1A transcription. The IC50 range for all compounds against HAdV5 and HAdV16 was from 0.45 μM to 2.3 μM with low cytotoxicity. Furtheromore, salicylanilide drugs caused a virus yeld reduction from 10 to 186 fold [71]. 24 CHAPTER 2: NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY Figure 7. Salicylanilide drugs with anti-HAdV activity. 2.3 Novel nucleoside or nitrogen bases analogues Considering that current drugs CDV, GCV and ribavirin are moderately effective to arrest Ad infections but provide no satisfactory results in terms of safety, many novel synthetic nucleoside or nitrogen bases analogues have been developed in order to reach better activity and reduced adverse effects in vitro and in vivo models [72]. Sets of ether lipid-ester of CDV and of (S)‐9‐(3‐hydroxy‐2‐ phosphonylmethoxypropyl)adenine (HPMPA) were evaluated against five HAdV serotypes (3, 5, 7, 8, 31). These acyclic nucleoside phosphonates (ANPs) presented several linkers and alkyl chain lengths on phosphonate group, resulting orally bioavailable and from 15 to 2500-fold more active than the parent compounds in in vitro experiments. Among best active compounds, 15 (hexadecyloxypropyl CDV, HDP-CDV) and 16 (octadecyloxyethyl HPMPA, ODE-HPMPA) (Figure 8) showed low IC50 values (from 0.009 to 0.28 mmol/L) towards all HAdV serotypes. In animal experiments these compounds demonstrated an efficacy similar to CDV, thus requesting further modifications for their use as anti-HAdV agents [73]. Based on these in vitro promising results, other octadecyloxyehtyl derivatives (ODE) of acyclic nucleoside phosphonates was prepared to evaluate the effect of different nitrogen bases. All compounds were able to inhibit HAdV14 and one of them, the octadecyloxyethyl derivative of 2,6-diaminopurine (ODE-HPMP-DAP, 17, Figure 8) resulted to be the most effective compound, with IC50 value of 1.7 nM and a virus yield reduction of 90% at 4.1 nM [74]. This compound could be further investigated to evaluate its efficacy in the treatment of Ad infections. Figure 8. Ether lipid-esters of acyclic nucleoside phosphonates with HAdV activity. 25 CHAPTER 2: NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY 6-Azacytidine derivatives were described by I. Alexeeva et al as new potential antiviral agents against HAdV types 2 and 5. Several substituents on N-4 of azacytidine nucleus were evaluated as well as the functionalization of hydroxyl groups. The thio-analogue 18 and N,O-tetracylated compound 19 (Figure 9) demonstrated the most potent anti-viral effect against HAdV2, with IC50 values of 0.8 μМ and 0.3 μМ respectively. Compounds 19, the tetracetylated derivative, presented high selectivity index. Further studies demonstrated that these molecules can inhibit the formation of intranuclear DNA-containing inclusion bodies, suggesting that they could be interfere with the viral genome expression [75]. A series of new 5-aminouracil derivatives were synthesized and in vitro evaluated against HAdV5. The effect of several substitutents on the aromatic moiety as well as on the uracil scaffold were explored. Compound with a morpholine ring (20, Figure 9) afforded the best inhibitory activity (IC50 = 0.5 μM) and a suitable selectivity index. Additional investigation highlighted the ability of these molecules to block viral replication thorough the inhibition of DNA polymerase and E1A gene expression [76]. The main advantage of DNA/RNA component analogues potentially used as antiviral agent is that they can be easily inserted into the viral genome suspending the replication. Figure 9. 6-Azacytidineand 5-aminouracil-derived compounds as new potential HAdV agents. 2.4 Novel non-nucleoside small molecules Over the years, structural diversified small molecules with non-nucleoside core have been described in order to obtain new effective antiviral candidates useful in the management of HAdV-induced diseases. Common features were present in a lot of new described compounds, mainly amide/urea functions and aromatic moieties. Many benzoic acid derivatives were found to inhibit Ad infections in vitro. In a study, three generations of 2-[2-benzoylamino)benzoylamino]benzoic acids were prepared and the structureactivity relationships were identified. Changes in the position of carboxylic acid moiety resulted to decrease the activity against HAdV 5, while electron-withdrawing substituents such as chlorine or 26 CHAPTER 2: NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY fluorine on terminal and central aromatic rings were well tolerated. The most active compounds 21 and 22 (Figure 10) inhibited viral replication with IC50 values of 0.57 μM and 0.58 μM respectively and low cytotoxicity [77]. A set of optimized salicylamide derivatives as potent anti-HAdV agents was recently described. The effect of several modifications on both aromatic moieties as well as a linker insertion between the benzamide and the other phenyl ring were evaluated through in vitro assay. Many halogenated molecules showed an improved antiviral activity against HAdV5 compared to lead compound niclosamide (600 nM). Compound 23 and 24 were the best of the series (Figure 10), reaching IC50 values of nanomolar concentrations (50 nM and 80 nM respectively) and high selectivity index. The proposed mechanism for compound 24 was the inhibition of viral replication whereas 23 affected later steps. A di-amide series was also prepared but resulted to be less active than mono-amide ones. Compounds 23 and 24 are suitable candidates to evaluate their efficacy in animal model and develop an alternative antiviral therapy for HAdV infections [78]. Benzoic acid esters with anti-HAdV activity were also developed. This compounds demonstrated inhibition of cytopathic effect of HAdV7 in host cell and reduction of virus progeny production. Moreover, they were able to block apoptosis of host cell caused by virus. For compound 25 (Figure 10) the inhibition rate was 82.4 % at 40 μg / mL [79]. Figure 10. Benzoic acid amides and esters with anti-HAdV activity. Several new biologically active compounds with nitrogen heterocycle scaffolds were discovered as anti-HAdV agents. Hamdy et al developed different sets of pyrazoles, pyrazolopyridazines, enaminones, and sulphonamides and examined their antiviral activity against adenovirus and rotavirus. Among the modification at N-1 of pyrazolo-pyridazine core, compound with a methoxy group on the aryl moiety resulted to be the most active (26, Figure 11), while the presence of electron withdrawing groups suppressed the activity. Compound 26 displayed an IC50 value of 0.06 mg/mL and suitable therapeutic index. Further investigations are needed to identify the mechanism of action of these new molecules [80]. Since there are no small molecules described as adenain inhibitors, a 27 CHAPTER 2: NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY set of pyrimidine nitrile derivatives was developed by a molecular hybridization strategy from two lead compounds. Compound 27 demonstrated to be the most effective inhibitor (Figure 11) toward adenovirus protease 8 and 5 (AVP), achieving IC50 values of 0.003 μM and 0.002 μM respectively. High-resolution X-ray co-crystal structures of these derivatives in complex with adenain illustrated that a nitrile group covalently connected with Cys122 and all amide functions were implicated in noncovalent hydrogen bonds with several protease residues [81]. In another study, privileged structureguided scaffold refining strategy was used to prepare new triazolyl-quinazoline-diones with in vitro activity towards vaccinia virus and HAdV2 (28, 29, Figure 11). The most active compounds were those with a methoxy group at para position or a fluorine atom at orto position of the phenyl ring conneted to triazol moiety. Active compounds presented IC50 values ranging from 6.2 μM to 13 μM exhibiting no cytotoxicity [82]. Figure 11. Several nitrogen heterocycle derivatives with anti-HAdV activity. A tri-substituted piperazin-2-one derivative (30, Figure 12) was identified among more than 25.000 synthetic small molecules screened against Ad infections, using high-throughput screening (HTS). Compound 30 inhibited HAdV 5 in a dose-dependent manner, reaching 100% in vitro inhibition at concentrations higher than 3 μM and a virus yield reduction of 12–17-fold. Mechanistic studies suggested that 30 affected viral replication, possibly targeting DNA–VII complex or involved replication proteins [83]. Starting from this selected hit compound, a new set of piperazine derivatives have been designed and synthesized by our research group to obtain new effective agents. Several substituents were evaluated on both nitrogen atoms, introducing urea and amide functions. From the structure-activity relationship point of view, the presence of a benzofuran group on amide function seemed to be relevant for the antiviral activity against HAdV5; in addition, halogen substituents on the urea phenyl ring generally increased the infection inhibition. In particular, compound 31 influenced later steps of viral DNA replication (Figure 12) and resulted to be the best compound of 28 CHAPTER 2: NEW PERSPECTIVES IN ADENOVIRUS DRUG DISCOVERY the series (IC50 = 1.1 μM) with no cytotoxicity. These compounds represent suitable candidate for further in vivo experiments and development of new potential anti-HAdV agents [84,85]. Figure 12. Piperazine-derived compounds with HAdV-activity. 29 AIMS OF THE WORK AIMS OF THE WORK HAdV usually infects the epithelium of respiratory tract causing severe pneumonia and other infections, mostly in immunosuppressed patients and young children. Despite its clinical relevance, there is no approved antiviral therapy for the treatment of HAdV infections and repurposed nonspecific antiviral drugs reached no satisfactory results. Since the find of novel specific anti-HAdV is an important topic for the medicinal chemistry, this PhD thesis aimed to discover new structural diversified small molecules useful for HAdV-induced diseases. This project was carried out in the University of Seville and was focused on the preparation of new nitrogen compounds based on cyclic and acyclic scaffolds as direct inhibitors of HAdV infection. The main tasks were: 1) Design of new small molecules based on piperazine and aminoalcohol scaffolds. An optimization process of piperazine-derived ureas privileged structures, by introducing slight modifications in the general piperazine backbone (amide and urea/thiourea functions), has been developed in order to improve the inhibition of HAdV infection and the safety profile. Since our interest in the discovery of new interesting structures with antiviral activity, the acyclic scaffolds 2-amino-1,3-propanediol and 3-amino-1,2-propanediol have also been employed. Firstly, a set of symmetric esters and carbamates from 2-amino-1,3-propanediol (serinol) have been designed, based on the typical features of reported antiviral agents. Serinol has been then replaced with its positional isomer 3-amino-1,2propanediol. In order to introduce diversified functionalization of hydroxyl and amino groups, urea, ester, carbamate, triazole derivatives have been planned. Figure 13. Cyclic and acyclic scaffolds employed for the development of new anti-HAdV agent. 2) Synthesis and structural characterization of designed compound libraries. A short and high yielded synthetic methodology has been employed for most compounds of these series. The selective O-acylation reactions of primary/secondary alcohol group (3-amino-1,2-propanediol) and the introduction of triazole moiety (click chemistry) have been performed through a multi-step synthesis. 36 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES Scheme 1. Chemical synthesis of 4-acyl-2-substituted piperazine thiourea derivatives (50–92). The thiourea function was introduced at N-1 in a second reaction between the monoacylated compound (41–49) and the corresponding phenyl isothiocyanate, which proceeded in DCM at rt (Scheme 1). Due to the variability of employed substituted phenyl isothiocyanates, a collection of 43 thiourea derivatives were generated (50–92, Table 1) Table 1. 4-Acyl-2-substituted-piperazine thiourea derivatives from pathway A. Comp R R1 R3 R4 R5 Yield (%) 50 Me Ot-Bu H Cl H 73 51 Me Ot-Bu H CN H 76 52 Me Ot-Bu H F H 79 53 Me Ot-Bu H CF3 H 73 54 Me Ot-Bu H OCH3 H 75 55 Me Ot-Bu H CH3 H 75 56 Me Ot-Bu CF3 H CF3 71 37 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES Comp R R1 R3 R4 R5 Yield (%) 57 Me CH2t-Bu H NO2 H 68 58 Me CH2t-Bu H Cl H 70 59 Me CH2t-Bu H CN H 66 60 Me CH2t-Bu H F H 60 61 Me CH2t-Bu H CF3 H 73 62 Me CH2t-Bu H OCH3 H 62 63 Me CH2t-Bu H CH3 H 76 64 Me CH2t-Bu CF3 H CF3 68 65 Me CH2c-Hex H NO2 H 76 66 Me CH2c-Hex H Cl H 75 67 Me CH2c-Hex H CN H 73 68 Me CH2c-Hex H F H 77 69 Me CH2c-Hex H CF3 H 68 70 Me CH2c-Hex H OCH3 H 70 71 Me CH2c-Hex H CH3 H 66 72 Me CH2c-Hex CF3 H CF3 78 73 Me CH2Ph H NO2 H 57 74 Me CH2Ph H Cl H 61 75 Me CH2Ph H CN H 67 76 Me CH2Ph H F H 56 77 Me CH2Ph H CF3 H 70 78 Me CH2Ph H OCH3 H 55 79 Me CH2Ph H CH3 H 62 80 Me CH2Ph CF3 H CF3 65 81 Me Benzofuran-2-yl H NO2 H 92 82 Me Benzofuran-2-yl H CN H 72 83 Me Benzofuran-2-yl H F H 85 84 Me Benzofuran-2-yl H CF3 H 84 85 Ph Ot-Bu H F H 70 86 Ph CH2t-Bu CF3 H CF3 96 38 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES Comp R R1 R3 R4 R5 Yield (%) 87 Ph CH2c-Hex H CN H 65 88 Ph CH2c-Hex H F H 60 89 Ph CH2c-Hex H CH3 H 60 90 Ph CH2Ph H NO2 H 65 91 Ph CH2Ph H CN H 72 92 Ph CH2Ph CF3 H CF3 93 -Pathway B: Exchange the acyl groups at N-4 in 2-phenyl piperazine urea derivatives (93–101). The preparation of compounds 93–99 (Scheme 2, Table 2) followed the previously described short synthetic route. The formation of urea function at N-1 occurred due to the reaction between 2-phenyl piperazine mono-amide 47–49 (2-tert-butyl acetyl, 2-cyclohexylacetyl, 2-phenylacetyl) and p-NO2, o-NO2 or o-Cl-m-CF3 phenyl isocyanate. Scheme 2. Chemical synthesis of 4-acyl-2-phenylpiperazine urea derivatives (93–101). 39 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES Compounds 100 (di-amide derivative) and 101 (di-urea derivative) were synthesized directly from 2phenyl piperazine, using an excess of reactive agent (acyl chloride/pyridine or isocyanate, respectively) in DCM at rt (Scheme 2). Table 2. 4-Acyl-2-phenylpiperazine urea derivatives from pathway B. Comp. R1 R2 R4 R5 Yield (%) 93 t-Bu H NO2 H 90 94 t-Bu NO2 H H 88 95 c-Hex H NO2 H 83 96 c-Hex NO2 H H 88 97 Ph H NO2 H 98 98 Ph NO2 H H 96 99 Ph Cl H CF3 96 100 83 101 90 -Pathway C: Replacement of 2-substituted piperazine core with 2,6-dimethylpiperazine and unsubstituted piperazine (104–110, 112–114, 118–121). A series of benzofurane-2-carbonyl-derived ureas (104–110, Table 3) with 2,6-dimethylpiperazine central ring were prepared as analogues of lead compounds 31–35, through the same procedure used for the other substituted piperazine derivatives. NO2, CN, Cl, CF3, CH3 substituted phenylisocyanates were implicated in the urea formation together with the intermediate 103 (Scheme 3). 40 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES Scheme 3. Chemical synthesis of 4-(benzofurane-2-carbonyl)-2,6-dimethylpiperazine urea derivatives (104–110). To prepare those analogues with an unsubstituted piperazine core, the Boc-piperazine, commercially available, was employed as the starting material. Firstly, the urea function was introduced by reaction with appropriate isocyanates, afforded compounds 112–114 which were deprotected in acid condition (CF3COOH) at rt, using DCM as solvent. The intermediates 115 and 116 reacted with the acylating agent (benzofuran-2-carbonyl chloride) to obtain final products 118 and 119. Finally, piperazine derivatives 120 (di-amide) and 121 (di-urea) were prepared through the same reaction used for 2methyl piperazine analogues (100, 101) (Scheme 4, Table 3). Scheme 4. Chemical synthesis of 4-acyl-piperazine urea derivatives (112–114, 118, 119). 41 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES Table 3. 2,6-disubstituted and unsubstituted piperazine urea derivatives from pathway C. Comp R R1 R2 R3 R4 R5 Yield (%) 104 Me Benzofuran-2-yl H H NO2 H 75 105 Me Benzofuran-2-yl H H Cl H 70 106 Me Benzofuran-2-yl H H CN H 85 107 Me Benzofuran-2-yl NO2 H H H 85 108 Me Benzofuran-2-yl H H CH3 H 63 109 Me Benzofuran-2-yl Cl H H CF3 69 110 Me Benzofuran-2-yl H CF3 Cl H 70 112 H Ot-Bu H H NO2 H 94 113 H Ot-Bu NO2 H H H 89 114 H Ot-Bu Cl H H CF3 92 118 H Benzofuran-2-yl H H NO2 H 80 119 H Benzofuran-2-yl NO2 H H H 88 120 91 121 98 All new piperazine-derived compounds were characterized by NMR and Mass Spectrometry and through the determination of melting points. Representative resonance assignments from 1H NMR and 13C NMR of some selected compounds were illustrated in the Table 4. 42 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES Table 4. Selected piperazine derivatives and some representative resonance assignments (1H NMR and 13C NMR). Compound 1H NMRa (ppm) 13C NMRb (ppm) NHSO CHCH3/CHPh CH2CO/ C(CH3)3a C=O/C=S CHCH3/CHPh CH3/OCH3 52 7.10 5.14-4.75 1.47a 183.6, 155.0 52.3 15.1 67 9.59 5.11 2.38-2.15 181.3, 170.7 51.7 15.1 68 9.23 5.06 2.40-2.11 181.9, 170.7 51.3 15.1 71 9.19 5.18-4.97 2.26-2.14 181.9, 170.7 51.2 20.5, 15.1 73 9.769.74 5.13 3.26-3.14, 3.08-2.86 181.2, 169.7 51.9 15.0 75 9.599.57 5.14 3.32-3-15, 3.08-2.89 181.3,169.7 51.7 15.0 80 9.729.69 5.12 3.29-3.17, 3.06-2.94 180.9,169.8 51.6 15.1 92 9.88 4.94 3.21-3.12 181.9,169.5 58.4 - 96 9-429.35 5.43-5.33 3.22-3.16 170.5,154.2 54.3 - 98 9.40 5.47 3.02-2.94 169.5,154.2 54.0 - 112 9.28 - 1.42a 153.9,153.8 - - 114 8.49 - 1.43a 154.5,153.8 - - a500 MHz, DMSO-d6; b125MHz, DMSO-d6 43 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES 3.2 Biological evaluation New synthesized compounds were assessed for their antiviral activity against HAdV5 as well as for their cytotoxicity. 3.2.1. In vitro antiviral activity and effect on cellular viability Firstly, the anti-HAdV activity of the new piperazine derivatives at 10 μM was evaluated in plaque assay (293β5 cell line). Since cidofovir current represent the only therapeutic option for HAdV infection, it was also evaluated and compared to our results. The effect on cellular viability was examined in A549 cell line and the 50% cytotoxic concentration (CC50) was determined for those compounds that reached a percentage of inhibition in the plaque assay >80%, in order to ascertain their safety profile. Among the compounds included in the pathway A (50–92) we have identified many piperazine thiourea derivatives which reached HAdV5-GFP plaque-formation inhibition >80% together with low cytotoxicity (CC50 > 100 μM) (Table 5). Compounds containing electronwithdrawing substituents (NO2, Cl, CN, CF3) in para position on the phenyl ring showed greater inhibition (51, 52, 65, 66, 67, 68, 69, 73, 74, 75 and 76); NO2 and Cl resulted to be the most present groups. Also the presence of two trifluoromethyl groups in 3 and 5 positions (not previously evaluated) increased the activity (72 and 80), while compound 71 with p-CH3 represented an exception of the series (Table 5). With regard to the acyl function at N-4, compounds with 2-tertbutyl group did not achieve a percentage of inhibition more than 80%. On the contrary, the major part of highly active compounds contained 2-cyclohexylacetyl moiety (65–69, 71 and 72). Thiourea derivatives with benzofuran-2-carbonyl group at N-4 (81, 82 and 84) did not represent potentially interesting analogues. They displayed high inhibition (100%), but showed low CC50 values. Neither 2-phenyl piperazine analogues have demonstrated high percentage of plaque-formation inhibition, with the exception of 86, 87 and 92 that showed better anti-HAdV activity (percentage range 80-100 %). It is important to note that compound 86 (2-tert-butylacetyl derivative from 2-phenyl piperazine) was an analogue of 64 (2-tert-butylacetyl derivative from 2-methy piperazine) which inhibited 4.8% of HAdV plaque formation. In this case, the presence of phenyl group in position 2 of piperazine core improved the antiviral activity profile. 44 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES Table 5. Inhibition of HAdV infection in the plaque assay and effects on cellular viability for compounds 50-92 from pathway A. Comp % of plaqueformation inhibitiona CC50b Comp. % of plaqueformation inhibitiona CC50b 50 73.43 ± 4.19 - 72 100.00 ± 0.0 82.4 ± 5.5 51 88.91 ± 15.92 175.0 ± 8.8 73 98.21 ± 3.57 210.4 ± 17.8 52 92.91 ± 3.82 200.0 ± 10.8 74 84.56 ± 15.72 175.0 ± 10.2 53 73.43 ± 22.49 - 75 98.36 ± 2.13 174.7 ± 4.8 54 36.55 ± 30.13 - 76 100 ± 0.0 26.3 ± 1.6 55 49.11 ± 4.73 - 77 73.43 ± 4.19 - 56 11.11 ± 19.25 - 78 88.91 ± 15.92 175.0 ± 8.8 57 76.23 ± 22.02 - 79 92.91 ± 3.82 200.0 ± 10.8 58 61.64 ± 25.29 - 80 73.43 ± 22.49 - 59 51 ± 4.13 - 81 100 ± 0.0 20.0 ± 15.5 60 7.50 ± 15.00 - 82 100 ± 0.0 25.5 ± 10.0 61 36.33 ± 32.43 - 83 85.0 ± 6.10 75.4 ± 22.8 62 58.11 ± 17.20 - 84 100 ± 0.0 46.3 ± 20.8 63 76.83 ± 13.56 - 85 27.5 ± 10.6 - 64 4.81 ± 9.34 - 86 100.0 ± 0.0 91.8 ± 0.9 65 82.56 ± 15.63 148.1 ± 12.5 87 80.0 ± 7.1 65.3 ± 10.5 66 89.33 ± 10.08 200.0 ± 0.0 88 5.0 ± 6.3 - 67 100.00 ± 0.0 193.0 ± 4.9 89 15.0 ± 8.5 - 68 100.00 ± 0.0 143.4 ± 6.6 90 5.5 ± 7.8 - 69 88.94 ± 10.32 142.2 ± 7.9 91 64.0 ± 12.7 - 70 25.53 ± 36.11 - 92 100.0 ± 0.0 104.3 ± 15.4 71 95.79 ± 4.82 122.2 ± 12.5 Cidofovic 3.51 ± 4.97 50.6 ± 9.8 a Percentage of control HAdV5-GFP inhibition in a plaque assay at 10 mM using the 293β5 cell line b Cytotoxic concentration 50%. The results represent means ± SD of triplicate samples from three independent experiments c Data of cidofovir, as positive clinical drug candidate, have been list. The effect of different acyl groups at N-4 keeping the presence of the urea function at N-1 in 2-phenyl piperazine was examined (Table 6). Compounds 93–99 from pathway B were all active, with 45 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES percentages of plaque-formation inhibition ranging from 74% to 100%, independently of the acyl nature (2-tert-butylacetyl, 2-cyclohexylacetyl, 2-phenylacetyl) and of the phenylaminocarbonyl substituents (p-NO2, o-NO2, o-Cl-m-CF3) Compounds 100 (di-amide derivative) showed a weak antiviral activity (19% inhibition in plaque assay), whereas compound 101 (di-urea derivative) resulted to be a good inhibitor of HAdV infection (90.4%). These results suggest the relevance of the urea function for the antiviral activity. Table 6. Inhibition of HAdV infection in the plaque assays and effects on cellular viability for compounds 93-101 from pathway B. Comp. % of plaqueformation inhibitiona CC50b 93 74.4 ± 29.0 72.0 ± 9.2 94 100 ± 0.0 63.3 ± 5.7 95 98.9 ± 1.5 71.8 ± 5.0 96 100 ± 0.0 112.1 ± 10.1 97 82.6 ± 12.3 174.0 ± 12.8 98 96.9 ± 1.4 120.5 ± 10.6 99 100 ± 0.0 64.5 ± 5.3 100 19.4 ± 6.4 - 101 90.4 ± 12.4 200 ± 0.0 Cidofovirc 3.51 ± 4.97 50.6 ± 9.8 a Percentage of control HAdV5-GFP inhibition in a plaque assay at 10 mM using the 293b5 cell line b Cytotoxic concentration 50%. The results represent means ± SD of triplicate samples from three independent experiments c Data of cidofovir, as positive clinical drug candidate, have been list. The impact of an additional substituent on the piperazine scaffold and of the use of unsubstituted piperazine core was also analysed (pathway C). Among the 2,6-dimetilpiperazine urea derivatives with benzofuranyl acyl group, only compounds 105 and 107 (p-Cl and o-NO2 respectively) displayed 52 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES a closely-related DNA polymerase belonging to the same family of viral DNA polymerase, the family B. Compound 71 showed significant inhibition of the Phi29 DNA polymerase activity (Figure 20C), suggesting that its preferential target may be the HAdV DNA polymerase. Compounds 52, 67, 68, 92 and 98 did not demonstrate inhibition of the HAdV genome accessibility to the nucleus or of the HAdV DNA production. The mechanism of action for these compounds may be related to later steps in the HAdV replicative cycle, such as assembly, maturation or release of the new viral particles. The twelve derivatives influenced different steps in the HAdV life cycle such as the HAdV entry process, the transcription of the E1A gene, viral DNA replication or later steps. Additional studies are needed to clarify the specific mechanisms for the inhibition of HAdV infection by these seven compounds. Since they have shown high variability regarding their potential mechanism of action, these compounds could be useful as a tool to clarify the complex events involved in the HAdV replicative cycle. Figure 20. Effect of the selected compounds on HAdV DNA replication. (A) De novo production of HAdV DNA copies compared to the positive control 24-h post-infection in a quantitative PCR assay. (B) Expression of the immediate early gene E1A compared to the positive control 6-h post-infection in a quantitative PCR assay. (C) Impact on the amplification efficiency of the Phi29 DNA polymerase. The results are expressed as the relative copy number of HAdV DNA and E1A mRNA normalized to GAPDH copy number, and they are presented as the mean ± SD of triplicate assays. *P < 0.05. 53 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES 3.2.4. Synergistic activity evaluation Since these new compounds presented different ways to explicate their activity, the effect on the HAdV infection inhibition of compound combinations with diversified mechanisms of action was investigated. A representative piperazine derivative for each mechanism type was selected to perform a combination study based on the Chou-Talalay method, using the CalcuSyn software [88]. Compound 68 was selected due to its action in later steps after DNA replication, compound 71 as an inhibitor of the HAdV DNA replication process (DNA polymerase) and compound 80 as inhibitor of the E1A transcription. All compounds were 4-acyl-1-phenylaminothiocarbonyl-2-methylpiperazine derivatives from pathway A. The ratio for each combination was selected based on IC50 values of selected derivatives. All the combinations demonstrated good conformity to the mass-action law as shown in Table 9 (r ranged from 0.937 to 0.967). A very strong synergism was observed for the combination 68 + 71 (1:4) at all three levels of inhibition (IC50, IC75 and IC90) and for 68 +71 + 80 (1:4:8) at IC90. The combination 71 + 80 (1:2) at IC90, 68 + 80 (1:8) at all the levels of inhibition and 68 + 71 + 80 (1:4:8) at IC50 and IC75 levels were considered synergism (Table 9). The different proposed ways of action of these compounds were supported by the significant combinatory index values CI (a pharmacological interaction estimation which uses the IC50 and the dose-response curve's shape of each individual compound and their combinations) obtained using the CalcuSyn software for all the combinations. Table 9. Synergistic activity of different combinations three selected anti-HAdV compounds. Comp ratio Combinatory index (CI) values at ra IC50 IC75 IC90 68 + 71 (1:4) 0.095 0.092 0.091 0.937 68 + 80 (1:8) 0.359 0.350 0.353 0.959 71 + 80 (1:2) 0.202 0.254 0.319 0.939 68 +71 + 80 (1:4:8) 0.443 0.474 0.091 0.937 a The r value for each combination is also reported to indicate the correlation coefficient of the data to the mass-action law. 54 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES 3.2.5. Hamster serum stability We examined the stability of selected compounds in Syrian hamster serum as a previous step to the evaluation of their efficacy and safety in the Syrian hamster model of HAdV infection. This assay allowed to select the antiviral compounds with structural moieties resistant to degradation by serum proteins and with low binding to serum proteins. Compounds included in this test were those SI values >100 and with different modes of action to block HAdV infection (52, 68, 73 and 92), together with 80 due to its ability to inhibit the E1A transcription. A significant degradation significant degradation after 2 hours of incubation in hamster serum was observed for compounds 52 and 68, tertbutoxycarbonyl and 2-cyclohexylacetyl derivatives respectively, with percentages of remaining compound <70%. Compounds 73 and 92 didn’t incur in any degradation after 2 hours of incubation, displaying percentages of remaining compound of 106.7 and 100.0 respectively. At last, compound 80 showed a percentage of remaining compound of 92.8% (Table 10). Table 10. Selected derivatives serum stability in a graphical representation of the percentage of the derivatives remaining at different incubation time points. Time 52 68 73 80 92 0 100.0±8.6a 100.0±25.3 100.0±11.2 100.0±3.0 100.0±8.4 15 97.8±6.0 116.7±20.5 142.7±8.5 108.9±6.2 97.2±3.6 30 102.4±14.3 96.4±31.2 139.4±2.3 106.6±4.2 109.6±13.5 60 86.5±30.3 85.0±24.1 114.2±6.5 96.5±3.6 90.5±16.9 120 75.4±5.0 67.3±27.7 106.7±3.8 92.8±13.3 100.0±9.2 a Percentages of remaining compound at different time points (minutes). The results represent means ± SD from three independent experiments. 3.3 In silico prediction of physicochemical properties: drug-likeness evaluation During the drug discovery and the development of compounds with biological activity, the preliminary estimation of the absorption, distribution, metabolism and excretion (ADME) properties for potential drug candidates reduces pharmacokinetic-related failures in the later phases of the development [89]. Some physicochemical properties of twelve selected compounds were predicted using a free online software (http://www.molinspiration.com) to assess their compliance with the Lipinski's rule of five [90]. Eleven compound resulted well conformed to the Lipinski's rule of five, 55 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES while compound 92 failed with two violations, LogP and Molecular Weight values that surpassed the accepted ranges (Table 11). The bioavailability is mainly related to gastrointestinal absorption. Drug-likeness is a concept used in drug discovery to identify and exclude compounds with an inadequate pharmacokinetic profile. it is based on the analysis of the physicochemical properties and structural features of drug candidates and allows to evaluate if a molecule can become an oral drug with respect to their bioavailability [91]. Also the freely accessible web tool SwissADME (http://www.swissadme.ch) was employed to evaluate pharmacokinetics and drug-likeness of these small molecules [92]. Also in this case, compound 92 showed the same Lipinski's rules violations; in addition, the Bioavailability Radar, a rapid drug-likeness evaluation based on physicochemical properties lipophilicity, size, polarity, solubility, flexibility and saturation, predicted only compound 92 as no orally bioavailable due to be too lipophilic, insoluble and high sized. On the contrary, other compounds conformed to both Lipinski's and Veber's rules. According to Brain Or IntestinaL EstimateD permeation method (BOILED-egg) [92] the predicted human gastrointestinal absorption was high for all compounds except for compound 92. Table 11. Prediction of physicochemical properties of selected compounds. Nº NViola Natomsb miLogPc MW/Dad nONe nOHNHf Nrotbg TPSA/A2, h MVi ARl <5 <500 <10 <5 ≤10 <140 52 0 24 3.01 356.46 5 1 5 44.81 321.91 67 0 27 3.63 384.55 5 1 5 59.37 365.47 68 0 26 4.04 377.53 4 1 5 35.57 353.54 71 0 26 4.33 373.57 4 1 5 35.57 365.17 73 0 28 2.90 398.49 7 1 6 81.40 353.36 75 0 27 2.69 378.50 5 1 5 59.37 346.88 80 0 33 4.66 489.49 4 1 7 35.57 392.62 92 2 38 5.91 551.56 4 1 8 35.57 447.46 96 1 33 5.14 450.54 8 1 5 98.47 417.91 98 0 33 4.20 444.49 8 1 5 98.47 399.32 112 0 25 2.58 350.38 9 1 4 107.70 314.85 114 0 27 4.12 407.82 6 1 4 61.868 336.35 a nViol: no. of violations; b natoms: no. of atoms; c miLogP: molinspiration predicted LogP; d MW: molecular weight; e nON: no. of hydrogen bond acceptors; f nOHNH: no. of hydrogen bond donors; g nrotb: no. of rotatable bonds; h TPSA: topological polar surface area; i MV: molar volume; l AR: accepted range These results have been recently published [93]. 56 CHAPTER 3: 4-ACYL-1-PHENYLAMINO(THIO)CARBONYL PIPERAZINE DERIVATIVES 57 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES CHAPTER 4 O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES 4.1 Chemistry 4.1.1 Design With the aim to identify novel privileged structures for the development of potential anti-adenovirus agents, our interest was focused on aminoalcohols as employed scaffolds for the preparation of new compounds libraries. In the first place, we have selected the symmetric aminoalcohol serinol as central backbone and precursor on new molecules. Many of reported antiviral acyclic nucleoside analogues presented an aminoalcohol or glycerol skeleton, such as cidofovir and ganciclovir (1, Figure 21), that represent the current therapeutic options for severe HAdV infections acting as inhibitors of viral DNA replication; even if they resulted not very effective and associated with several adverse effects (section 1.4)[56]. Also the antiviral drug penciclovir (122, Figure 21), used for the treatment of various herpesvirus infections, consist of a guanine base connected to an aminoalcohol five-carbon chain [94]. Figure 21. Design of new set of serinol derivatives. 58 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES In this context, non-nucleoside compounds with isopropanolamine core and a simple heterocyclic structure have been discovered as antiviral agents against HIV (123, Figure 21) [95]. An important structural consideration that prompted us to choose this scaffold consists in the symmetry. Diversified symmetric compounds have been described as potential antiviral candidates, such as novel symmetrical phenylenediamines targeting the viral HCV NS3 helicase[96], symmetrically disposed stilbenes as potent inhibitors of NS5A proteins (124, Figure 21) [97], and complex homodimeric structures derived from daclastavir and other related symmetric compounds [98,99]. In this field, no symmetric compounds have been described as potential anti-HAdV agents. From a chemical point of view, serinol scaffold allow us to quickly design and generate new molecules keeping some important features present in reported anti-adenovirus compounds, mainly urea [76,84] and amide/ester [78,79,81] functions that have been identified as relevant for the antiviral activity (20, 24, 25, 112, Figure 21). The general structure of new compounds shared the urea function at position 2 of serinol chain and an aromatic moiety connected to both primary hydroxyl groups through an acyl function (ester or carbamate, Figure 22). The points of structural variability that have been investigated regarded the introduction of several substituents on the phenyl ring of the ester function as well as of the phenylaminocarbonyl group, in order to assess different electronic properties, and the replacement of the ester moiety with a carbamate one. The N-aryl urea function was decorated with three different groups (p-CF3, p-CH3, 3-CF3-4-Cl), in order to evaluate the effect of mono and di-substitutions as well as different electronic behaviours. In the collection of diester derivatives, we have explored the presence of a wider variety of substituents on the phenyl ring, having electron-withdrawing (CN and NO2) or donating properties (CH3, N(CH3)2, OCH3). In particular, our attention was focused on methoxy groups (mono, di and trimethoxy), due to these benzoyl derivatives represent interesting scaffolds found in several anticancer and antiviral compounds, in the form of amides or esters [82,100,101]. In this field and aimed to further explore the presence of methoxy groups, a trimethoxycynnamic moiety was also inserted, in order to examine the presence of a spacer between the acyl function and the terminal 3,4,5-trimetoxyphenyl ring (Figure 22). Over the years, several reported antiviral natural products and molecular hybrids with trimethoxycynnamic portions have been discovered, such as compound 125 (Figure 21) which demonstrated in vitro anti-hepatitis B activity [102]. Some monoester serinol derivatives were also designed (Figure 22) considering that many antiviral acyclic nucleosides displayed at least one free primary hydroxyl group. Finally, we performed a change in the acyl function, introducting a carbamate in the pleace of the ester. These 59 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES compounds were prepared due to their potential to engage in additional hydrogen interactions through both urethane functions. Figure 22. General structures of new designed diester, dicarbamate and monoester derivatives from serinol. 4.1.2 Synthesis The synthetic pathways for the preparation of new set of serinol-derived diesters, monoesters and dicarbamates included two reaction starting from commercially available 2-amino-1,3-propanediol (serinol), which shared the first step providing the insertion of urea function, while they differed in the second one (ester/carbamate formation). 60 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES -Pathway A: Synthesis of N-phenylaminocarbonyl serinol diester derivatives (131–155) Diester derivatives of serinol (131–135) were obtained following the synthetic route depicted in the Scheme 5. Primarily, the urea function was introduced an N-2 of serinol skeleton by the reaction of 126 with appropriate substituted phenyl isocyanate (p-CF3, p-CH3, 3-CF3-4-Cl, p-Cl) in DCM at rt (127–130). Subsequently, aromatic esters 131–152 were synthesized by an acylation reaction of both primary hydroxyl groups, using corresponding acyl chloride and DMAP, in DCM at rt. At this step, the diversity was introduced on the phenyl ring through substituents with different electronic properties (CN, NO2, CH3, N(CH3)2, OCH3, di-OCH3, tri-OCH3). Scheme 5. Synthetic routes for the preparation of N-phenylaminocarbonyl diesters derivatives from serinol (131–155). For the preparation of trimethoxycynnamic ester derivatives 153–155 the condensation occurred using carboxylic acid as acylating in DCM and following Steglich condition (EDCI, DMAP, Scheme 5) [103]. 61 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES Table 12. Serinol-derived aromatic esters and cynnamic acid esters from pathway A. Comp R1 R2 R3 R4 R5 R6 Yield (%) 131 (A) H CF3 H H CH3 H 75 132 (A) H CF3 CH3 H H H 68 133 (A) H CF3 H H OCH3 H 67 134 (A) H CF3 H H CN H 65 135 (A) H CF3 H H NO2 H 71 136 (A) H CF3 OCH3 H OCH3 H 70 137 (A) H CF3 H OCH3 OCH3 OCH3 82 138 (A) H CH3 H H CH3 H 66 139 (A) H CH3 CH3 H H H 92 140 (A) H CH3 H H OCH3 H 79 141 (A) H CH3 H H NO2 H 62 142 (A) H CH3 OCH3 H OCH3 H 65 143 (A) H CH3 H OCH3 OCH3 OCH3 61 144 (A) CF3 Cl H H CH3 H 85 145 (A) CF3 Cl CH3 H H H 62 146 (A) CF3 Cl H H OCH3 H 56 147 (A) CF3 Cl H H CN H 70 148 (A) CF3 Cl H H NO2 H 68 149 (A) CF3 Cl OCH3 H OCH3 H 74 150 (A) CF3 Cl H OCH3 OCH3 OCH3 76 151 (A) CF3 Cl H H N(CH3)2 H 67 152 (A) H Cl H H OCH3 H 86 153 (B) CF3 Cl - - - - 57 154 (B) H CF3 - - - - 61 155 (B) H CH3 - - - - 64 -Pathway B: Synthesis of N-phenylaminocarbonyl serinol monoester derivatives (156–158) For the preparation of monoester derivatives 156–158, the urea intermediates 128 and 129 was previously prepared with the same procedure described above. A selective O-acylation reaction of serinol ureas with the corresponding acyl chloride (mono, di and trimethoxy substituted) in DCM and 68 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES infection in a dose-dependent manner (Figure 23) showing IC50 values ranging from 2.05 μM to 9.74 μM (Table 17). Figure 23. Dose-dependent activity of representative compounds in a plaque assay. For all panels, the DMSO control is a positive control with cells infected at the same MOI but in the absence of drugs. The results represent means ± SD of triplicate samples from three independent experiments. IC50 values for cidofovir from previously reported studies [106] and from our methodology were significantly higher than those shown by our compounds, as it can be observed in the Table 17. Among selected active diesters with p-CF3 phenyl urea (131–133), compound 133 (pmethoxybenzoyl derivative) showed the lowest IC50 value (2.63 µM), whereas the presence of methyl group in para (131) or orto (132) position reduced the activity (5.35 µM and 4.53 µM respectively). In the case of compound 132, if an additional substituent was introduced on the phenyl urea (3-CF34-Cl, analogue 145), an increased inhibition was observed (2.82 µM). Compound 146 (4- 69 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES methoxybenzoyl derivative) with the same urea function, showed IC50 value of 8.96 µM, whereas the trimetoxybenzoyl analogue (150) showed better inhibitory activity (3.67 µM). Among the small set of active carbamate derivatives, 163 reached the best results with an IC50 value of 2.05 µM. Table 17. IC50, CC50, SI and virus yield reduction values for selected compounds compared to drug cidofovir. Comp IC50 (µM)a CC50 (µM) Selectivity Index (SI)b Yield reduction (fold-reduction)c 131 5.35 ± 0.66 93.24 ± 7.7 17.42 7.23 ± 5.38 132 4.53 ± 0,64 200.00 ± 33.87 44.15 92.28 ± 33.77 133 2.63 ± 0.26 11.73 ± 0.26 4.46 - 145 2.82 ± 0.31 25.10 ± 0.16 8.90 42.83 ± 15.68 146 8.96 ± 1.12 11.24 ± 4.48 1.25 - 150 3.67 ± 1.46 63.73 ± 0.50 17.37 1.22 ± 0.63 160 3.76 ± 0.23 19.8 ± 0.16 5.27 - 162 7.05 ± 2.65 14.4 ± 0.63 2.04 - 70 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES Comp IC50 (µM)a CC50 (µM) Selectivity Index (SI)b Yield reduction (fold-reduction)c 163 2.05 ± 0.02 11.56 ± 4.30 5.64 - 165 7.78 ± 2.02 18.4 ± 0.60 2.37 - 168 9.74 ± 0.90 41.16 ± 3.17 4.23 - Cidofovird 24.06 ± 5.9 50.6 ± 9.8 7.5 82.5 ± 21.4 aInhibitory concentration 50 at low MOI in a plaque assay. bSelectivity Index value was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) in a plaque assay for each compound. cFold-reduction in virus yield as the ratio of particles produced in the presence of DMSO divided by the yield in the presence of each of compounds (50 µM). The results represent means ± SD of triplicate samples from three independent experiments d Data of cidofovir as positive clinical drug candidate. Compounds with selectivity index SI >10 were selected for further evaluation to obtain some knowledge regarding their potential mechanism of action. Only compounds 131, 132, 145 and 150 showed a CC50 value at least 10-times over their IC50, with selectivity indexes ranging from 8.9 to 44.15 (Table 17) The anti-HAdV potency were assessed using a virus burst assay, measuring their efficacy in blocking the production of new HAdV particles. The presence of these compounds was associated with reductions in virus yield (from 1.22-fold tu 92.3-fold), similar to cidofovir (82.5-fold). (Table 17). 4.2.3. Insights into the antiviral mechanism of action Impact on HAdV entry To explore if selected compounds interfere with some steps of HAdV entry and HAdV DNA transport into the nucleus, we carried out a nuclear association assay to quantify the HAdV genome accessibility to the host nucleus. If the mechanism of action of these molecules was directed to HAdV entry this would be reflected in the amount of HAdV genomes that reach the nucleus. As depicted in Figure 24, the treatment with selected compounds did not show a significant difference in the number of HAdV genomes into the nucleus compared with those treated with DMSO. This study suggests that any of selected compounds interfered with the entry phase of the HAdV viral particles. 71 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES Figure 24. Effect of the selected compounds on nuclear association of HAdV5 genomes. Results are expressed as the mean ± SD of duplicate assays. Impact on HAdV DNA replication The DNA copy number of the cellular housekeeping gene GAPDH in both the nucleus and the cytoplasm were also measured as a control for the purity of nuclear isolation, indicating that we were specifically measuring the HAdV genomes that reached the nuclear membrane. This study confirms that any of selected compounds affected the early steps that span the entry phase of the HAdV viral particles. Since the mechanism of action of these four compounds seems to be related with early steps after HAdV entry into the nucleus we next evaluate the capacity of these compounds to block the HAdV entry or the DNA replication process. A real-time PCR was carried out to evaluate the HAdV DNA replication efficiency in the presence of these compounds, in a single round of infection for 24 h. Compound 132 blocked 99.2% of the synthesis of new HAdV DNA copies, while compounds 131 and 145 showed slight lower inhibitions, 95.1% and 92.1%, respectively. Compound 150 also showed a significant inhibition of HAdV DNA replication (73.5%), even if less then the other ones (Figure 25A). 72 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES Figure 25. Effect of the selected compounds on HAdV DNA replication. A) HAdV copies number; B) Expression of the E1A gene. Results are expressed as the relative copy number of HAdV DNA and E1A mRNA normalized to GAPDH copy number, and they are presented as the mean ± SD of duplicate assays. * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. We also quantified the mRNA copy number of the E1A gene using quantitative reverse transcription (RT-PCR). As shown in Figure 25B, 131, 132 and150 significatly blocked the expression of the E1A gene in a 6 h assay. Compound 145 did not show any decrease in the expression of the E1A gene compared with a control treated with DMSO. The ability of compounds 131, 132 and150 to interfere HAdV early gene transcription may be the cause of the inhibition of the DNA replication showed by these compounds. On the other hand, since any inhibition of the early gene transcription was observed 73 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES by compound 145, this compound could interact with viral proteins essential for HAdV DNA replication, including the HAdV DNA polymerase, the precursor of the terminal protein (pTP), or the DNA-binding protein (DBP). Further studies will be needed to clarify their specific mechanism for antiviral activity. 74 CHAPTER 4: O-ACYL-N-PHENYLAMINOCARBONYL SERINOL DERIVATIVES 75 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES CHAPTER 5 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES 5.1 Chemistry 5.1.1 Design Due to the preliminary and promising results demonstrated by some serinol-derived aromatic diesters in the inhibition of HAdV infection, our interest was focused in the replacement of serinol scaffold (2-amino-1,3-propanediol) with its regioisomer, 3-amino-1,2-propanediol (Figure 26) to identify the largest number of lead compounds for further optimization processes. This skeleton has been found in several antiviral acyclic nucleoside and non-nucleoside analogues. CDV (3, Figure 26) can be considered as constituted by an aminopropanediol central core, with a phosphonate group on the secondary alcohol and a cytosine base at N-3. Other acyclic nucleoside phosphonate analogues with aminoalcohol skeleton have been discovered as potent and selective inhibitors of herpesvirus replication [107]. Figure 26. Design of new 3-amino-1,2-propanediol derivatives. 76 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES Among non-nucleoside compounds, a series of novel aminopropanediol-derived small molecules have been described as inhibitors of human immunodeficiency virus 1 (HIV-1). In these molecules the propyl chain linked the sulphonamide function with an heterocyclic moiety (169, Figure 26) [108]. Also in this case, we designed a set of new compounds preserving the urea function as in many antiviral agents [76,84], including our piperazine and serinol derivatives (112, 150, Figure 26). Firstly, keeping in mind the antiviral effect of some serinol-derived aromatic diesters (IC50 from 2.05 μM to 9.74 μM) and other described compounds with acyl moieties in their structures (24, 25, Figure 26) [78,79,81], a set of diester derivatives from 3-amino-1,2-propanediol were designed in order to explore the effect of this functionality (Figures 26 and 27). Regarding the aromatic acyl moiety, the same structural variability was examined on the new scaffold: methyl and methoxy as representative electron releasing substituents due to their presence in most active serinol diesters (131–133, 145, 146, 150); CN and NO2 were selected as electron-withdrawing groups, with the addition of CF3 (A, Figure 27). The next step of our design was directed to a small set of monoester derivatives in order to evaluate the presence of a free hydroxyl group (primary or secondary, as in the case of cidofovir). Variously substituted aromatic acyl function was located at the position 1 or 2 of the three-carbon chain, preserving the urea function at N-3 (B, Figure 27). At last, we have explored the effect of 1,2,3-triazole ring into the structure; this interesting heterocycle is associated with a wide range of biological targets due to its ability to establish hydrogen-bonding and dipole interactions [109]. Furthermore, 1,2,3-triazole resulted stable in acidic and basic hydrolytic conditions as well as in oxidative and reductive ones, demonstrating a suitable resistance to metabolic degradation [110]. Many compounds with antiviral properties presented a triazole moiety in their structure; the drug ribavirin (2, Figure 26) and other synthetic compounds identified as inhibitors of influenza A virus were decorated with a 1,2,4-triazole ring (170, Figure 26) [111]. In other instances, the triazole ring represented a passive linker between the pharmacophore and other portions of the molecule. The antiviral set of 3-hydroxyquinazolinedione derivatives described by Kang et al consisted of a 1,2,3-triazole connecting units between the quinazoline core and a substituted aromatic moiety (28, 29, Figure 26) [82]. Other studies on compounds with 1,2,3-triazole nucleus bearing a phosphate group have been reported [112], such as compound 171 (Figure 26) which demonstrated moderate activity against herpes simplex viruses [113]. In our subset of aminopropanediol-derived 1,2,3-triazoles we introduced several functions at position 4 of the heterocyclic nucleus in order to verify their impact on the antiviral activity. In addition to a phosphonate group, other polar and non- 77 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES polar functionalities were evaluated such as a short alcohol chain, aromatic aldehyde or acid and substituted phenyl rings (C, Figure 27). Figure 27. General structures of new designed diester, monoester and 1,2,3-triazole derivatives of 3amino-1,3-propanediol. 5.1.2 Synthesis For the preparation of new set of diester, monoester and triazole derivatives several synthetic strategies consisting of two or more reaction steps were employed starting from 3-amino-1,2propanediol. 84 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES (232). Once the p-Cl substituted urea derivative 223 has been prepared, an oxidation reaction of olefin, using meta-chloroperbenzoyc acid (mCPBA) in dry DCM, furnished the corresponding oxirane 234 (Scheme 10). In the next step the acid-catalysed ring opening reaction using NH4Cl and sodium azide under EtOH reflux, gave the azide derivatives 235. Finally, the synthesis of 1,2,3triazole ring (1,4 adduct) was performed through a click chemistry approach, the copper(I)-catalysed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. The reaction proceeded using the appropriate terminal alkyne, CuSO4 as a source of pre-catalyst CuII and sodium ascorbate as reducing agent, in t-BuOH-H2O at room temperature. This reaction represents the most used “click” reaction to obtain 1,2,3 triazole, due to its reliability, specificity, and biocompatibility [114,115]. Compounds 236-241 were characterised by NMR Spectroscopy (Table 22), Mass Spectrometry and melting points determination Scheme 10. Synthetic route for the preparation of N-phenylaminocarbonyl-1,2,3-triazole derivatives 236–241. The mechanism proposed by Sharpless and co-workers for the CuAAC is illustrated in the Figure 28. The reaction requires copper at the oxidation state 1; the pre-catalyst could be a CuII salt such as CuSO4 and it needs the presence of a reducing agent (sodium ascorbate), or a CuI compound (CuBr/CuOAc) with a base and a reducing agent to avoid the oxidation to CuII. It started with the formation of a copper (I) acetylide and subsequent coordination of substituted azide to copper (I) at the alkylated nitrogen atom and the formation of a ternary complex (azide-alkyne-copper(I), A, step 1). The intermediate A involved into a six-membered metallacycle, in wich the copper was oxidized to the state 3, and provided the first covalent C-N bond (B, step 2). The reductive ring contraction 85 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES afforded the triazole nucleus with the contemporary reduction of copper (III) to copper (I) (C, step 3). Finally, the triazolide captures a proton from an alkyne molecule to ultimate its formation [116]. Figure 28. The mechanism of CuAAC proposed by Sharpless and co-workers. -Pathway C 2: Synthesis of 1,2,3-triazole derivatives at secondary position (245–250) For the introduction of the triazole ring at secondary position more steps are required and depicted in Scheme 11. From the urea derivative (p-Cl, 137) the first step involves the selective protection of primary alcohol with the benzoyl group (212). The acylated product 212 was mesyled in THF (242) for the subsequent nucleophilic substitution with sodium azide (in DMF at 85 ºC), furnishing the introduction of the azide group in position 2 (243, Scheme 11). The click chemistry reaction between azide derivative 243 and the appropriate substituted alkyne, in the same conditions described above, afforded final 1,2,3-triazole derivatives 245–250, that were characterised by NMR Spectroscopy (Table 21), Mass Spectrometry and melting points determination. 86 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES Scheme 11. Synthetic route for the preparation of N-phenylaminocarbonyl 1,2,3-triazol derivatives 245–250. Table 21. 1,2,3-Triazole derivatives and representative resonance assignments (1H NMR and 13C NMR). Comp R Yield (%) 1H NMRa (ppm) 1C NMRc (ppm) NHAr CH triaz OH C=O CH triaz CH prop 236 (A)b 79 - 7.95 - 158.2 128.3 70.7 245 (B)b 64 - 8.02 - 158.2 128.4 70.6 237 (A) 82 8.03 7.79 5.10 155.2 130.6 68.3 246 (B) 69 8.10 7.93-7.87 5.10 154.9 128.4 69.8 238 (A) 81 8.78 8.17 5.46 155.2 128.4 65.1 247 (B) 72 8.71 8.18 5.16 155.5 128.9 65.7 87 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES Comp R Yield (%) 1H NMRa (ppm) 1C NMRc (ppm) NHAr CH triaz OH C=O CH triaz CH prop 239 (A) 60 8.80 8.52 5.47 155.2 128.5 68.7 248 (B) 84 8.70 8.65 5.21 155.0 128.4 63.1 240 (A) 71 8.80 8.41 5.47 155.2 128.4 68.7 249 (B) 64 8.70 8.51 5.22 155.2 128.3 68.7 241 (A) 62 8.87 7.83-7.78 5.22 155.7 128.9 69.1 250 (B) 60 8.82 8.62 5.13 155.7 128.9 69.1 a500 MHz, DMSO-d6 b500 MHz, MeOD4 c125MHz, DMSO-d6 The formation of 1,4-adduct in the click chemistry reaction was confirmed by 2D NMR techniques HSQC and HMBC. -Synthesis of terminal akynes used for the click chemistry reaction (253, 254) For the synthesis of 1,2,3-triazole derivatives (1,4 adduct), six different terminal alkynes were employed but only four of them were commercially available (propargyl alcohol, 4-ethynylanisole, 1-ethynyl-4-fluorobenzene and 2-ethynylbenzaldehyde). With the aim to introduce an aromatic acid function, the ring-opening of phthalic anhydride were performed by an aminolysis reaction with propargyl amine, in THF at room temperature (253, Scheme 12). For the preparation of alkynyl phosphonate 254 the propargyl alcohol reacted with the diethyl(tosyloxy)methyl phosphonate in basic condition of NaH at 25 ºC. Both compounds were characterised by NMR Spectroscopy (experimental section 6.1.4). 88 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES Scheme 12. Synthesis of terminal alkynes 253 and 254. 5.2 Biological evaluation As for previous synthesized derivatives (sections 3.2 and 4.2), new compounds were submitted to biological assays. 5.2.1. In vitro antiviral activity and effect on cellular viability From a structure-activity relationship point of view, similar results to previous serinol-derived diacylated compounds were obtained by new set of diester derivatives of 3-amino-1,2-propanediol. The impact of electron-withdrawing substituents of the aromatic acyl moiety (CN, NO2, CF3) was generally detrimental for the antiviral activity. In fact, the plaque formation inhibition range were from 0% to 25.83 %, with the exception of compounds 193 (p-NO2 with p-CH3 substituted phenyl urea) and 200 (p-CN with CF3 substituted phenyl urea), which showed a moderate activity (44.65% and 40.41% respectively). Conversely, electron donor groups (methyl and methoxy) achieved hiher levels of inhibition (46.69–81.29%). However, p-CH3 derivatives (181, 189 and 197) gave low inhibtion values (Table 23). Specifically, compounds with a methyl group in orto position on the aromatic ester with p-Cl and p-CH3 phenyl urea (182 and 190), demonstrated significant antiviral activity (75.08% and 60.70% respectively). The dimethoxybenzoyl derivatives (187, 195 and 203) gave similar percentage of plaque formation inhibition (29.70%, 47.01%, 46.69% respectively), regardless of the type of substituent on the phenylaminocarbonyl function. The introduction of an additional methoxy group (188, 196 and 204, trimetoxybenzoyl derivatives) provided an improved antiviral activity, inhibiting plaque formation from 57.85% to 81.29% (Table 23). Compound 196 89 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES resulted the most active diester derivative (81.29% of plaque formation inhibition), demonstrating the relevance of methoxy groups. On the other hand, the alternative presence of trimethoxycinnamoyl function 210 and 211 abolished the activity (0% plaque formation). Considering the in vitro inhibition demonstrated by compounds 182 and 196, their analogues with different substituted phenyl ureas were generated, in order to attempt an improvement of the antiviral activity. For compound 182 (o-CH3 benzoyl derivative with p-Cl phenyl urea), the effect of an additional electron-withdrawing substituent (2-Cl-5-CF3, 205) was evaluated and resulted in a totally loss of the activity (0% in plaque assay). With regard to the compound 196 (trimethoxybenzoyl derivative with p-CH3 substituted phenyl urea function), the alternative presence of the methyl group in orto position (206, 20.29 %) decreased the activity as well as a disubstitution 2Cl-5-CF3 (208, 0% respectively). On the contrary, compound with the trimethoxy benzoyl group in combination with trimethoxy phenyl urea (209) resulted less active then 196 (44.11 % vs 81.29), while 207 (p-OCH3 phenyl urea) resulted in an almost complete inhibition (99%). These results suggest the importance of an electron donor group in para position of the phenyl urea. Table 22. Table 22. Inhibition of HAdV infection in the plaque assay for diester, monoester, monocarbamate and triazole derivatives (180–211, 212–215, 225–231, 236–241, 245–250). Comp. % of plaqueformation inhibitiona CC50b Comp. % of plaqueformation inhibitiona CC50b 180 (A) 0.00 ± 0.00 - 208 (A) 0.00 ± 0.00 - 181 (A) 0.00 ± 0.00 - 209 (A) 44.11 ± 1.70 - 182 (A) 75.08 ± 15.71 28.70 ± 3.10 210 (A) 0.00 ± 0.00 - 183 (A) 0.00 ± 0.00 - 211 (A) 0.00 ± 0.00 - 184 (A) 0.00 ± 0.00 - 212 (B) 0.00±0.00 - 185 (A) 0.00 ± 0.00 - 213 (B) 15.2 ± 1.39 - 186 (A) 26.47 ± 8.32 - 214 (B) 25.99 ± 3.65 - 187 (A) 49.70 ± 6.77 - 215 (B) 5.70 ± 8.06 - 188 (A) 60.22 ± 8.43 97.25 ± 16.56 225 (C) 20.92 ± 10.17 - 189 (A) 31.00 ± 10.43 - 226 (C) 100 >200 90 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES 190 (A) 60.70 ± 16.65 51.33 ± 19.04 227 (C) 0.00 ± 0.00 - 191 (A) 53.07 ± 16.18 - 228 (C) 48.03 ± 6.79 - 192 (A) 4.43 ± 6.26 - 229 (C) 40.21 ± 7.11 - 193 (A) 44.65 ± 18.79 - 230 (C) 0.00 ± 0.00 - 194 (A) 21.79 ± 30.81 - 231 (C)d 19.57 ± 2.84 - 195 (A) 47.01 ± 8.79 - 236 (D) 10.77 ± 15.23 - 196 (A) 81.29 ± 1.10 175.16 ± 2.97 237 (D) 4.78 ± 74.93 - 197 (A) 0.00 ± 0.00 - 238 (D) 33.33 ± 6.10 - 198 (A) 7.30 ± 11.04 - 239 (D) 21.54 ± 26.11 - 199 (A) 18.51 ± 1.92 - 240 (D) 0.00 ± 0.00 - 200 (A) 40.41 ± 13.31 - 241 (D) 0.00 ± 0.00 - 201 (A) 2.12 ± 3.00 - 245 (E) 5.80 ± 15.48 - 202 (A) 26.83 ± 24.33 - 246 (E) 0.00 ± 0.00 - 203 (A) 46.69 ± 3.36 - 247 (E) 0.00 ± 0.00 - 204 (A) 57.85 ± 22.21 - 248 (E) 37.45 ± 0.56 - 205 (A) 0.00 ± 0.00 - 249 (E) 21.57 ± 4.21 - 206 (A) 20.29 ± 2.82 - 250 (E) 12.63 ± 7.36 - 207 (A) 99.86 ± 0.13 136.62 ± 6.54 Cidofovirc 3.51 ± 4.97 50.6 ± 9.8 a Percentage of control HAdV5-GFP inhibition in a plaque assay at 10 μM using the 293β5 cell line b Cytotoxic concentration 50%. The results represent means ± SD of triplicate samples from three independent experiments b Data of cidofovir as positive clinical drug candidate. d monocarbamate To evaluate the presence of a free hydroxyl group on the antiviral activity a small collection of monoester derivatives of 3-amino-1,2-propanediol, in both position 1 and 2, were prepared. Two representative electron-withdrwing and donating groups were selected for the aromatic acyl moiety (CH3 and CF3), with the p-Cl phenyl urea (212–215). The monoester derivatives on the primary alcohol were not well tolerated, giving percentages of plaque formation inhibition from 0% to 25.99%, regardless of the electronic properties of the aromatic ester function (Table 23). When the acyl moiety is located on the secondary alcohol, the presence of a methyl group (225) furnished similar results to its analogue 213 (20.92% vs 15.2% respectively), while the trifluoromethyl group in para position (226) reached a complete plaque formation inhibition (100%). Other electronattracting groups such as NO2 (227) did not give active compounds (0%), while the presence of a trimethoxy group (228) achived a moderate activity (48.03%). For compound 226, which demonstrated the highest level of inhibition, the analogue with p-CH3 phenyl urea (229) were prepared but it was less active than 226 (40.21%). In the similar way, the replacement of the ester function with a carbamate one (231) decreased the antiviral activity (19.57%). 91 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES Finally, the biological evaluation of the synthesized of 1,2,3-triazole derivatives (at both positions) did not offered a suitable inhibitory activity (Table 23). Among triazole derivatives at position 1, compound 238 (phosphonate group) showed a moderate activity, with 33.33% of plaque formation inhibition, and compound 239 (p-F phenyl ring) with 21.54% of inhibition. Its analogue from the serie of triazole derivatives at position 2 (compound 248) showed higher level of inhibition (37.45%). The p-OMe phenyl ring triazole derivative 249 gave 21.57% of inhibition. In spite of not displaying high levels of inhibion, four of them offered moderate activity, and become interesting compounds for further optimization process, searching for new scaffolds. The effect on cellular viability in A549 cell line was examined for those compounds with percentage of inhibition in the plaque assay >60% (six compounds) in order to evaluate their safety profile determining the 50% cytotoxic concentration (CC50) (Table 22). 5.2.2. Determination of IC50 values At the time to present this manuscript the selected six compounds are being submitted to further biological assays. Firstly, the determination of their half maximal inhibitory concentration, in order to calculate the SI and to chose compounds with SI > 10. The data available showed that these compounds dose-dependently reduced HAdV5 infection (Figure 29) showing IC50 values ranging from 2.47 μM to 4.19 μM and SI > 10 (Table 23). Also in this instance, new compounds resulted to be more active than cidofovir. 92 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES Figure 29. Dose-dependent activity of representative compounds in a plaque assay. For all panels, the DMSO control is a positive control with cells infected at the same MOI but in the absence of drugs. The results represent means ± SD of triplicate samples from three independent experiments. Table 23. IC50, CC50, SI values for selected compounds compared to drug cidofovir. Comp IC50 (µM)a CC50 (µM)b Selectivity Index (SI)c 182 2.47 ± 0.07 28.70 ± 3.10 11.63 188 4.19 ± 2.59 97.25 ± 16.56 23.21 93 CHAPTER 5: 3-PHENYLAMINOCARBONYL-1,2-PROPANEDIOL DERIVATIVES 190 NEd 51.33 ± 19.04 NE 196 2.82 ± 0.31 175.16 ± 2.97 62.68 207 NE 136.62 ± 6.54 - 226 3.02 ± 0.08 >200 66.23 Cidofovir - 24.06 ± 5.9 50.6 ± 9.8 7.5 aInhibitory concentration 50 at low MOI in a plaque assay. bCytotoxic concentration 50. cSelectivity Index value was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) in a plaque assay for each compound. d Not yet evaluated. Secondly, biological assays to explore their mechanism of action are being carried out as for previously studied libraries. The main goal it is to contribute to improve the structural diversity of this novel class of antiadenovirus agents. One consideration should be metioned. In spite of the fact that these selected 3-amino-1,3-propanediol derivatives did not show percentages of plaque inhibition as high as those from our previous scaffolds (piperazine and serinol derivatives), they are an important contribution in the development of new antiadenovirus agents. They displayed promising IC50 for lead compounds (2.47-4.19 μM), they could inhibit virus replication through different ways to our other compounds (data from studies wil clarify this aspect) so they can be considered promising initial point to obtain optimized analogues with increased biological properties. 100 CHAPTER 6: EXPERIMENTAL SECTION 383.1500. Anal. Calcd C18H24N4O2S: C, 59.97; H, 6.71; N, 15.54; S, 8.90. Found: C, 60.03; H, 6.81; N, 15.20; S, 9.15. 4-tert-Butoxycarbonyl-1-[(4-fluorophenyl)aminothiocarbonyl]-2-methylpiperazine (52). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (3:1) as eluent (209 mg, 79% yield), mp 74–76 ºC. MS (FAB): m/z 376 (100%) M+Na+. 1H NMR (500 MHz, CDCl3)  7.22–7.14 (m, 2H), 7.10 (s, 1H), 7.06–7.02 (m, 2H), 5.14–4.75 (m, 1H), 4.51– 3.69 (m, 3H), 3.41–3.32 (m, 1H), 3.24–3.16 (m, 1H), 3.14–2.75 (m, 1H), 1.47 (s, 9H), 1.28 (d, J = 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3)  183.6, 155.0, 135.9, 126.6, 115,9. 80.4, 52.3, 43.6, 28.4, 15.1. HRMS (m/z): calcd. for C17H24FN3O2SNa 376.1465 M+Na+; found 376.1456. Anal. Calcd C17H24FN3O2S: C, 57.77; H, 6.84; N, 11.89; S, 9.07. Found: C, 58.00; H, 6.82; N, 11.68; S, 9.48. 4-tert-Butoxycarbonyl-2-methyl-1-[(4-trifluoromethylphenyl)aminothiocarbonyl]piperazine (53). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (4:1) as eluent (220 mg, 73% yield), mp 141–143 ºC. MS (FAB): m/z 426 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.52 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 8.3 Hz, 2H), 5.11 (br s, 1H), 4.45–4.38 (m, 1H), 3.98–3.84 (m, 1H), 3.82–3.75 (m, 1H), 3.25–3.12 (m, 1H), 3.09– 2.93 (m, 1H), 1.45 (s, 9H), 1,20 (d, J = 6.7 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)  181.7, 154.2, 144.9, 125.1, 125.0, 124.5, 79.2, 51.4, 42.7, 28.0, 14.8. HRMS (m/z): calcd for C18H24F3N3O2SNa 426.1434 M+Na+; found 426.1428. Anal. Calcd C18H24F3N3O2S: C, 53.58; H, 6.00; N, 10.41. Found: C, 53.12; H, 6.41; N, 10.31. 4-tert-Butoxycarbonyl-1-[(4-methoxyphenyl)aminothiocarbonyl]-2-methylpiperazine (54). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (3:1) as eluent (205 mg, 75% yield), mp 127–129 ºC. MS (FAB): m/z 388 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.15 (s, 1H), 7.16 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 5.05 (br s, 1H), 4.48–4.38 (m, 1H), 3.95–3.80 (m, 1H), 3.76 (s, 3H), 3.28–2.92 (m, 3H), 1.44 (s, 9H), 1,16 (d, J = 6.8 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)  182.0, 156.6, 154.3, 133.8, 127.6, 113.2, 79.1, 55.2, 50.8, 42.1, 28.0, 14.8. HRMS (m/z): calcd for C18H27FN3O3SNa 388.1665 M+Na+; found 388.1669. 4-tert-Butoxycarbonyl-2-methyl-1-[(4-methylphenyl)aminothiocarbonyl]piperazine (55). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (6:1) as eluent (149 mg, 75% yield), mp 155–157 ºC. MS (FAB): m/z 372 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.18 (s, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 5.06 (br s, 1H), 4.46–4.37 (m, 1H), 3.95–3.81 (m, 1H), 3.79–3.72 (m, 1H), 3.21–2.88 (m, 2H), 2.29 (s, 3H), 1.44 101 CHAPTER 6: EXPERIMENTAL SECTION (s, 9H), 1,16 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)  181.9, 154.2, 138.4, 133.7, 128.5, 125.8, 79.1, 50.9, 42.2, 28.0, 20.5, 15.1. HRMS (m/z): calcd for C18H27N3O2SNa 372.1716 M+Na+; found 372.1710. 1-[{3,5-Bis(trifluoromethyl)phenyl}aminothiocarbonyl]-4-tert-butoxycarbonyl-2methylpiperazine (56). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (7:1) as eluent (251 mg, 71% yield), mp 134–136 ºC. MS (FAB): m/z 494 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.72 (s, 1H), 8.09 (s, 2H), 7.78 (s, 1H), 5.10 (br s, 1H), 4.55–4.42 (m, 1H), 4.04–3.74 (m, 2H), 3.26–2.91 (s, 2H), 1.45 (s, 9H), 1,21 (d, J = 6.7 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)  181.0, 154.2, 143.1, 130.1, 129.8, 129.5, 129.3, 126.5, 124.8, 124.4, 122.2, 116.7, 79.2, 51.5, 42.7, 28.0, 14.9. HRMS (m/z): calcd for C19H23F6N3O2SNa 494.1307 M+Na+; found 494.1300. Anal. Calcd C19H23F6N3O2S: C, 48.40; H, 4.92; N, 8.91. Found: C, 48.56; H, 4.64; N, 8.88. 4-(3,3-Dimethylbutanoyl)-2-methyl-1-[(4-nitrophenyl)aminothiocarbonyl]piperazine (57). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (193 mg, 68% yield), mp 195–198 ºC. MS (FAB): m/z 401 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.76 (s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 5.12 (br s, 1H), 4.48–4.43 (m, 1H), 4.27 (d, J = 13.5 Hz, 1H), 4.08–3.88 (m, 1H), 3.02–2.85 (m, 1H), 2.42–2.08 (m, 2H), 1,22, 1,15 (2d, J = 6.7 Hz, 3H), 1.03, 1.02 (2s, 9H). 13C NMR (125 MHz, DMSO-d6)  181.5, 170.4, 170.3, 147.8, 142.2, 123.9, 123.0, 52.1, 51.8, 49.1, 45.1, 44.4, 43.6, 43.4, 43.1, 40.7, 31.0, 30.9, 29.7, 15.1, 14.7. HRMS (m/z): calcd for C18H26N4O3SNa 401.1618 M+Na+; found 401.1615. Anal. Calcd C18H26N4O3S: C, 57.12; H, 6.92; N, 14.80. Found: C, 57.47; H, 7.07; N, 14.56. 1-[(4-Chlorophenyl)aminothiocarbonyl]-4-(3,3-dimethylbutanoyl)-2-methylpiperazine (58). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (193 mg, 70% yield), mp 77–80 ºC. MS (FAB): m/z 390 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.76 (s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 5.12 (br s, 1H), 4.48–4.43 (m, 1H), 4.27 (d, J = 13.5 Hz, 1H), 4.08–3.88 (m, 1H), 3.02–2.85 (m, 1H), 2.42– 2.08 (m, 2H), 1,22, 1,15 (2d, J = 6.7 Hz, 3H), 1.03, 1.02 (2s, 9H). 13C NMR (125 MHz, DMSO-d6)  181.6, 181.4, 170.4, 170.2, 128.4, 127.8, 127.2, 51.5, 51.2, 49.1, 45.6, 44.4, 43.6, 43.2, 43.0, 42.6, 40.7, 31.0, 30.9, 29.7, 15.1, 14.7. HRMS (m/z): calcd for C18H26ClN3OSNa 390.1377 M+Na+; found 390.1373. Anal. Calcd C18H26ClN3OS: C, 58.76; H, 7.12; N, 11.42. Found: C, 58.45; H, 7.03; N, 11.12. 102 CHAPTER 6: EXPERIMENTAL SECTION 1-[(4-Cyanophenyl)aminothiocarbonyl]-4-(3,3-dimethylbutanoyl)-2-methylpiperazine (59). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (177 mg, 66% yield), mp 158–160 ºC. MS (FAB): m/z 381 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.59 (s, 1H), 7.76–7.72 (m, 2H), 7.58–7.53 (m, 2H), 5.10 (br s, 1H), 4.46–4.33 (m, 1H), 4.26 (d, J = 13.3 Hz, 1H), 4.04–3.87 (m, 1H), 3.02–2.87 (m, 1H), 2.42–2.08 (m, 2H), 1,22, 1,15 (2d, J = 6.2 Hz, 3H), 1.03, 1.02 (2s, 9H). 13C NMR (125 MHz, DMSO-d6)  , 170.0, 145.6, 132.2, 123.9, 119.1, 105.3, 51.9, 51.6, 49.1, 45.1, 44.6, 43.6, 43.4, 42.9, 40.7, 31.0, 30.9, 29.7, 15.2, 14.8. HRMS (m/z): calcd for C19H26N4OSNa 381.1720 M+Na+; found 381.1714. Anal. Calcd C19H26N4OS: C, 63.65; H, 7.31; N, 15.63. Found: C, 63.28; H, 7.35; N, 15.24. 1-[(4-Fluorophenyl)aminothiocarbonyl]-4-(3,3-dimethylbutanoyl)-2-methylpiperazine (60). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (158 mg, 60% yield), mp 76–80 ºC. MS (FAB): m/z 374 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.26 (br s, 1H), 7.33–7.26 (m, 2H), 7.18–7.11 (m, 2H), 5.06 (br s, 1H), 4.51–4.38 (m, 1H), 4.22 (d, J = 13.5 Hz, 1H), 4.05–3.84 (m, 1H), 3.06–2.88 (m, 1H), 2.43–2.09 (m, 2H), 1,20, 1,14 (2d, J = 6.6 Hz, 3H), 1.04, 1.02 (2s, 9H). 13C NMR (125 MHz, DMSO-d6)  181.9, 170.4, 170.2, 160.3, 158.3, 137.3, 128.0, 127.9, 114.7, 114.5, 51.4, 51.1, 49.2, 45.1, 44.4, 43.6, 43.4, 43.0, 42.3, 40.7, 31.0, 30.9, 29.7, 15.2, 14.8. HRMS (m/z): calcd. for C18H26FN3OSNa 374.1673 M+Na+; found 374.1670. Anal. Calcd C18H26FN3OS: C, 61.51; H, 7.46; N, 11.96. Found: C, 61.30; H, 7.29; N, 11.69. 4-(3,3-Dimethylbutanoyl)-2-methyl-1-[(4-trifluoromethylphenyl)aminothiocarbonyl] piperazine (61). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (220 mg, 73% yield), mp 79–82 ºC. MS (FAB): m/z 424 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.53 (s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 5.12 (br s, 1H), 4.49–4.36 (m, 1H), 4.25 (d, J = 13.3 Hz, 1H), 4.05–3.87 (m, 1H), 3.05–2.86 (m, 1H), 2.45–2.08 (m, 2H), 1,22, 1,16 (2d, J = 6.7 Hz, 3H), 1.04, 1.03 (2s, 9H). 13C NMR (125 MHz, DMSO-d6)  181.6, 181.4, 170.4, 170.2, 144.9, 125.5, 125.0, 124.6, 124.5, 124.0, 123.8, 123.3, 51.8, 51.5, 49.1, 45.1, 44.4, 43.6, 43.4, 43.3, 42.8, 40.7, 31.0, 30.9, 29.7, 15.2, 14.8. HRMS (m/z): calcd for C19H26F3N3OSNa 424.1641 M+Na+; found 424.1631. Anal. Calcd C19H26F3N3OS: C, 56.84; H, 6.53; N, 10.47. Found: C, 56.86; H, 6.33; N, 10.12. 4-(3,3-Dimethylbutanoyl)-1-[(4-methoxyphenyl)aminothiocarbonyl]-2-methylpiperazine (62). The product was obtained as a solid and purified by column chromatography using hexane-ethyl 103 CHAPTER 6: EXPERIMENTAL SECTION acetate (1:1) as eluent (169 mg, 62% yield), mp 138–140 ºC. 1H NMR (500 MHz, DMSO-d6)  9.15 (s, 1H), 7.16 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 5.05 (br s, 1H), 4.53–4.34 (m, 1H), 4.20 (d, J = 12.9 Hz, 1H), 4.01–3.83 (m, 1H), 3.76 (s, 3H), 3.04–2.87 (m, 1H), 2.41–2.08 (m, 2H), 1,19, 1,13 (2d, J = 6.4 Hz, 3H), 1.03, 1.02 (2s, 9H). 13C NMR (125 MHz, DMSO-d6)  182.0, 181.9, 170.4, 170.2, 156.7, 133.8, 127.6, 113.2, 55.2, 51.2, 50.9, 49.2, 45.1, 44.4, 43.6, 43.4, 42.8, 42.2, 40.7, 31.0, 30.9, 29.7, 26.8, 15.2, 14.8. HRMS (m/z): calcd for C19H29N3O2SNa 386.1873 M+Na+; found 386.1870. Anal. Calcd C19H29N3O2S: C, 62.78; H, 8.04; N, 11.56. Found: C, 62.50; H, 7.81; N, 11.25. 4-(3,3-Dimethylbutanoyl)-2-methyl-1-[(4-methylphenyl)aminothiocarbonyl]piperazine (63). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (3:2) as eluent (197 mg, 76% yield), mp 136–138 ºC. 1H NMR (500 MHz, DMSO-d6)  9.19, 9.18 (ds, 1H), 7.16 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 5.06 (bs, 1H), 4.50–4.34 (m, 1H), 4.21 (d, J = 13.0 Hz, 1H), 4.02–3.82 (m, 1H), 3.04–2.87 (m, 1H), 2.29 (s, 3H), 2.41–2.10 (m, 2H), 1.19, 1.13 (2d, J = 6.4 Hz, 3H), 1.03, 1.02 (2s, 9H). 13C NMR (125 MHz, DMSO-d6)  181.8, 181.6, 170.4, 170.3, 138.3, 133.7, 128.5, 125.8, 51.3, 51.0, 49.2, 48.7, 45.1, 44.4, 43.6, 43.4, 42.9, 42.3, 40.7, 31.0, 30.9, 29.7, 26.8, 20.5, 15.1, 14.8. HRMS (m/z): calcd for C19H29N3OSNa 370.1924 M+Na+; found 370.1920. Anal. Calcd C19H29N3OS: C, 65.67; H, 8.41; N, 12.09. Found: C, 65.38; H, 8.25; N, 11.99. 1-[{3,5-Bis(trifluoromethyl)phenyl}aminothiocarbonyl]-4-(3,3-dimethylbutanoyl)-2-methyl piperazine (64). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (239 mg, 68% yield), mp 87–90 ºC. MS (FAB): m/z 492 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.71 (s, 1H), 8.10 (br s, 2H), 7.77 (br s, 1H), 5.12 (br s, 1H), 4.59–4.42 (m, 1H), 4.26 (d, J = 13.3 Hz, 1H), 4.08–3.87 (m, 1H), 3.07–2.92 (m, 1H), 2.45–2.11 (m, 2H), 1.24, 1.18 (2d, J = 6.4 Hz, 3H), 1.04, 1.03 (2s, 9H). 13C NMR (125 MHz, DMSO-d6)  181.0, 180.8, 170.4, 170.3, 143.1, 130.1, 129.9, 129.5, 129.3, 124.8, 124.4, 122.2, 116.7, 51.8, 51.6, 49.1, 45.0, 44.4, 43.6, 43.4, 43.2, 40.6, 31.0, 30.9, 29.7, 15.2, 14.8. HRMS (m/z): calcd for C20H25F6N3OSNa 492.1515 M+Na+; found 492.1500. Anal. Calcd C20H25F6N3OS: C, 51.17; H, 5.37; N, 8.95. Found: C, 51.10; H, 5.17; N, 8.83. 4-(2-Cyclohexylacetyl)-2-methyl-1-[(4-nitrophenyl)aminothiocarbonyl]piperazine (65). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:3) as eluent (230 mg, 76% yield), mp 87–90 ºC. MS (FAB): m/z 427 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.77 (s, 1H), 8.18 (d, J = 9.1 Hz, 2H), 7.61 (d, J = 9.1 Hz, 2H), 5.12 (br s, 104 CHAPTER 6: EXPERIMENTAL SECTION 1H), 4.46–4.34 (m, 1H), 4.30–4.20 (m, 1H), 4.08–3.81 (m, 1H), 3.03–2.85 (m, 1H), 2.37–2.14 (m, 2H), 1.79–1.58 (m, 6H), 1.31–0.90 (m, 9H). 13C NMR (125 MHz, DMSO-d6)  181.2, 181.1, 170.7, 147.8, 142.2, 123.9, 122.9, 59.7, 51.9, 51.8, 48.5, 44.5, 44.4, 43.7, 43.1, 34.4, 34.3, 32.6, 32.5, 25.9, 25.7, 15.1, 14.6. HRMS (m/z): calcd for C20H28N4O3SNa 427.1774 M+Na+; found 427.1768. Anal. Calcd C20H28N4O3S: C, 59.38; H, 6.98; N, 13.85. Found: C, 59.53; H, 7.09; N, 13.67. 1-[(4-Chlorophenyl)aminothiocarbonyl]-4-(2-cyclohexylacetyl)-2-methylpiperazine (66). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (3:2) as eluent (221 mg, 75% yield), mp 77–80 ºC. MS (FAB): m/z 416 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.32 (s, 1H), 7.38–7.31 (m, 4H), 5.09 (br s, 1H), 4.50–4.36 (m, 1H), 4.25– 4.16 (m, 1H), 3.95–3.78 (m, 1H), 3.05–2.85 (m, 1H), 2.36–2.15 (m, 2H), 1.81–1.56 (m, 6H), 1.30– 0.88 (m, 9H). 13C NMR (125 MHz, DMSO-d6)  181.7, 181.5, 170.7, 140.0, 128.4, 127.8, 127.1, 51.4, 51.2, 44.5, 44.4, 43.1, 42.4, 40.7, 34.4, 34.3, 32.6, 32.5, 25.8, 25.7, 15.1, 14.7. HRMS (m/z): calcd for C20H28ClN3OSNa 416.1534 M+Na+; found 416.1527. Anal. Calcd C20H28ClN3OS: C, 60.97; H, 7.16; N, 10.67. Found: C, 60.66; H, 6.97; N, 10.28. 1-[(4-Cyanophenyl)aminothiocarbonyl]-4-(2-cyclohexylacetyl)-2-methylpiperazine (67). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (210 mg, 73% yield), mp 84–87 ºC. MS (FAB): m/z 407 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.59 (s, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 5.11 (br s, 1H), 4.47–4.33 (m, 1H), 4.29–4.18 (m, 1H), 3.98–3.79 (m, 1H), 3.04–2.83 (m, 1H), 2.38–2.15 (m, 2H), 1.82–1.57 (m, 6H), 1.32–0.90 (m, 9H). 13C NMR (125 MHz, DMSO-d6)  181.3, 181.2, 170.7, 145.6, 132.2, 119.1, 105.2, 51.7, 51.6, 48.5, 44.5, 44.4, 43.5, 42.9, 40.7, 34.4, 34.3, 32.6, 32.5, 25.9, 25.7, 15.1, 14.6. HRMS (m/z): calcd for C21H28N4OSNa 407.1876 M+Na+; found 407.1866. Anal. Calcd C21H28N4OS: C, 65.59; H, 7.34; N, 14.57. Found: C, 65.52; H, 7.09; N, 14.15. 4-(2-Cyclohexylacetyl)-1-[(4-fluorophenyl)aminothiocarbonyl]-2-methylpiperazine (68). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (218 mg, 77% yield), mp 87–90 ºC. MS (FAB): m/z 400 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.23 (s, 1H), 7.33–7.26 (m, 2H), 7.18–7.10 (m, 2H), 5.06 (br s, 1H), 4.52– 4.37 (m, 1H), 4.25–4.13 (m, 1H), 3.97–3.75 (m, 1H), 3.06–2.85 (m, 1H), 2.40–2.11 (m, 2H), 1.83– 1.53 (m, 6H), 1.33–0.89 (m, 9H). 13C NMR (125 MHz, DMSO-d6)  181.9, 181.8, 170.7, 160.3, 158.3, 137.2, 128.0, 127.9, 114.7, 114.4, 51.3, 51.1, 48.5, 44.5, 44.4, 42.9, 42.2, 40.7, 34.4, 34.3, 32.7, 32.6, 32.5, 25.9, 25.7, 15.1, 14.7. HRMS (m/z): calcd for C20H28FN3OSNa 400.1829 M+Na+; 105 CHAPTER 6: EXPERIMENTAL SECTION found 400.1826. Anal. Calcd C20H28FN3OS: C, 63.63; H, 7.48; N, 11.13. Found: C, 63.38; H, 7.17; N, 10.77. 4-(2-Cyclohexylacetyl)-2-methyl-1-[(4-trifluoromethylphenyl)aminothiocarbonyl] piperazine (69). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (218 mg, 68% yield), mp 153–156 ºC. MS (FAB): m/z 450 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.53 (s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 5.11 (br s, 1H), 4.50–4.35 (m, 1H), 4.28–4.17 (m, 1H), 3.99–3.78 (m, 1H), 3.05–2.84 (m, 1H), 2.39– 2.14 (m, 2H), 1.81–1.56 (m, 6H), 1.34–0.89 (m, 9H). 13C NMR (125 MHz, DMSO-d6)  181.6, 181.5, 170.7, 144.9, 125.1, 125.0, 124.5, 124.0, 123.8, 123.5, 123.3, 51.6, 51.5, 48.5, 44.5, 44.4, 43.4 42.7, 40.7, 34.4, 34.3, 32.7, 32.6, 32.5, 25.9, 25.7, 15.1, 14.7. HRMS (m/z): calcd for C21H28F3N3OSNa 450.1797 M+Na+; found 450.1788. Anal. Calcd C21H28F3N3OS: C, 59.00; H, 6.60; N, 9.83. Found: C, 59.27; H, 6.35; N, 9.60. 4-(2-Cyclohexylacetyl)-1-[(4-methoxyphenyl)aminothiocarbonyl]-2-methylpiperazine (70). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (3:2) as eluent (204 mg, 70% yield), mp 65–68 ºC. MS (FAB): m/z 412 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.14 (s, 1H), 7.17 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 5.16–4.97 (m, 1H), 4.57–4.36 (m, 1H), 4.29–4.14 (m, 1H), 3.95–3.78 (m, 1H), 3.75 (s, 3H), 3.07–2.83 (m, 1H), 2.37–2.17 (m, 2H), 1.81–1.56 (m, 6H), 1.32–0.89 (m, 9H). 13C NMR (125 MHz, DMSO-d6)  181.9, 181.8, 170.6, 156.6, 133.8, 127.7, 113.2, 55.2, 51.1, 50.9, 48.5, 44.5, 44.4, 42.8, 42.2, 40.7, 34.4, 34.3, 32.7, 32.6, 32.5, 25.8, 25.7, 15.1, 14.7. HRMS (m/z): calcd for C21H31N3O2SNa 412.2029 M+Na+; found 412.2025. Anal. Calcd C21H31N3O2S: C, 64.75; H, 8.02; N, 10.79. Found: C, 64.65; H, 7.91; N, 10.61. 4-(2-Cyclohexylacetyl)-2-methyl-1-[(4-methylphenyl)aminothiocarbonyl]piperazine (71). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (3:2) as eluent (185 mg, 66% yield), mp 72–75 ºC. MS (FAB): m/z 396 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.19 (s, 1H), 7.19–7.08 (m, 4H), 5.18–4.97 (m, 1H), 4.55–4.33 (m, 1H), 4.28–4.09 (m, 1H), 3.98–3.72 (m, 1H), 3.06–2.82 (m, 1H), 2.30 (s, 3H), 2.26–2.14 (m, 2H), 1.82– 1.54 (m, 6H), 1.34–0.82 (m, 9H). 13C NMR (125 MHz, DMSO-d6)  181.9, 181.7, 170.7, 138.3, 133.7, 128.5, 125.8, 51.2, 51.1, 48.6, 44.5, 44.4, 42.9, 42.3, 40.8, 34.4, 34.3, 32.7, 32.6, 32.5, 25.8, 25.7, 20.5, 15.1, 14.7. HRMS (m/z): calcd for C21H31N3OSNa 396.2080 M+Na+; found 396.2075. Anal. Calcd C21H31N3OS: C, 67.52; H, 8.36; N, 11.25. Found: C, 67.24; H, 8.16; N, 10.95. 106 CHAPTER 6: EXPERIMENTAL SECTION 1-[{3,5-Bis(trifluoromethyl)phenyl}aminothiocarbonyl]-4-(2-cyclohexylacetyl)-2-methyl piperazine (72). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (290 mg, 78% yield), mp 156–159 ºC. MS (FAB): m/z 518 (90%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.71 (s, 1H), 8.09 (s, 2H), 7.77 (s, 1H), 5.12 (br s, 1H), 4.56–4.39 (m, 1H), 4.31–4.15 (m, 1H), 4.01–3.78 (m, 1H), 3.10–2.87 (m, 1H), 2.39–2.17 (m, 2H), 1.82–1.56 (m, 6H), 1.27–0.91 (m, 9H). 13C NMR (125 MHz, DMSO-d6)  181.0, 180.9, 170.7, 143.1, 130.1, 129.8, 129.5, 129.3, 126.5, 124.7 124.4, 122.2. 120.0, 116.7, 51.7, 51.6, 48.4, 44.4, 44.3, 43.3, 42.7, 40.7, 34.4, 34.3, 32.7, 32.6, 32.5, 25.9, 25.7, 15.1, 14.7. HRMS (m/z): calcd for C22H27F6N3OSNa 518.1671 M+Na+; found 518.1654. Anal. Calcd C22H27F6N3OS: C, 53.32; H, 5.49; N, 8.48. Found: C, 53.61; H, 5.34; N, 8.40. 2-Methyl-1-[(4-nitrophenyl)aminothiocarbonyl]-4-(2-phenylacetyl)piperazine (73). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:3) as eluent (170 mg, 57% yield), mp 81–84 ºC. MS (FAB): m/z 421 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.76, 9.74 (2s, 1H), 8.23–8.14 (m, 2H), 7.66–7.56 (m, 2H), 7.40–7.20 (m, 5H), 5.13 (br s, 1H), 4.47–4.32 (m, 1H), 4.29–4.18 (m, 1H), 4.08–3.91 (m, 1H), 3.89–3.70 (m, 2H), 3.26– 3.14 (m, 1H), 3.08–2.86 (m, 1H), 1.15–1.09 (m, 3H). 13C NMR (125 MHz, DMSO-d6)  181.2, 181.1, 169.7, 147.8, 142.2, 135.7, 135.6, 129.1, 128.9, 128.4, 128.3, 126.4, 123.9, 122.9, 51.9, 51.8, 48.7, 44.7, 43.4, 42.9, 40.9, 15.0, 14.4. HRMS (m/z): calcd for C20H22N4O3SNa 421.1305 M+Na+; found 421.1298. Anal. Calcd C20H22N4O3S: C, 60.28; H, 5.56; N, 14.06. Found: C, 59.99; H, 5.70; N, 13.82. 1-[(4-Chlorophenyl)aminothiocarbonyl]-2-methyl-4-(2-phenylacetyl)piperazine (74). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:3) as eluent (177mg, 61% yield), mp 168–171 ºC. MS (FAB): m/z 410 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.31, 9.30 (2s, 1H), 7.43–7.17 (m, 9H), 5.09 (br s, 1H), 4.49–4.33 (m, 1H), 4.27–4.15 (m, 1H), 4.04–3.86 (m, 1H), 3.84–3.71 (m, 2H), 3.28–3.12 (m, 1H), 3.07–2.88 (m, 1H), 1.09 (d, J = 6.9 Hz. 3H). 13C NMR (125 MHz, DMSO-d6)  181.2, 181.1, 169.7, 169.6, 140.0, 135.8, 135.6, 129.1, 128.9, 128.4, 128.3, 128.2, 127.8, 127.1, 126.4, 51.3, 48.7, 44.7, 42.8, 42.3, 40.9, 15.0, 14.5. HRMS (m/z): calcd for C20H22ClN3OSNa 410.1064 M+Na+; found 410.1059. Anal. Calcd C20H22ClN3OS: C, 61.92; H, 5.72; N, 10.83. Found: C, 61.53; H, 5.69; N, 10.60. 1-[(4-Cyanophenyl)aminothiocarbonyl]-2-methyl-4-(2-phenylacetyl)piperazine (75). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:2) as eluent (190 mg, 67% yield), mp 79–82 ºC. MS (FAB): m/z 401 (100%) M+Na+. 1H NMR 107 CHAPTER 6: EXPERIMENTAL SECTION (500 MHz, DMSO-d6)  9.59, 9.57 (2s, 1H), 7.79–7.72 (m, 2H), 7.61–7.53 (m, 2H), 7.40–7.24 (m, 5H), 5.14 (br s, 1H), 4.47–4.33 (m, 1H), 4.31–4.22 (m, 1H), 4.09–3.71 (m, 4H), 3.32–3.15 (m, 1H), 3.08–2.89 (m, 1H), 1.12 (d, J = 6.6 Hz. 3H). 13C NMR (125 MHz, DMSO-d6)  181.3, 181.2, 169.7, 169.6, 145.6, 135.7, 135.6, 132.2, 129.1, 128.9, 128.4, 128.3, 126.5, 126.4, 123.9, 119.1, 105.2, 51.7, 51.6, 48.7, 44.7, 43.3, 42.8, 40.9, 15.0, 14.5. HRMS (m/z): calcd for C21H22N4OSNa 401.1407 M+Na+; found 401.1402. Anal. Calcd C21H22N4OS: C, 66.64; H, 5.86; N, 14.80. Found: C, 66.53; H, 5.88; N, 14.65. 1-[(4-Fluorophenyl)aminothiocarbonyl]-2-methyl-4-(2-phenylacetyl)piperazine (76). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:3) as eluent (156 mg, 56% yield), mp 132–136 ºC. MS (FAB): m/z 394 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.25, 9.24 (2s, 1H), 7.37–7.10 (m, 9H), 5.07 (br s, 1H), 4.49–4.37 (m, 1H), 4.26–4.16 (m, 1H), 4.04–3.69 (m, 3H), 3.30–3.10 (m, 1H), 3.07–2.89 (m, 1H), 1.09 (d, J = 6.6 Hz. 3H). 13C NMR (125 MHz, DMSO-d6)  181.9, 181.8, 169.7, 169.6, 160.2, 158.3, 137.3, 137.2, 135.8, 135.7, 129.1, 128.9, 128.3, 128.2, 127.9, 127.8, 126.4, 126.3, 114.7, 114.5, 51.1, 48.7, 44.7, 42.7, 42.2, 40.9, 15.0, 14.5. HRMS (m/z): calcd for C20H22FN3OSNa 394.1360 M+Na+; found 394.1355. Anal. Calcd C20H22FN3OS: C, 64.67; H, 5.97; N, 11.31. Found: C, 64.45; H, 5.77; N, 10.95. 2-Methyl-4-(2-phenylacetyl)-1-[(4-trifluoromethylphenyl)aminothiocarbonyl]piperazine (77). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:3) as eluent (221 mg, 70% yield), mp 161–164 ºC. MS (FAB): m/z 444 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.52, 9.51 (2s, 1H), 7.68-7.61 (m, 2H), 7.59–7.51 (m, 2H), 7.39– 7.22 (m, 5H), 5.12 (br s, 1H), 4.47–4.33 (m, 1H), 4.29–4.18 (m, 1H), 4.06–3.90 (m, 1H), 3.89–3.69 (m, 2H), 3.28–3.13 (m, 1H), 3.06–2.89 (m, 1H), 1.11 (d, J = 6.4 Hz. 3H). 13C NMR (125 MHz, DMSO-d6)  181.6, 181.5, 169.7, 169.6, 144.9, 135.8, 135.7, 129.1, 128.9, 128.4, 128.3, 126.4, 125.1, 125.0, 124.5, 51.5, 48.7, 44.7, 43.1, 42.7, 42.2, 40.9, 26.82, 15.0, 14.5. HRMS (m/z): calcd for C21H22F3N3OSNa 444.1328 M+Na+; found 444.1316. Anal. Calcd C21H22F3N3OS: C, 59.84; H, 5.26; N, 9.97. Found: C, 59.56; H, 5.46; N, 9.68. 1-[(4-Methoxyphenyl)aminothiocarbonyl]-2-methyl-4-(2-phenylacetyl)piperazine (78). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:3) as eluent (158 mg, 55% yield), mp 70–73 ºC. MS (FAB): m/z 406 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.14, 9.13 (2s, 1H), 7.38–7.22 (m, 5H), 7.19–7.13 (m, 2H), 6.88 (d, J = 8.0 Hz, 2H), 5.06 (br s, 1H), 4.48–4.36 (m, 1H), 4.26–4.13 (m, 1H), 4.02–3.78 (m, 2H), 3.76 (s, 3H), 108 CHAPTER 6: EXPERIMENTAL SECTION 3.30–3.09 (m, 2H), 3.06–2.89 (m, 1H), 1.08 (d, J = 6.6 Hz. 3H). 13C NMR (125 MHz, DMSO-d6)  181.9, 181.8, 169.7, 169.6, 156.6, 135.8, 135.7, 133.8, 129.1, 128.9, 128.3, 128.2, 127.6, 126.4, 113.2, 55.2 50.9, 48.7, 44.7, 42.6, 42.0, 40.7, 15.0, 14.5. HRMS (m/z): calcd for C21H25N3O2SNa 406.1560 M+Na+; found 406.1554. Anal. Calcd C21H25N3O2S: C, 65.77; H, 6.57; N, 10.96. Found: C, 65.52; H, 6.23; N, 10.76. 2-Methyl-1-[(4-methylphenyl)aminothiocarbonyl]-4-(2-phenylacetyl)piperazine (79). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (171mg, 62% yield), mp 66–70 ºC. MS (FAB): m/z 390 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.18, 9.17 (2s, 1H), 7.39–7.22 (m, 5H), 7.19–7.08 (m, 4H), 5.08 (br s, 1H), 4.45–4.33 (m, 1H), 4.25–4.15 (m, 1H), 4.04–3.85 (m 1H), 3.83–3.68 (m, 2H), 3.30–3.07 (m, 2H), 3.05–2.87 (m, 1H), 2.29 (s, 3H), 1.08 (d, J = 6.6 Hz. 3H). 13C NMR (125 MHz, DMSO-d6)  181.8, 181.7, 169.7, 169.6, 138.3, 135.8, 135.7, 133.7, 129.1, 128.9, 128.4, 128.3, 128.2, 126.4, 125.7, 51.0, 48.7, 44.7, 42.7, 42.2, 40.9, 20.5, 15.0, 14.5. HRMS (m/z): calcd for C21H25N3OSNa 390.1611 M+Na+; found 390.1605. Anal. Calcd C21H25N3OS: C, 68.63; H, 6.86; N, 11.43. Found: C, 68.33; H, 6.69; N, 11.33. 1-[{3,5-Bis(trifluoromethyl)phenyl}aminothiocarbonyl]-2-methyl-4-(2-phenylacetyl)piperazine (80). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (3:2) as eluent (238 mg, 65% yield), mp 178–180 ºC. MS (FAB): m/z 512 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  9.72, 9.69 (2s, 1H), 8.09, 8.07 (2s, 2H), 7.79 (s, 1H), 7.37–7.24 (m, 5H), 5.12 (br s, 1H), 4.52–4.38 (m, 1H), 4.24 (t, J = 12.5 Hz, 1H), 4.07–3.91 (m, 1H), 3.89–3.72 (m, 2H), 3.50–3.43 (m, 1H), 3.29–3.17 (m, 1H), 3.06–2.94 (m, 1H), 1.13, 1.12 (2d, J = 6.5 Hz. 3H). 13C NMR (125 MHz, DMSO-d6)  180.9, 180.8, 169.8, 169.7, 143.0, 135.7, 135.6, 130.0, 129.8, 129.5, 129.3, 129.1, 128.9, 128.4, 128.3, 126.5, 126.4, 124.8, 124.4, 122.2, 116.8, 51.6, 48.6, 44.7, 43.0, 42.6, 40.9, 15.1, 14.6. HRMS (m/z): calcd for C22H21F6N3OSNa 512.1202 M+Na+; found 512.1188. Anal. Calcd C22H21F6N3OS: C, 53.98; H, 4.32; N, 8.58. Found: C, 53.90; H, 4.29; N, 8.67. 4-(Benzofuran-2-carbonyl)-2-methyl-1-[(4-nitrophenyl)aminothiocarbonyl]piperazine (81). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (292 mg, 92% yield), mp 189–191 ºC. 1H NMR (500 MHz, DMSO-d6)  9.83 (s, 1H), 8.19 (d, J = 9.2 Hz, 2H), 7.79 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.64 (d, J = 9.1 Hz, 2H), 7.51–7.47 (m, 2H), 7.37 (t, J = 7.5 Hz, 1H), 5.23 (br s, 1H), 4.55–4.46 (m, 1H), 4.40–4.33 (m, 1H), 4.31–4.25 (m, 1H), 3.61–3.53 (m, 2H), 1.29 (t, J = 6.7 Hz, 3H). 13C RMN (125 MHz, DMSO- 109 CHAPTER 6: EXPERIMENTAL SECTION d6)  181.3, 159.7, 154.0, 148.0, 147.7, 142.3, 126.7, 123.9, 123.7, 122.9, 122.5, 111.8, 111.4, 51.9, 15.0 HRMS (m/z): calcd for C21H20N4O4SNa 447.1077 M+Na+; found 447.1094. Anal. Calcd C21H20N4O4S: C, 59.42; H, 4.75; N, 13.20. Found: C, 59.50; H, 4.92; N, 13.12. 4-(Benzofuran-2-carbonyl)-1-[(4-cyanophenyl)aminothiocarbonyl]-2-methylpiperazine (82). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (218 mg, 72% yield), mp 174–175 ºC. 1H NMR (500 MHz, DMSO-d6)  9.66 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.52–7.46 (m, 2H), 7.37 (t, J = 7.4 Hz, 1H), 5.23 (br s, 1H), 4.55–4.45 (m, 1H), 4.40–4.32 (m, 1H), 4.30–4.23 (m, 1H), 3.60–3.49 (m, 2H), 1.27 (t, J = 6.4 Hz, 3H). 13C RMN (125 MHz, DMSOd6)  181.3, 159.7, 154.0, 147.9, 145.6, 132.2, 126.7, 123.9, 123.7, 122.5, 119.1, 111.8, 111.4, 105.3, 51.7, 15.1. HRMS (m/z): calcd for C22H20N4O2SNa 427.1199 M+Na+; found 427.1193. Anal. Calcd C22H20N4O2S: C, 65.33; H, 4.98; N, 13.85. Found: C, 65.42; H, 5.15; N, 13.91. 4-(Benzofuran-2-carbonyl)-1-[(4-fluorophenyl)aminothiocarbonyl]-2-methylpiperazine (83). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (253 mg, 85% yield), mp 171–173 ºC. 1H NMR (500 MHz, DMSO-d6)  9.32 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.52–7.46 (m, 2H), 7.37 (t, J = 7.4 Hz, 1H), 7.34–7.29 (m, 2H), 7.15 (t, J = 8.8 Hz, 2H), 5.18 (br s, 1H), 4.58–4.49 (m, 1H), 4.37–4.29 (m, 1H), 4.26–4.19 (m, 1H), 3.57–3.48 (m, 2H), 1.26 (t, J = 6.6 Hz, 3H). 13C RMN (125 MHz, DMSOd6)  181.9, 160.2, 159.6, 158.4, 154.0, 148.0, 137.2, 128.0, 127.9, 126.7, 123.7, 122.5, 114.7, 114.5, 111.8, 111.4, 51.2, 15.1. HRMS (m/z): calcd for C21H20FN3O2SNa 420.1152 M+Na+; found 420.1148. Anal. Calcd C21H20FN3O2S: C, 63.46; H, 5.07; N, 10.57. Found: C, 63.78; H, 5.20; N, 10.44. 4-(Benzofuran-2-carbonyl)-2-methyl-1-[(4-trifluoromethyl)aminothiocarbonyl]piperazine (84). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (282 mg, 84% yield), mp 186–188 ºC. 1H NMR (500 MHz, DMSO-d6)  9.59 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.2Hz, 2H), 7.52–7.46 (m, 2H), 7.37 (t, J = 7.4 Hz, 1H), 5.22 (br s, 1H), 4.52 (m, 1H), 4.39–4.32 (m, 1H), 4.29–4.23 (m, 1H), 3.60–3.51 (m, 2H), 1.28 (t, J = 6.6 Hz, 3H). 13C RMN (125 MHz, DMSO-d6)  181.6, 159.7, 154.0, 148.0, 144.9, 126.7, 125.0, 123.7, 122.5, 111.8, 111.3, 51.6, 15.1. HRMS (m/z): calcd for C22H20F3N3O2SNa 470.1121 M+Na+; found 470.1115. Anal. Calcd C22H20F3N3O2S: C, 59.05; H, 4.51; N, 9.39. Found: C, 59.20; H, 4.58; N, 9.26. 116 CHAPTER 6: EXPERIMENTAL SECTION 8.5 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.54–7.45 (m, 3H), 7.41–7.34 (m, 1H), 4.49–4.27 (m, 4H), 1.30 (d, J = 6.7 Hz, 6H). 13C RMN (125 MHz, DMSO-d6)  160.0, 154.0, 153.9, 148.2, 137.7, 132.1, 130.5, 129.1, 126.6, 123.8, 122.8, 122.7, 122.5, 121.8, 111.8, 111.4, 46.6, 19.9. HRMS (m/z): calcd for C23H21ClF3N3O3Na 502.1116 M+Na+; found 502.1110. Anal. Calcd C23H21ClF3N3O3: C, 57.57; H, 4.41; N, 8.76. Found: C, 57.54; H, 4.45; N, 8.76. 4-(Benzofuran-2-carbonyl)-1-[(4-chloro-3-trifluoromethylphenyl)aminocarbonyl]-2,6dimethylpiperazine (110). The product was obtained as a solid and purified by column using chromatography hexane-ethyl acetate (2:1) as eluent (252 mg, 70% yield), mp 218–220 ºC. 1H NMR (500 MHz, DMSO-d6)  8.84 (s, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.86 (d, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.53–7.45 (m, 2H), 7.40– 7.34 (m, 1H), 4.49–4.38 (m, 2H), 4.37–4.28 (m, 2H), 1.27 (d, J = 6.7 Hz, 6H). 13C RMN (125 MHz, DMSO-d6)  160.0, 154.0, 153.9, 148.2, 140.1, 131.5, 126.6, 124.5, 123.7, 122.5, 121.8, 118.8, 111.8, 111.3, 46.1, 20.1. HRMS (m/z): calcd for C23H21ClF3N3O3Na 502.1116 M+Na+; found 502.1111. Anal. Calcd C23H21ClF3N3O3: C, 57.57; H, 4.41; N, 8.76. Found: C, 57.59; H, 4.65; N, 8.54. 1-tert-Butoxycarbonyl-4-[(4-nitrophenyl)aminocarbonyl]piperazine (112). The product was obtained as a solid and purified by column using chromatography dichloromethane-methanol (100:1) as eluent (247 mg, 94% yield), mp 190–192 ºC. 1H NMR (500 MHz, DMSO-d6)  9.27 (s, 1H), 8.17 (d, J = 9.5 Hz, 2H), 7.37 (d, J = 9.5 Hz, 2H), 3.50–3.48 (m, 4H), 3.40–3.38 (m, 4H), 1.43 (m, 9H). 13C RMN (125 MHz, DMSO-d6)  154.0, 153.8, 147.3, 140.9, 124.7 118.3, 79.1, 43.6, 28.0. HRMS (m/z): calcd for C16H22N4O5Na 373.1482 M+Na+; found 373.1476. Anal. Calcd C16H22N4O5: C, 54.85; H, 6.33; N, 15.99. Found: C, 54.79; H, 6.21; N, 15.87. 1-tert-Butoxycarbonyl-4-[(2-nitrophenyl)aminocarbonyl]piperazine (113). The product was obtained as a solid and purified by column using chromatography dichloromethane-methanol (100:1) as eluent (234 mg, 89% yield), mp 91–94 ºC. 1H NMR (500 MHz, DMSO-d6)  9.34 (s, 1H), 7.96 (dd, J = 8.2 Hz, J = 1.2 Hz, 1H), 7.71–7.63 (m, 2H), 7.26–7.22 (m, 1H), 3.48–3.46 (m, 4H), 3.40– 3.38 (m, 4H), 1.44 (m, 9H). 13C RMN (125 MHz, DMSO-d6)  154,2, 153,9, 140,6, 134,5, 134,0, 125,0, 123,8, 123,0. 79.2, 43.6, 28,0. HRMS (m/z): calcd for C16H22N4O5Na 373.1482 M+Na+; found 373.1476. Anal. Calcd C16H22N4O5: C, 54.85; H, 6.33; N, 15.99. Found: C, 55.11 H, 6.46; N, 15.94. 4-tert-Butoxycarbonyl-1-[(2-chloro-5-trifluoromethylphenyl)aminocarbonyl]piperazine (114). The product was obtained as a solid and purified by column using chromatography dichloromethane- 117 CHAPTER 6: EXPERIMENTAL SECTION methanol (150:1) as eluent (285 mg, 92% yield), mp 153–155 ºC. 1H NMR (500 MHz, DMSO-d6)  8.45 (s, 1H), 7.96 (dd, J = 1.9 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.49 (dd, J = 1.9 Hz, J = 8.3 Hz, 1H), 3.48–3.46 (m, 4H), 3.41–3.39 (m, 4H), 1.44 (m, 9H). 13C RMN (125 MHz, DMSO-d6)  154.5, 153.9, 137.6, 131.3, 130.4, 122.3, 121.5 79.1, 43.6, 28.0. HRMS (m/z): calcd for C17H21ClF3N3O3Na 430.1116 M+Na+; found 430.1110. Anal. Calcd C17H21ClF3N3O3: C, 50.07; H, 5.19; N, 10.30. Found: C, 50.16, H, 5.06; N, 10.27. B) Synthesis of diurea derivatives from 2-phenyl piperazine or piperazine (101 and 121). To a solution of 2-phenyl piperazine or piperazine (1 mmol) in dry DCM (10 mL) was added the corresponding isocyanate (2 mmol). The reaction mixture was stirred at rt until TLC showed that all the starting material had reacted (12 hours) and then it was evaporated to dryness. The compound was purified by flash chromatography on silica gel using the appropriate eluent. 1,4-Bis(2-chloro-5-trifluoromethylphenyl)aminocarbonyl-2-phenylpiperazine (101). The product was obtained as a solid and purified by column using chromatography hexane-ethyl acetate (4:1) as eluent (408 mg, 90% yield), mp 134–137 ºC. 1H NMR (500 MHz, DMSO-d6)  8.35 (s, 1H), 8.27 (s, 2H), 8.06 (dd, J = 1.9 Hz, 1H), 7.84 (m, 1H), 7.70 (t, J = 7.5 Hz, 2H), 7.50–7.45 (m, 4H), 7.40 (t, J = 7.5 Hz, 2H) 7.30 (t, J = 7.2 Hz, 1H), 5.45 (t, J = 4.0 Hz, 1H), 4.36 (dd, J = 4.1 Hz, J = 13.9 Hz, 1H), 4.18–4.12 (m, 1H), 3.93–3.88 (m, 1H), 3.75 (dd, J = 4.4 Hz, J = 13.9 Hz, 1H), 3.54– 3.40 (m, 2H). 13C RMN (125 MHz, DMSO-d6)  154.5, 154.4, 139.2, 137.4, 137.3, 131.3, 130.4, 128.6, 127.3, 126.6, 122.6, 122.2, 121.6, 121.3, 54.9, 45.4, 43.5. HRMS (m/z): calcd for C26H20Cl2F6N4O2Na 627.0760 M+Na+; found 627.0752. Anal. Calcd C26H20Cl2F6N4O2: C, 51.59; H, 3.33; N, 9.26. Found: C, 51.61, H, 3.56; N, 9.06. 1,4-Bis(2-chloro-5-trifluoromethylphenyl)aminocarbonylpiperazine (121). The product was obtained as a solid and purified by column using chromatography hexane-ethyl acetate (3:1) as eluent (388 mg, 98% yield), mp 249–250 ºC. 1H NMR (500 MHz, DMSO-d6)  8.49 (s, 2H), 7.98 (d, J = 1.6 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.50 (dd, J = 8.4 Hz, J = 1.7 Hz, 2H), 3.56–3.59 (m, 8H). 13C RMN (125 MHz, DMSO-d6)  154.6, 137.6, 131.4, 130.5, 128.3, 128.0, 127.8, 124.8, 122.6, 122.4, 121.7, 43.6. Anal. Calcd C20H16Cl2F6N4O2: C, 46.43; H, 3.34; N, 10.31 Found: C, 46.74, H, 3.33; N, 10.45. 118 CHAPTER 6: EXPERIMENTAL SECTION -General Procedure 3. Deprotection reaction and synthesis of compounds 118 and 119. According with a reported procedure [117], CF3COOH (10 mmol) was added to a solution of 112 and 113 (1 mmol) in DCM (20 mL) at 0 ºC and the reaction mixture was warmed and stirred at rt. The reaction was concentrated under vacuum, the residue was dissolved in DCM, washed with saturated NaHCO3 and brine. The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure, to afford N-deprotected compounds (115 and 116) which were used in the next reaction without further purification. Compounds 115 and 116 (1 mmol) were dissolved in dry DCM (30 mL) and cooled to 0 ºC, then benzofurane-2-carbonyl chloride (1 mmol) and pyridine (2.5 mmol) were added. The reaction mixture was kept into an ice-water bath with stirring 6 hours and left at rt until TLC showed that all the starting material had reacted (12 hours). The reaction mixture was evaporated to dryness to obtain the corresponding acylderivatives. The compound was further purified by flash column chromatography on silica gel using the appropriate eluent. 1-(Benzofurane-2-carbonyl)-4-[(4-nitrophenyl)aminocarbonyl]piperazine (118). The product was obtained as a solid and purified by column using chromatography dichloromethane-methanol (80:1) (315 mg, 80% yield), mp 165–167 ºC. 1H NMR (500 MHz, DMSO-d6)  9.34 (s, 1H), 8.19– 8.15 (m, 2H), 7.81–7.65 (m, 4H), 7.52–7.45 (m, 2H), 7.38–7.33 (m, 1H), 3.93–3.75 (m, 4H), 3.52– 3.22 (m, 4H). 13C RMN (125 MHz, DMSO-d6)  159.1, 154.1, 153.9, 148.9, 147.3, 141.0, 126.7, 126.5, 124.7, 123.7, 122.5, 118.4, 111.7, 111.1, 43.8. HRMS (m/z): calcd for C20H18N4O5Na 417.1169 M+Na+; found 417.1163. Anal. Calcd C20H18N4O5: C, 60.91; H, 4.60; N, 14.21. Found: C, 60.77, H, 4.73 N, 13.83. 1-(Benzofurane-2-carbonyl)-4-[(2-nitrophenyl)aminocarbonyl]piperazine (119). The product was obtained as a solid and purified by column using chromatography hexane-ethyl acetate (1:1) (346 mg, 88% yield), mp 105–108 ºC. 1H NMR (500 MHz, DMSO-d6)  9.41 (s, 1H), 8.00–7.94 (m, 1H), 7.85–7.64 (m, 4H), 7.57–7.21 (m, 4H), 3.89–3.73 (m, 4H), 3.69–3.58 (m, 4H). 13C RMN (125 MHz, DMSO-d6)  159.1, 154.1, 153.9, 137.6, 148.1, 147.3, 141.1, 126.7, 126.6, 124.7, 123.7, 122.5, 118.4, 111.8, 111.1, 43.8. HRMS (m/z): calcd for C20H18N4O5Na 417.1169 M+Na+; found 417.1164 Anal. Calcd C20H18N4O5: C, 60.91; H, 4.60; N, 14.21. Found:C, 60.99, H, 4.50; N, 13.97. 119 CHAPTER 6: EXPERIMENTAL SECTION 6.1.2 O-Acyl-N-phenylaminocarbonyl serinol derivatives -General Procedure 4. Synthesis of urea derivatives from serinol (127-130). To a solution of appropriate isocyanate (3.6 mmol) in dry DCM (20 mL), a solution of the aminoalcohol (3 mmol) in methanol (1 mL) was added dropwise. A white precipitate appeared that was filtered at vacuum and washed with fresh DCM to give urea derivative. N-(1,3-Dihydroxyprop-2-yl)-N’-[4-(trifluoromethyl)phenyl]urea (127). The compound was obtained as a white solid (717 mg, 86% yield); mp 191–193 ºC. 1H NMR (500 MHz, DMSO-d6)  9.03 (s, 1H, NHAr), 7.57–7.44 (bs, 4H, Ar), 6.19 (d, J = 8.3 Hz, 1H, CHNH), 4.73 (t, J = 5.3 Hz, 2H, OH), 3.67–3.61 (m, 1H, CH), 3.56–3.41 (m, 4H, 2CH2OH). 13C NMR (125 MHz, DMSO-d6)  154.6, 144.2, 126.0, 125.9, 117.1, 117.0, 60.0, 52.6. HRMS (m/z): calcd for C11H13N2F3O3Na 301.0770 M+Na+; found 301.0770. Anal. Calcd for C11H13N2F3O3: C, 47.49; H, 4.71, N, 10.07. Found: C, 47.37; H, 4.72; N.10.03. N-(1,3-Dihydroxyprop-2-yl)-N’-(4-methylphenyl)urea (128). The product was obtained as a white resin (511 mg; 77 % yield). 1H NMR (500 MHz, DMSO-d6) δ 8.48 (s, 1H, NHAr), 7.27 (d, J = 8.4 Hz, 2H, Ar), 7.03 (d, J = 8.2 Hz, 2H, Ar), 5.99 (d, J = 8.0 Hz, 1H, CHNH), 4.69 (t, J = 5.1 Hz, 2H, OH), 3.65–3.58 (m, 1H, CH), 3.54–3.39 (m, 4H, 2CH2OH), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ 155.0, 138.0, 129.1, 129.0, 117.5, 60.2, 52.4, 20.2. HRMS (m/z): calcd. for C11H16N2O3Na 247.1053 [M+Na]+; found 247.1054. N-[4-chloro-3-(trifluoromethyl)phenyl]-N’-(1,3-dihydroxyprop-2-yl)urea (129). The product was obtained as a white solid. (750 mg, 80% yield); mp 176–178 ºC. 1H NMR (500 MHz, DMSOd6)  9.06 (s, 1H, NHAr), 8.03 (d, J = 2.1 Hz, 1H, Ar), 7.59–7.44 (m, 2H, Ar), 6.2 (d, J = 8.2 Hz, 1H, CHNH), 4.87 (bs, 2H, OH), 3.51–3.39 (m, 5H, HOCH2CHCH2OH). 13C NMR (125 MHz, DMSOd6)  154.7, 139.8, 131.9, 122.3, 121.5, 116.0, 115.9, 60.1, 52.5. HRMS (m/z): calcd for C11H12ClN2F3ClO3Na 335.0380 M+Na+; found 335.0381. Anal. Calcd for C11H12ClN2F3ClO3: C, 42.26; H, 3.87; N, 8.68. Found: C, 42.75; H, 3.65; N, 8.68. N-(4-Chlorophenyl)-N’-(1,3-dihydroxyprop-2-yl)urea (130) [118]. The compound was obtained as a pure white solid (606 mg, 83% yield); mp 181–184 ºC. 1H NMR (500 MHz, DMSO-d6)  8.75 (s, 1H, NHAr), 7.41 (d, J = 8.9 Hz, 2H, Ar), 7.26 (d, J = 8.7 Hz, 2H, Ar), 6.07 (d, J = 7,8 Hz, 1H, CHNH), 4.71 (t, J = 5,2 Hz, 2H, OH), 3.65–3.59 (m, 1H, CH), 3.53–3.40 (m, 4H, 2CH2OH). 13C NMR (125 MHz, DMSO-d6)  154.7, 139.5, 128.5, 124.3, 118.9, 60.1, 52.4. HRMS (m/z): calcd for 120 CHAPTER 6: EXPERIMENTAL SECTION C10H13ClN2O3Na 267.0507 M+Na+; found 267.0508. Anal. Calcd for C10H13ClN2O3: C, 49.09; H, 5.36; N, 11.45. Found: C, 49.47; H, 5.38; N, 11.40. -General Procedure 5. Acylation reaction of N-(substituted)-N’-(1,3-dihydroxyprop-2yl)phenylureas from acyl chloride (131-152, 156-158) A) O,O’-Diacylation reaction (131-152). To a solution of the urea derivative (127-130) (0.54 mmol) in dry DCM (20 mL) and DMAP (1.35 mmol), the appropriate acylating agent (1.1 mmol) in dry DCM (5 mL) was added. The reaction mixture was stirred at rt until TLC showed that all the starting material had reacted (24 h), then was washed with HCl 1N aqueous solution (2 x 20mL), saturated NaHCO3 solution (2 x 20 mL) and brine (20 mL). The organic layer was dried over Na2SO4, filtered and evaporated under reduced pressure. The compound was further purified by flash column chromatography on silica gel using the appropriate eluent. N-[1,3-Bis(4-methylbenzoyloxy)prop-2-yl]-N’-[4-(trifluoromethyl)phenyl]urea (131). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (4:1) as eluent (209 mg; 75% yield); mp 158–162 ºC. 1H NMR (500 MHz, DMSO-d6)  9.03 (s, 1H, NHAr), 7.90 (d, J = 8.1 Hz, 4H, Ar), 7.62–7.56 (m, 4H, Ar), 7.32 (d, J = 8.1 Hz, 4H, Ar), 6.71 (d, J = 8.5 Hz, 1H, CHNH), 4.57–4.40 (m, 5H, OCH2CHCH2O), 2.39 (s, 6H, CH3). 13C NMR (125 MHz, DMSO-d6)  165.5, 154.5, 143.8, 129.3, 129.2, 126.7, 126.0, 125.9, 121.4, 121.1, 117.4, 63.9, 47.3, 21.1. HRMS (m/z): calcd for C27H25F3N2O5Na 537.1608 M+Na+; found 537.1600. Anal. Calcd for C27H25F3N2O5: C, 63.03; H, 4.90; N, 5.44. Found: C, 62.90; H, 4.91; N, 5.46. N-[1,3-Bis(2-methylbenzoyloxy)prop-2-yl]-N’-[4-(trifluoromethyl)phenyl]urea (132). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (5:1) as eluent (190 mg; 68% yield); mp 158–160 ºC. 1H NMR (500 MHz, DMSO-d6)  9.00 (s, 1H, NHAr), 7.85 (d, J = 7.2 Hz, 2H, Ar), 7.61–7.52 (m, 4H, Ar), 7.50–7.43 (m, 2H, Ar), 7.35– 7.22 (m, 4H, Ar), 6.66 (d, J = 8.0 Hz, 1H, CHNH), 4.57–4.40 (m, 5H, OCH2CHCH2O), 2.50 (s, 6H, CH3). 13C NMR (125 MHz, DMSO-d6)  166.7, 154.6, 143.5, 139.2, 132.3, 131.6, 130.1, 129.0, 125.9, 117.5, 63.8, 47.3, 20.9. HRMS (m/z): calcd for C27H25F3N2O5Na 537.1608 M+Na+; found 537.1598. Anal. Calcd for C27H25F3N2O5: C, 63.03; H, 4.90; N, 5.44. Found: C, 62.85; H, 4.92; N, 5.42. N-[1,3-Bis(4-methoxylbenzoyloxy)prop-2-yl]-N’-[4-(trifluoromethyl)phenyl]urea (133). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent. (199 mg; 67 % yield); mp 118–119ºC. 1H NMR (500 MHz, DMSO-d6) δ 9.04 121 CHAPTER 6: EXPERIMENTAL SECTION (s, 1H, NHAr), 7.94 (d, J = 8.4 Hz, 4H, Ar), 7.62–7.52 (m, 4H, Ar), 7.00 (d, J = 8.4 Hz, 4H, Ar), 6.71 (d, J = 7.1 Hz, 1H, CHNH), 4.52–4.35 (m, 5H, OCH2CHCH2O), 3.81 (s, 6H, OCH3). 13C NMR (125 MHz, DMSO-d6) δ 165.2, 163.3, 154.6, 143.9, 131.4, 125.9, 125.6, 123.5, 121.6, 117.4, 113.9, 63.8, 55.5, 47.4. HRMS (m/z): calcd for C27H25F3N2O7Na 569.1506 [M+Na]+; found 569.1498. Anal. Calcd for C27H25F3N2O7: C, 59.34; H, 4.61; N, 5.13. Found: C, 59.12; H, 4.59; N, 5.11. N-[1,3-Bis(4-cyanobenzoyloxy)prop-2-yl]-N’-[4-(trifluoromethyl)phenyl]urea (134). The product was obtained as an amorphous solid and purified by column chromatography using hexaneethyl acetate (2:1) as eluent. (194 mg, 65% yield); 1H NMR (500 MHz, DMSO-d6)  8.98 (s, 1H, NHAr), 8.10 (d, J = 8.4 Hz, 4H, Ar), 7.92 (d, J = 8.4 Hz, 4H, Ar), 7.58–7.50 (m, 4H, Ar), 6.75 (d, J = 7.7 Hz, 1H, CHNH), 4.58–4.47 (m, 5H, OCH2CHCH2O). 13C NMR (125 MHz, DMSO-d6)  164.4, 143.4, 133.2, 132.6, 129.9, 125.9, 117.9, 117.5, 115.5, 64.7, 47.1. HRMS (m/z): calcd for C27H19F3N4O5Na 559.1200 M+Na+; found 559.1193. Anal. Calcd for C27H19F3N4O5: C, 60.45; H, 3.57; N, 10.44. Found: C, 60.49; H, 3.59; N, 10.40. N-[1,3-Bis(4-nitrobenzoyloxy)prop-2-yl]-N’-[4-(trifluoromethyl)phenyl]urea (135). The product was obtained as a yellow solid and purified by flash chromatography using hexane-ethyl acetate (2:1) as eluent. (223mg; 71% yield); mp 208–210 ºC. 1H NMR (500 MHz, DMSO-d6) δ 9.04 (s, 1H, NHAr), 8.27 (d, J = 8.5 Hz, 4H, Ar), 8.18 (d, J = 8.8 Hz, 4H, Ar), 7.55–7.51 (m, 4 H, Ar), 6.80 (d, J = 7.3 Hz, 1H, CHNH), 4.61–4.50 (m, 5H, OCH2CHCH2O). 13C NMR (125 MHz, DMSO-d6) δ 164.2, 154.7, 150.2, 143.5, 134.7, 130.7, 126.0, 125.9, 123.7, 117.5, 64.9, 47.1. HRMS (m/z): calcd for C25H19F3N4O9Na 599.0996 [M+Na]+; found 599.0989. Anal. Calcd for C25H19F3N4O9: C, 52.05; H, 3.22; N, 9.72. Found: C, 51.95; H, 3.23; N, 9.68. N-[1,3-Bis(2,4-dimethoxylbenzoyloxy)prop-2-yl]-N’-[4-(trifluoromethyl)phenyl]urea (136). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent. (233mg; 70% yield); mp 177–178 ºC. 1H NMR (500 MHz, DMSO-d6) δ 9.05 (s, 1H, NHAr), 7.74 (d, J = 8.7 Hz, 2H, Ar), 7.60–7.54 (m, 4H, Ar), 6.64–6.60 (m, 1H, CHNH), 6.57– 6.51 (m, 4H, Ar), 4.43–4.29 (m, 5H, OCH2CHCH2O), 3.84–3.75 (m, 12H, OCH3). 13C NMR (125 MHz, DMSO-d6) δ 164.6, 164.1, 160.9, 154.4, 143.9, 133.2, 125.9, 125.6, 123.5, 117.3, 111.4, 105.3, 98.9, 63.2, 55.8, 55.5, 47.3. HRMS (m/z): calcd for C29H29F3N2O9Na 629.1717 [M+Na]+; found 629.1711. Anal. Calcd for C29H29F3N2O9: C, 57.43; H, 4.82; N, 4.62. Found: C, 57.17; H, 4.80; N, 4.60. N-[1,3-Bis(3,4,5-trimethoxylbenzoyloxy)prop-2-yl]-N’-[4-(trifluoromethyl)phenyl]urea (137). The product was obtained as a white solid and purified by column chromatography using hexane- 122 CHAPTER 6: EXPERIMENTAL SECTION ethyl acetate (1:2) as eluent. (295 mg; 82% yield); mp 188–189 ºC. 1H NMR (500 MHz, DMSO-d6) δ 9.05 (s, 1H, NHAr), 7.63–7.56 (m, 4H, Ar), 7.28 (s, 4H, Ar), 6.70 (d, J = 8.80 Hz, 1H, CHNH), 4.63-4.55 (m, 1H, CH), 4.53–4.42 (m, 4H, OCH2CHCH2O), 3.84–3.72 (m, 18H, OCH3). 13C NMR (125 MHz, DMSO-d6) δ 165.1, 154.6, 152.7, 143.8, 141.9, 125.9, 125.6, 124.5, 121.7, 121.3, 117.4, 106.8, 64.3, 60.1, 55.9, 47.4. HRMS (m/z): calcd for C31H32F3N2O11Na 689.1929 [M+Na]+; found 689.1920. Anal. Calcd for C31H33F3N2O11: C, 55.86; H, 4.99; N, 4.20. Found: C, 55.61; H, 4.97; N, 4.22. N-[1,3-Bis(4-methylbenzoyloxy)prop-2-yl]-N’-(4-methylphenyl)urea (138). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent. (165 mg; 66% yield); mp 164–165 ºC. 1H NMR (500 MHz, DMSO-d6) δ 8.50 (s, 1H, NHAr), 8.00 (d, J = 8.1 Hz, 4H, Ar), 8.21 (d, J = 8.9 Hz, 4H, Ar), 7.44–7.35 (m, 2H, Ar), 7.13 (d, J = 8.3 Hz, 2H, Ar), 6.61 (d, J = 8.1 Hz, 1H, CHNH), 4.63–4.50 (m, 5H, OCH2CHCH2O), 2.49 (s, 6H, CH3), 2.32 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ 165.5, 154.9, 143.7, 137.5, 130.1, 129.3, 129.2, 129.0, 126.7, 117.9, 64.0, 47.2, 21.1, 20.2. HRMS (m/z): calcd for C27H28N2O5Na 483.1880 [M+Na]+; found 483.1887. Anal. Calcd for C27H28N2O5: C, 70.42; H, 6.13; N, 6.08. Found: C, 70.09; H, 6.15; N, 6.10. N-[1,3-Bis(2-methylbenzoyloxy)prop-2-yl]-N’-(4-methylphenyl)urea (139). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent. (230 mg; 92 % yield); mp 122–123 ºC. 1H NMR (500 MHz, DMSO-d6) δ 8.43 (s, 1H, NHAr), 7.85 (d, J = 7.7 Hz, 2H, Ar), 7.78 (d, J = 7.6 Hz, 2H, Ar), 7.49–7.41 (m, 2H, Ar), 7.33–7.19 (m, 4H, Ar), 7.00 (d, J = 8.2 Hz, 2H, Ar), 6.42 (d, J = 8.5 Hz, CHNH), 4.51–4.33 (m, 5H, OCH2CHCH2O), 2.49 (s, 6H, CH3), 2.19 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ 166.6, 154.8, 139.3, 137.5, 132.2, 131.9, 131.5, 130.2, 130.1, 129.9, 129.8, 129.0, 125.9, 117.9, 64.0, 60.4, 47.2, 21.0, 20.2. HRMS (m/z): calcd for C27H28N2O5Na 483.1890 [M+Na]+; found 483.1883. Anal. Calcd for C27H28N2O5: C, 70.42; H, 6.13; N, 6.08. Found: C, 70.08; H, 6.10; N, 6.06. N-[1,3-Bis(4-methoxylbenzoyloxy)prop-2-yl]-N’-(4-methylphenyl)urea (140). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent. (210 mg; 79% yield); mp 165–166 ºC. 1H NMR (500 MHz, DMSO-d6) δ 8.49 (s, 1H, NHAr), 7.99–7.94 (m, 4H, Ar), 7.28 (d, J = 8.4 Hz, 2H, Ar), 7.08–6.99 (m, 6H, Ar), 6.49 (d, J = 8.3 Hz, 1H, CHNH), 4.52–4.38 (m, 5H, OCH2CHCH2O), 3.85 (s, 6H, OCH3), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ 165.2, 163.2, 154.9, 137.5, 131.4, 130.1, 129.8, 121.6, 117.7, 113.9, 63.7, 55.4, 47.3, 20.2HRMS (m/z): calcd. for C27H28N2O7Na 515.1789 [M+Na]+; ]+; 515.1782. Anal. Calcd for C27H28N2O7: C, 65.84; H, 5.73; N, 5.69. Found: C, 65.54; H, 5.71; N, 5.70. 123 CHAPTER 6: EXPERIMENTAL SECTION N-[1,3-Bis(4-nitrobenzoyloxy)prop-2-yl]-N’-(4-methylphenyl)urea (141). The product was obtained as a light yellow solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent. (177 mg; 62% yield); mp 195–196 ºC. 1H NMR (500 MHz, DMSO-d6) δ 8.48 (s, 1H, NHAr), 8.30 (d, J = 8.9 Hz, 4H, Ar), 8.21 (d, J = 8.9 Hz, 4H, Ar), 7.25 (d, J = 8.4 Hz, 2H, Ar), 7.02 (d, J = 8.3 Hz, 2H, Ar), 6.58 (d, J = 6.4 Hz, 1H, CHNH), 4.59–4.49 (m, 5H, OCH2CHCH2O), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ 164.1, 154.9, 150.3, 137.5, 134.9, 130.7, 130.1, 129.0, 123.7, 117.9, 64.9, 47.0, 20.2. HRMS (m/z): calcd for C25H22N4O9Na 545.1279 [M+Na]+; found 545.1272. Anal. Calcd for C25H22N4O9: C, 57.47; H, 4.24; N, 10.72. Found: C, 57.69; H, 4.26; N, 10.71. N-[1,3-Bis(2,4-dimethoxylbenzoyloxy)prop-2-yl]-N’-(4-methylphenyl)urea (142). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:2) as eluent. (195 mg; 65% yield); mp 158–159 ºC. 1H NMR (500 MHz, DMSO-d6) δ 8.52 (s, 1H, NHAr), 7.77 (d, J = 8.7 Hz, 2H, Ar), 7.3 (d, J = 8.4 Hz, 2H, Ar), 7.28 (d, J = 8.2 Hz, 2H, Ar), 7.03 (d, J = 8.0 Hz, 2H, Ar), 6.69–6.51 (m, 2H, Ar), 6.32 (d, J = 7.6 Hz, 1H, CHNH), 4.48–4.29 (m, 5H, OCH2CHCH2O), 3.95–3.85 (m, 12H, OCH3), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ 164.6, 164.1, 160.9, 154.8, 137.6, 133.2, 130.0, 129.0, 117.8, 111.5, 105.3, 98.9, 63.4, 55.8, 55.5, 47.2, 20.2. HRMS (m/z): calcd for C29H32N2O9Na 575.2000 [M+Na]+; found 575.1993. Anal. Calcd for C29H32N2O9: C, 63.04; H, 5.84; N, 5.07. Found: C, 62.74; H, 5.85; N, 5.09. N-[1,3-Bis(3,4,5-trimethoxylbenzoyloxy)prop-2-yl]-N’-(4-methylphenyl)urea (143). The product was obtained as a white solid and purified through column chromatography using hexane-ethyl acetate (1:2) as eluent. (202mg; 61% yield); mp 204–205 ºC. 1H NMR (500 MHz, DMSO-d6) δ 8.48 (s, 1H, NHAr), 7.31–7.24 (m, 6H, Ar), 7.03 (d, J = 8.2 Hz, 2H, Ar), 6.47 (d, J = 8.70 Hz, 1H, CHNH), 4.60–4.40 (m, 5H, OCH2CHCH2O), 3.80 (s, 12H, OCH3), 3.75 (s, 6H, OCH3), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ 165.1 (2C), 154.9, 152.7, 141.9, 137.5, 130.1, 128.9, 124.5, 117.9, 106.8, 64.5, 60.1, 55.9, 47.2, 20.2. HRMS (m/z): calcd for C31H36N2O11Na 635.2211 [M+Na]+; found 635.2204. N-[1,3-Bis(4-methylbenzoyloxy)prop-2-yl]-N’-[4-chloro-3-(trifluoromethyl)phenyl]urea (144) [118]. The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (3.5:1) as eluent. (248 mg; 85% yield); mp 170–173 ºC. 1H NMR (500 MHz, DMSO-d6)  9.07 (s, 1H, NHAr), 7.98 (d, J = 1.8 Hz, 1H, Ar), 7.98–7.80 (d, J = 8.2 Hz, 4H, Ar), 7.52–7.47 (m, 2H, Ar), 7.26 (d, J = 8.1 Hz, 4H, Ar), 6.68 (d, J = 8.7 Hz, 1H, CHNH), 4.56–4.35 (m, 5H, OCH2CHCH2O), 2.34 (s, 6H, CH3). 13C NMR (125 MHz, DMSO-d6)  165.6, 154.7, 143.9, 139.4, 131.8, 129.3, 129.2, 129.0, 126.7, 122.5, 121.6, 116.4, 63.8, 47.5, 21.0. HRMS (m/z): calcd 124 CHAPTER 6: EXPERIMENTAL SECTION for C27H25ClF3N2O5 549.1399 M+H+; found 549.1391. Anal. Calcd for C27H24ClF3N2O5: C, 58.08; H, 4.41; N, 5.10. Found: C, 58.18; H, 4.42; N, 5.08. N-[1,3-Bis(2-methylbenzoyloxy)prop-2-yl]-N’-[4-chloro-3-(trifluoromethyl)phenyl]urea (145). The product was obtained as a white solid and purified by column chromatography using hexaneethyl acetate (3.5:1) as eluent (186 mg; 62% yield); mp 132–136 ºC. 1H NMR (500 MHz, DMSO-d6)  9.06 (s, 1H, NHAr), 8.00 (d, J = 2.1 Hz, 1H, Ar), 7.85 (d, J = 7.1 Hz, 2H, Ar), 7.55–7.43 (m, 4H, Ar), 7.33–7.21 (m, 4H, Ar), 6.67 (d, J = 8.7 Hz, 1H, CHNH), 4.54–4.48 (m, 1H, CH), 4.45–4.49 (m, 4H, OCH2CHCH2O), 2.49 (s, 6H, CH3). 13C NMR (125 MHz, DMSO-d6)  166.7, 154.6, 139.4, 139.2, 132.2, 131.8, 131.6, 130.1, 129.0, 125.8, 122.6, 63.8, 47.4, 20.9. HRMS (m/z): calcd for C27H24ClF3N2O5Na 571.1218 M+Na+; found 571.1213. Anal. Calcd for C27H24ClF3N2O5: C, 58.08; H, 4.41; N, 5.10. Found: C, 58.32; H, 4.39; N, 5.12. N-[1,3-Bis(4-methoxylbenzoyloxy)prop-2-yl]-N’-[4-chloro-3-(trifluoromethyl)phenyl]urea (146) [118]. The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:2) as eluent. (177 mg; 56% yield); mp 133–134 ºC. 1H NMR (500 MHz, DMSO-d6) δ 9.19 (s, 1H, NHAr), 8.50 (s, 1H, Ar), 7.95 (d, J = 8.5 Hz, 4H, Ar), 7.58–7.53 (m, 2H, Ar), 7.04–6.96 (m, 4H, Ar), 6.80 (d, J = 8.2 Hz, 1H, CHNH), 4.55–4.38 (m, 5H, OCH2CHCH2O), 3.82 (s, 6H, OCH3). 13C NMR (125 MHz, DMSO-d6) δ 165.2, 163.3, 154.7, 139.8, 131.9, 131.4, 126.5, 123.9, 122.5, 121.7, 121.6, 116.3, 113.9, 63.7, 55.8, 47.5. HRMS (m/z): calcd for C27H24ClF3N2O7Na 603.1116 [M+Na]+; found 603.1109. Anal. Calcd for C27H24ClF3N2O7: C, 55.82; H, 4.16; N, 4.82. Found: C, 55.56; H, 4.15; N, 4.84. N-[1,3-Bis(4-cyanobenzoyloxy)prop-2-yl]-N’-[4-chloro-3-(trifluoromethyl)phenyl]urea (147) [118]. The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (218 mg; 70% yield); mp 132–133 ºC. 1H NMR (500 MHz, DMSO-d6)  9.05 (s, 1H, NHAr), 8.1 (d, J = 8.3 Hz, 4H, Ar), 7.96–7.90 (m, 5H, Ar), 7.53–7.45 (m, 2H, Ar), 6.76 (d, J = 8.0 Hz, 1H, CHNH), 4.52 (m, 5H, OCH2CHCH2O). 13C NMR (125 MHz, DMSO-d6)  164.4, 154.7, 139.3, 133.2, 132.6, 131.8, 129.9, 122.6, 122.0, 117.9, 116.2, 115.4, 64.7, 47.2. HRMS (m/z): calcd for C27H18ClF3N4O5Na 593.0810 M+Na+; found 593.0809. Anal. Calcd for C27H18ClF3N4O5: C, 56.80; H, 3.18; N, 9.62. Found: C, 56.75; H, 3.20; N, 9.62. N-[1,3-Bis(4-nitrobenzoyloxy)prop-2-yl]-N’-[4-chloro-3-(trifluoromethyl)phenyl]urea (148). The product was obtained as a yellow solid and purified by column chromatography using hexaneethyl acetate (2:1) as eluent. (127mg; 68% yield); mp 134–136 ºC. 1H NMR (500 MHz, DMSO-d6) 125 CHAPTER 6: EXPERIMENTAL SECTION  9.05 (s, 1H, NHAr), 8.1 (d, J = 8.3 Hz, 4H, Ar), 7.55–7.43 (m, 4H, Ar), 7.94–7.91 (m, 1H, Ar), 7.53 (d, J = 8.8 Hz, 1H, Ar), 7.47 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H, Ar), 6.76 (d, J = 8.0 Hz, 1H, NHCH), 4.52 (m, 5H, OCH2CHCH2O). 13C NMR (125 MHz, DMSO-d6)  164.4, 154.7, 139.3, 133.2, 132.6, 131.8, 129.9, 122.6, 117.9, 115.4, 64.7, 47.2. HRMS (m/z): calcd for C25H18ClF3N4O9Na 633.0607 M+Na+; found 633.0599. Anal. Calcd for C25H18ClF3N4O9: C, 49.15; H, 2.97; N, 9.17. Found: C, 48.99; H, 2.98; N, 9.14. N-[1,3-Bis(2,4-dimethoxylbenzoyloxy)prop-2-yl]-N’-[4-chloro-3-(trifluoromethyl)phenyl]urea (149). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:2) as eluent. (255 mg; 74% yield); mp 178–179 ºC. 1H NMR (500 MHz, DMSO-d6) δ 9.11 (s, 1H, NHAr), 8.06–8.03 (m, 1H, Ar), 7.74 (d, J = 8.7 Hz, 2H, Ar), 7.55–7.52 (m, 2H, Ar), 6.63–6.59 (m, 2H, Ar), 6.56–6.50 (m, 3H, Ar, CHNH), 4.43–4.29 (m, 5H, OCH2CHCH2O), 3.87–3.76 (m, 12H, OCH3). 13C NMR (125 MHz, DMSO-d6) δ 164.6, 164.1, 160.9, 154.5, 139.8, 133.2, 131.8, 128.6, 126.5, 126.3, 123.9, 116.3, 111.4, 105.3, 98.9, 63.2, 55.5, 55.4, 47.4. HRMS (m/z): calcd for C29H28ClF3N2O9Na 663.1328 [M+Na]+; found 663.1321. Anal. Calcd for C29H28ClF3N2O9: C, 54.34; H, 4.40; N, 4.37. Found: C, 54.11; H, 4.39; N, 4.35. N-[1,3-Bis(3,4,5-trimethoxylbenzoyloxy)prop-2-yl]-N’-[4-chloro-3- (trifluoromethyl)phenyl]urea (150). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (287 mg; 76 % yeld); mp 159– 160 ºC. 1H NMR (500 MHz, DMSO-d6) δ 9.12 (s, 1H, NHAr), 8.08 (s, 1H, Ar), 7.53 (s, 2H, Ar), 7.26 (s, 4H, Ar), 6.73 (d, J = 8.9 Hz, 1H, CHNH), 4.62–4.54 (m, 1H, CH), 4.51–4.41 (m, 4H, OCH2CHCH2O), 3.83–3.71 (m, 18H, OCH3). 13C NMR (125 MHz, DMSO-d6) δ 165.1, 154.7, 152.7, 141.9, 139.7, 131.8, 126.7, 124.4, 123.9, 122.5, 121.8, 121.7, 106.7, 64.3, 60.1, 55.9, 47.5. HRMS (m/z): calcd for C31H32ClF3N2O11Na 723.1539 M+Na+; found 723.1524. Anal. Calcd for C31H32ClF3N2O11: C, 53.11; H, 4.60; N, 4.00. Found: C, 53.20; H, 4.59; N, 4.02. N-[1,3-Bis(4-dimethylaminobenzoyloxy)prop-2-yl]-N’-[4-chloro-3- (trifluoromethyl)phenyl]urea (151) The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent. (215mg; 66% yield). 1H NMR (500 MHz, DMSO-d6)  9.10 (s, 1H, NHAr), 8.07 (s, 1H, Ar), 7.80 (d, J = 8.9 Hz, 4H, Ar), 7.56 (s, 2H, , Ar), 6.66 (m, 5H, Ar + CHNH), 4.45-4.32 (m, 5H, OCH2CHCH2O). 13C NMR (125 MHz, DMSO-d6)  165.7 (2C), 154.7, 153.3 (2C), 139.8, 131.8, 130.9, 129.5, 122.4, 121.6, 116.3, 115.4, 110.7, 63.2, 42.7. 132 CHAPTER 6: EXPERIMENTAL SECTION 6.1.3 3-Phenylaminocarbonyl-1,2-propanediol derivatives -General procedure 8. Synthesis of urea derivatives of 3-amino-1,2-propanediol and allylamine (173-179, 233) A) Synthesis of urea derivatives from commerical isocyanates (173-176, 178, 233). Compounds were prepared following the general procedure 4, section 6.1.2. N-(4-Chlorophenyl)-N’-(2,3-dihydroxypropyl)urea (173) [119]. The product was obtained as a white solid (690 mg; 94% yield), mp 160–161 ºC. 1H NMR (500 MHz, DMSO-d6)  8.76 (s, 1H, NHAr), 7.37–7.35 (m, 2H, Ar), 7.25–7.23 (m, 2H, Ar), 6.19 (t, J = 5.5 Hz, 1H, CH2NH), 5.01 (d, J = 4.9 Hz, 1H. CHOH), 4.82 (t, J = 5,2 Hz, 1H, CH2OH), 3.56–3.50 (m, 2H, CH2OH), 3.38–3.30 (m, 2H, CH2NH), 3.03–2.98 (m, 1H, CH). 13C NMR (125 MHz, DMSO-d6)  155.6, 139.1, 128.4, 124.8, 119.4, 70.5, 63.5, 42.4. HRMS (m/z): calcd for C10H13ClN2O3Na 267.0507 M+Na+; found 267.0501. Anal. Calcd for C10H13ClN2O3: C, 49.09; H, 5.36; N, 11.45. Found: C, 49.51; H, 5.66; N, 11.34. N-(2,3-Dihydroxypropyl)-N’-(4-methylphenyl)urea (174). The product was obtained as a white solid (615 mg; 91% yield), mp 147–148 ºC. 1H NMR (500 MHz, DMSO-d6)  8.48 (s, 1H, NHAr), 7.26 (d, J = 8.5 Hz, 2H, Ar), 7.03 (d, J = 8.2 Hz, 2H, Ar), 6.10 (t, J = 5.6 Hz, 1H, CH2NH), 4.85 (d, J = 4.9 Hz, 1H, CHOH), 4.60 (t, J = 5.7 Hz, 1H, CH2OH), 3.51 (sex, J = 5.6 Hz, 1H, CH), 3.32–3.27 (m, 2H, CH2OH), 3.01-2.95 (m, 2H, CH2NH), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  155.6, 138.1, 129.6, 129.0, 117.6, 70.7, 63.7, 42.5, 20.3. HRMS (m/z): calcd for C11H16N2O3Na 247.1064 M+Na+; found 247.1056. Anal. Calcd for C11H16N2O3: C, 58.91; H, 7.19; N, 12.49. Found: C, 58.53; H, 7.03; N, 12.16. N-(2,3-Dihydroxypropyl)-N’-[4-(trifluoromethyl)phenyl]urea (175). The product was obtained as a white solid (673 mg; 80% yield) , mp 135–136 ºC 1H NMR (500 MHz, DMSO-d6)  9,04 (s, 1H, NHAr), 7.60–7.57 (m, 4H, Ar), 6.30 (t, J = 5,3 Hz, 1H, CH2NH), 4.88 (d, J = 5.0 Hz, 1H, CHOH), 4.61 (t, J = 5,6 Hz, 1H, CH2OH), 3.57–3.51 (m, 1H, CH), 3.35–3.30 (m, 2H, CH2OH), 3.04–2.99 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  155.0, 144.2, 125.9, 121.0, 120.8, 117.1, 70.5, 63.7, 42.5. HRMS (m/z): calcd for C11H13F3N2O3Na 301.0770 M+Na+; found 301,0769. Anal. Calcd for C11H13F3N2O3: C, 47.49; H, 4.71; N, 10.07. Found: C, 49.51; H, 5.66; N, 11.34. N-(2,3-Dihydroxypropyl)-N’-(4-methoxyphenyl)urea (176). The product was obtained as a white solid (635 mg; 88% yield), mp 155–156 ºC. 1H NMR (300 MHz, DMSO-d6)  8.39 (s, 1H, NHAr), 133 CHAPTER 6: EXPERIMENTAL SECTION 7.28 (d, J = 6.8 Hz, 2H, Ar), 6.81 (d, J = 6.8 Hz, 2H, Ar), 6.03 (t, J = 5.5 Hz, 1H, CH2NH), 4.85 (d, J = 4.8 Hz, 1H, CHOH), 4.60 (t, J = 5.7 Hz, 1H, CH2OH), 3.69 (s, 3H, OCH3), 3.53–3.44 (m, 1H, CH), 3.38–3.23 (m, 2H, CH2OH), 3.02–2.92 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  156.2, 154.1, 134.2, 119.7, 114.3, 71.2, 64.1, 55.6, 43.1. N-[2-Chloro-5-(trifluoromethyl)phenyl]-N’-(2,3-dihydroxypropyl)urea (178). The product was obtained as a white solid (680 mg; 72% yield), mp 129–130 ºC. MS (FAB): m/z 403 (65%) [M+Na]+. 1H NMR (500 MHz, DMSO-d6)  8.66 (s, 1H, NHAr), 8.50 (s, 1H, Ar), 7.65 (d, J = 5.3 Hz, 1H, Ar), 7.32-7.26 (m, 2H, Ar + CH2NH), 4.85 (m, 1H, CHOH), 4.58 (t, J = 5.7 Hz, 1H, CH2OH), 3.58-3.52 (m, 1H, CH), 3.41-3.34 (m, 2H, CH2OH), 3.05-2.99 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSOd6)  154.8, 137.7, 130.2, 128.2, 124.4, 118.2, 116.3, 70.5, 63.7, 42.5. N-Allyl-N’-(4-chlorophenyl)urea (233). The product was obtained as a white solid and purified by column chromatography using dichloromethane-methanol (140:1) as eluent (570 mg; 90% yield). 1H NMR (500 MHz, DMSO-d6)  8.66 (s, 1H, NHAr), 7.46 (d, J = 8.3 Hz, 2H, Ar), 7.30 (d, J = 8.7 Hz, 2H, Ar), 6.32 (t, J = 5.3 Hz, 1H, CH2NH), 5.91–5.87 (m, 1H, CH2=CH), 5.23–5.18 (m, 1H, CH2=CH), 5.13–5.08 (m, 1H, CH2=CH), 3.77 (t, J = 5.4 Hz, 1H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  154.9, 139.4, 136.2, 128.6, 128.4, 124.5, 119.8, 119.1, 114.7, 41.4. HRMS (m/z): calcd for C10H12ClN2O 211.0633 M+H+; found 211.0633. Anal. Calcd for C10H11ClN2O: C, 57.02; H, 5.26; N, 13.30. Found: C, 56.79; H, 5.03; N, 12.86. B) Synthesis of urea derivatives from substituted anilines (177 and 179). According to a reported procedure with some modifications [105], a solution of appropriate substituted aniline (1.2 mmol) in DCM (25 mL) was added to a solution of Na2CO3 (1.92 mmol) in water (25 mL) and the reaction mixture was vigorously stirred for 5 minutes at rt; then triphosgene (0.39 mmol) was added and the solution was stirred for additional 30 minutes. After this time, 3-amino-1,2-propanediol (1.8 mmol) was added dropwise to the flask and the solution was stirred at rt until TLC showed the full consumption of the starting material (2 hours). The mixture was separated and the inorganic layer was extracted with ethyl acetate (3 x 40 mL). The combined organic layers were dried over MgSO4, filtered and evaporated in vacuo to obtain a crude product. The compound was further purified by flash column chromatography on silica gel using the appropriate eluent or by filtration at vacuum. N-(2,3-Dihydroxypropyl)-N’-(2-methylphenyl)urea (177). The product was obtained as a white resin by filtration at vacuum. (455 mg; 67% yield). 1H NMR (300 MHz, DMSO-d6)  7.87–7.77 (m, 2H, NHAr, Ar), 7.13–7.04 (m, 1H, Ar), 6.85 (t, J = 7.3 Hz, 2H, Ar), 6.63 (t, J = 5.2 Hz, 1H, CH2NH), 134 CHAPTER 6: EXPERIMENTAL SECTION 4.89–4.84 (m, 1H, CHOH), 4.63–4.56 (m, 1H, CH2OH), 3.55–3.46 (m, 1H, CH), 3.33–3.25 (m, 2H, CH2OH), 3.03-2.93 (m, 2H, CH2NH), 2.18 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  156.2, 138.7, 130.5, 127.0, 126.5, 122.2, 120.8, 71.3, 64.1, 42.9, 18.4. N-(2,3-Dihydroxypropyl)-N’-(3,4,5-trimethoxyphenyl)urea (179). The product was obtained as a white solid and purified by flash column chromatography using dichloromethane-methanol (15:1) as eluent. (255 mg; 70% yield), mp 165–166 ºC. 1H NMR (500 MHz, DMSO-d6)  8.55 (s, 1H, NHAr), 6.73 (s, 2H, Ar), 6.08 (t, J = 5.65 Hz, 1H, CH2NH), 4.81 (d, J = 4.8 Hz, 1H, CHOH), 4.57 (t, J = 5.70 Hz, 1H, -CH2OH), 3.73 (s, 6H, OCH3), 3.60 (s, 3H, OCH3), 3.54–3.49 (m, 1H, CH), 3.39–3.26 (m, 2H, CH2OH), 3.03–2.95 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  155.5, 152.7, 136.7, 131.9, 95.4, 70.6, 63.7, 60.0, 55.6, 42.4. HRMS (m/z): calcd for C13H20N2O6Na 323.1214 M+Na+; found 323.1211. -General procedure 9. Acylation reaction from acyl chloride (180-209, 212-223) A) O,O’-Diacylation reaction of N-(2,3-dihydroxypropyl)-N’-(substituted)phenylureas (180-209). Compounds were prepared following the general procedure 5A section 6.1.2. N-[2,3-Bis(benzoyloxy)propyl]-N’-(4-chlorophenyl)urea (180). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (160 mg; 70% yield), mp 130–131 ºC. NMR (500 MHz, DMSO-d6)  8.72, (s, 1H, NHAr), 8.02 (d, J = 7.5 Hz, 2H, Ar), 7.95 (d, J = 7.5 Hz, 2H, Ar), 7.60–7.52 (m, 2H, Ar), 7.55–7.49 (m, 4H, Ar), 7.43 (d, J = 8.8 Hz, 2H, Ar), 7.28 (d, J = 8.7 Hz, 2H, Ar), 6.50 (t, J = 5.9 Hz, 1H, CH2NH), 5.52–5.45 (m, 1H, CH), 4.67 (dd, J = 3.4 Hz, J = 12.0 Hz, 1H, CH2O), 4.52 (dd, J = 6.6 Hz, J = 12.0 Hz, 1H, CH2O), 3.70–3.58 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  165.4, 165.3, 155.2, 139.3, 133.4,129.6, 129.4, 129.3, 129.1, 128.7, 128.6, 128.4, 124.7 119.2, 710.6, 63.7, 39.5. HRMS (m/z): calcd for C24H21ClN2O5Na 475.1031 M+Na+; found 475.1026. N-[2,3-Bis(4-methylbenzoyloxy)propyl]-N’-(4-chlorophenyl)urea (181) [119]. The product was obtained as a colourless oil and purified by column chromatography using hexane-ethyl acetate (1.5:1) as eluent (205 mg; 85% yield). MS (FAB): m/z 503 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  8.72, (s, 1H, NHAr), 7.89–7.87 (m, 2H, Ar), 7.83–7.81 (m, 2H, Ar), 7.42–7.40 (m, 2H, Ar), 7.33–7.29 (m, 4H, Ar), 7.27–7.25 (m, 2H, Ar), 6.28 (t, J = 5.8 Hz, 1H, CH2NH), 5.46–5.41 (m, 1H, CH), 4.61 (dd, J = 4.1 Hz, J = 12.0 Hz, 1H, CH2O), 4.46 (dd, J = 6.0 Hz, J = 11.6 Hz, 1H, CH2O), 3.62–3.54 (m, 2H, CH2NH), 2.38 (s, 3H, CH3), 2.37 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) 135 CHAPTER 6: EXPERIMENTAL SECTION  165.4, 165.2, 155.1, 143.8, 143.6, 139.3, 129.4, 129.3, 129.2, 129.1, 128.4, 126.8, 124.4, 119.2, 71.3, 65.7, 30.7, 21.2, 21.1. Anal. Calcd for C26H25ClN2O5: C, 64.93; H, 5.24; N, 5.82. Found: C, 64.82; H, 5.76; N, 5.74. N-[2,3-Bis(2-methylbenzoyloxy)propyl]-N’-(4-chlorophenyl)urea (182) [119]. The product was obtained as a colourless oil and purified by column chromatography hexane-ethyl acetate (1.5:1) as eluent (216 mg; 90% yield). MS (FAB): m/z 503 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  8.72, (s, 1H, NHAr), 7.88–7.86 (m, 1H, Ar), 7.83–7.81 (m, 1H, Ar), 7.50–7.46 (m, 2H, Ar), 7.43– 7.41 (m, 2H, Ar), 7.43–7.27 (m, 6H, Ar), 6.48 (t, J = 6.0 Hz, 1H, CH2NH), 5.50–5.45 (m, 1H, CH), 4.63 (dd, J = 3.4 Hz, J = 12.0 Hz, 1H, CH2O), 4.48 (dd, J = 6.5 Hz, J = 12.0 Hz, 1H, CH2O), 3.64– 3.55 (m, 2H, CH2NH), 2.49 (s, 3H, CH3), 2.48 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  167.0, 166.8, 155.6, 139.8, 139.7, 139.6, 132.8, 132.7, 132.1, 132.0, 130.7, 130.6, 129.9, 129.5, 128.9, 126.5, 126.3, 125.2, 119.8, 71.8, 64.1, 31.2, 21.5, 21.4. Anal. Calcd for C26H25ClN2O5: C, 64.93; H, 5.24; N, 5.82. Found: C, 64.75; H, 5.58; N, 5.60. N-[2,3-Bis(4-methoxybenzoyloxy)propyl]-N’-(4-chlorophenyl)urea (183) [119]. The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (190 mg; 74% yield), mp 202–203 ºC. 1H NMR (500 MHz, DMSO-d6)  8.71, (s, 1H, NHAr), 7.96–7.94 (m, 2H, Ar), 7.90–7.88 (m, 2H, Ar), 7.44–7.42 (m, 2H, Ar), 7.27–7.25 (m, 2H, Ar), 7.04– 7.01 (m, 4H, Ar), 6.48 (t, J = 5.7 Hz, 1H, CH2NH), 5.45–5.41 (m, 1H, CH), 4.59 (dd, J = 3.5 Hz, J = 12.1 Hz, 1H, CH2O), 4.46 (dd, J = 6.7 Hz, J = 11.8 Hz, 1H, CH2O), 3.63–3.55 (m, 2H, CH2NH), 3.83 (s, 3H, OCH3), 3.82 (s, 3H, OCH3). 13C NMR (125 MHz, DMSO-d6)  165.1, 165.0, 163.3, 163.2, 155.1, 139.3, 131.4, 131.3, 128.4, 124.7, 121.8, 121.5, 119.2, 114.0, 113.9, 71.2, 63.4, 55.5, 55.4, 31.2. HRMS (m/z): calcd for C26H25ClN2O7Na 535.1242 M+Na+; found 535.1247. N-[2,3-Bis(4-cyanobenzoyloxy)propyl]-N’-(4-chlorophenyl)urea (184) [119]. The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1.5:1) as eluent (150 mg; 60% yield), mp 205–206 ºC. 1H NMR (500 MHz, DMSO-d6)  8.70, (s, 1H, NHAr), 8.13–8.12 (m, 2H, Ar), 8.08–8.06 (m, 2H, Ar), 8.01–7.99 (m, 4H, Ar), 7.40–7.38 (m, 2H, Ar), 7.26–7.23 (m, 2H, Ar), 6.51 (t, J = 5.7 Hz, 1H, CH2NH), 5.50–5.47 (m, 1H, CH), 4.70 (dd, J = 3.3 Hz, J = 12.4 Hz, 1H, CH2O), 4.56 (dd, J = 6.6 Hz, J = 12.0 Hz, 1H, CH2O), 3.67-3.60 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  164.3, 164.2, 155.2, 139.2, 133.5, 133.2, 132.8, 132.7, 129.9, 129.8, 128.4, 124.7, 119.3, 118.0, 117.9, 115.6, 7.23, 64.3. HRMS (m/z): calcd for 136 CHAPTER 6: EXPERIMENTAL SECTION C26H19ClN4O5Na 525.0936 M+Na+; found 525.0933. Anal. Calcd for C26H19ClN4O5: C, 62.09; H, 3.82; N, 11.14. Found: C, 62.25; H, 3.81; N, 11.00. N-[2,3-Bis(4-nitrobenzoyloxy)propyl]-N’-(4-chlorophenyl)urea (185) [119]. The product was obtained as a yellow solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (244 mg; 90% yield), mp 216-217 ºC. mp 216–217 ºC. MS (FAB): m/z 565 (100%) M+Na+. 1H NMR (500 MHz, DMSO-d6)  8.70, (s, 1H, NHAr), 8.35–8.33 (m, 4H, Ar), 8.23–8.21 (m, 2H, Ar), 8.18–8.16 (m, 2H, Ar), 7.41–7.38 (m, 2H, Ar), 7.26–7.23 (m, 2H, Ar), 6.53 (t, J = 6.01 Hz, 1H, CH2NH), 5.55–5.51 (m, 1H, CH), 4.74 (dd, J = 3.3 Hz, J = 12.2 Hz, 1H, CH2O), 4.60 (dd, J = 6.7 Hz, J = 12.2 Hz, 1H, CH2O), 3.70–3.60 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  164.1, 163.0, 155.2, 150.4, 150.3, 139.2, 134.9, 134.7, 130.8, 130.6, 128.4, 124.7, 123.9, 123.8, 119.2, 72.5, 64.4. N-{2,3-Bis[(4-trifluoromethyl)benzoyloxy]propyl}-N’-(4-chlorophenyl)urea (186). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (198 mg; 67% yield), mp 156–157 ºC. 1H NMR (500 MHz, DMSO-d6)  8.73, (s, 1H, NHAr), 8.21 (d, J = 8.53 Hz, 2H, Ar), 8.16 (d, J = 8.62 Hz, 2H, Ar), 7.93–7.88 (m, 4H, Ar), 4.43– 4.41 (m, 2H, Ar), 7.28–7.24 (m, 2H, Ar), 6.54 (t, J = 5.90 Hz, 1H, CH2NH), 5.57–5.52 (m, 1H, CH), 4.74 (dd, J = 3.6 Hz, J = 12.3 Hz, 1H, CH2O), 4.61 (dd, J = 6.6 Hz, J = 12.0 Hz, 1H, CH2O), 3.72– 3.62 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  164.4, 164.3, 155.2, 139.2, 133.3, 133.1, 133.0, 132.9, 130.2, 130.0, 128.4, 125.8, 125.7, 125.6, 124.7, 122.6, 122.5, 119.2, 72.2, 64.2. HRMS (m/z): calcd for C26H19Cl F6N2O5Na 611.0779 M+Na+; found 611.0771. Anal. Calcd for C26H19ClF6N2O5: C, 53.03; H, 3.25; N, 4.76. Found: C, 52.75; H, 3.36; N, 4.77. N-[2,3-Bis(2,4-dimethoxybenzoyloxy)propyl]-N’-(4-chlorophenyl)urea (187) The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:1.5) as eluent (235 mg; 82% yield), mp 113–114 ºC. 1H NMR (500 MHz, DMSO-d6)  8.75, (s, 1H, NHAr), 7.78–7.72 (m, 2H, Ar), 7.45 (d, J = 8.5 Hz, 2H, Ar), 7.28 (d, J = 8.9 Hz, 2H, Ar), 6.65 (s, 2H, Ar), 6.62–6.57 (m, 2H, Ar), 6.42 (t, J = 5.7 Hz, 1H, CH2NH), 5.35–5.30 (m, 1H, CH), 4.47 (dd, J = 3.2 Hz, J = 12.1 Hz, 1H, CH2O), 4.37 (dd, J = 6.5 Hz, J = 12.2 Hz, 1H, CH2O), 3.85 (d, J = 3.3 Hz, 6H, OCH3), 3.80 (d, J = 4.5 Hz, 6H, OCH3), 3.55 (t, J = 5.5 Hz, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  164.5, 164.1, 164.0, 163.9, 161.0, 160.9, 155.1, 139.3, 133.2, 133.1, 128.4, 124.6, 119.2, 111.6, 111.3, 105.3, 105.2, 98.9, 70.5, 62.9, 55.7, 55.5. HRMS (m/z): calcd for C28H29ClN2O9Na 137 CHAPTER 6: EXPERIMENTAL SECTION 595.1454 M+Na+; found 595.1447. Anal. Calcd for C28H29ClN4O9: C, 58.69; H, 5.10; N, 4.89. Found: C, 58.25; H, 5.23; N, 4.44. N-[2,3-Bis(3,4,5-trimethoxybenzoyloxy)propyl]-N’-(4-chlorophenyl)urea (188) The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:1.5) as eluent (248 mg; 78% yield), mp 163–164 ºC. 1H NMR (500 MHz, DMSO-d6)  8.73 (s, 1H, NHAr), 7.41 (d, J = 8.7 Hz, 2H, Ar),7.28-7.25 (m, 4H, Ar), 7.22 (s, 2H, Ar), 6.49 (t, J = 8.2 Hz 1H, CH2NH), 5.48-5.42 (m, 1H, CH), 4.65 (dd, J = 3.4 Hz, , J = 11.9 Hz, 1H, CH2O), 4.49-4.42 (m, 1H, CH2O), 3.81-3.76 (m, 12H, OCH3) 3.74-3.71 (m, 6H, OCH3), 3.66-3.55 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  165.0, 164.9, 155.2, 152.7, 152.6, 141.9, 141.8, 139.2, 129.2, 128.4, 124.7, 124.6, 124.3, 119.2, 106.8, 106.4, 71.7, 63.6, 60.1, 55.9, 55.8. HRMS (m/z): calcd. for C30H33ClN2O11Na 655.1665 M+Na+; found 655.1653. N-[2,3-Bis(4-methylbenzoyloxy)propyl]-N’-(4-methylphenyl)urea (189). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2.5:1) as eluent (175 mg; 84% yield), mp 95–96 ºC. 1H NMR (500 MHz, DMSO-d6)  8.44 (s, 1H, NHAr), 7.89 (d, J = 8.2 Hz, 2H, Ar), 7.82 (d, J = 8.2 Hz, 2H, Ar), 7.31 (t, J = 6.7 Hz, 4H, Ar), 7.27 (d, J = 8.4 Hz, 2H, Ar), 7.02 (d, J = 8.3 Hz, 2H, Ar), 6.39 (t, J = 6.0 Hz, 1H, CH2NH), 5.44 (quint, J = 5.2 Hz, 1H, CH), 4.62 (dd, J = 3,5 Hz, J = 8,6 Hz, 1H, CH2O), 4.47 (dd, J = 6,8 Hz, J = 5,2 Hz, 1H, CH2O), 3.63–3.54 (m, 2H, CH2NH), 2.37 (d, J = 4,0 Hz, 6H, CH3), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  165.4, 165.3, 155.3, 143.8, 143.7, 137.7, 129.9, 129.4, 129.3, 129.2, 129.1(2C), 128.9, 126.9, 126.7, 117.9, 71.4, 63.6, 26.8, 21.1, 20.3. HRMS (m/z): calcd for C27H28N2O5Na 483.1890 M+Na+; found 483.1884. Anal. Calcd for C27H28N2O5: C, 70.42; H, 6.13; N, 6.08. Found: C, 69.40; H, 5.96; N, 5.89. N-[2,3-Bis(2-methylbenzoyloxy)propyl]-N’-(4-methylphenyl)urea (190). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2.5:1) as eluent (168 mg; 74% yield), mp 116–117 ºC. 1H NMR (500 MHz, DMSO-d6)  8.45 (s, 1H, NHAr), 7.87 (d, J = 7.7 Hz, 2H, Ar), 7.83 (d, J = 7.7 Hz, 2H, Ar),7.51–7.46 (m, 2H, Ar), 7.34–7.25 (m, 6H, Ar), 7.03 (d, J = 8.0 Hz, 2H, Ar), 6.39 (t, J = 5.9 Hz, 1H, CH2NH), 5.50–5.44 (m, 1H, CH), 4.63 (dd, J = 3.3 Hz, J = 12.1 Hz, 1H, CH2O), 4.48 (dd, J = 6.8 Hz, J = 12.1 Hz, 1H, CH2O), 3.63–3.53 (m, 2H, CH2NH), 2.49 (s, 6H, CH3), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  166.5, 166.3, 155.3, 139.2, 139.1, 137.7, 132.3, 132.2, 131.6, 131.5, 130.2, 130.1, 129.9, 129.3, 129.1, 129.0, 125.9, 138 CHAPTER 6: EXPERIMENTAL SECTION 125.8, 117.2, 71.3, 63.5, 21.0, 20.8, 20.2. HRMS (m/z): calcd for C27H28N2O5Na 483.1890 M+Na+; found 483.1886. N-[2,3-Bis(4-methoxybenzoyloxy)propyl]-N’-(4-methylphenyl)urea (191). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (150 mg; 61% yield), mp 144–145 ºC. 1H NMR (500 MHz, DMSO-d6)  8.42 (s, 1H, NHAr), 7.95 (d, J = 7.5 Hz, 2H, Ar), 7.89 (d, J = 9.1 Hz, 2H, Ar), 7.27 (d, J = 8.0 Hz, 2H, Ar), 7.05–7.00 (m, 6H, Ar), 6.37 (t, J = 6.0 Hz, 1H, CH2NH), 5.43–5.39 (m, 1H, CH), 4.58 (dd, J = 3.8 Hz, J = 11.5 Hz, 1H, CH2O), 4.45 (dd, J = 3.8 Hz, J = 11.5 Hz, 1H, CH2O), 3.83 (s, 6H, OCH3), 3.62–3.53 (m, 2H, CH2NH), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  165.1, 164.9, 163.3, 155.3, 137.7, 131.4, 131.3, 129.9, 128.9, 121.8, 121.5, 117.9, 114.0, 113.9, 71.3, 63.5, 55.5, 55.4, 20.2. HRMS (m/z): calcd for C27H28N2O7Na 515.1789 M+Na+; found 515.1783. N-[2,3-Bis(4-cyanobenzoyloxy)propyl]-N’-(4-methylphenyl)urea (192). The product was obtained as a solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (155 mg; 64% yield), mp 181–182 ºC. 1H NMR (500 MHz, DMSO-d6)  8.40 (s, 1H, NHAr), 8.13 (d, J = 8.2 Hz, 2H, Ar), 8.07 (d, J = 8.2 Hz, 2H, Ar), 8.03–7.98 (m, 4H, Ar), 7.23 (d, J = 8.4 Hz, 2H, Ar), 7.02 (d, J = 8.3 Hz, 2H, Ar), 6.40 (t, J = 6.0 Hz, 1H, CH2NH), 5.51–5.47 (m, 1H, CH), 4.69 (dd, J = 3,5 Hz, J = 8,6 Hz, 1H, CH2O), 4.56 (dd, J = 6,8 Hz, J = 5,2 Hz, 1H, CH2O), 3.66–3.57 (m, 2H, CH2NH), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  164.3, 164.2, 155.3, 137.6, 133.5, 133.2, 129.9, 129.8, 129.0, 117.9, 115.6, 72.4, 64.3, 39.5, 20.2. HRMS (m/z): calcd for C27H22N4O5Na 505.1482 M+Na+; found 505.1476. Anal. Calcd for C27H22N4O5: C, 67.21; H, 4.60; N, 11.61. Found: C, 66.22; H, 4.47; N, 11.14. N-[2,3-Bis(4-nitrobenzoyloxy)propyl]-N’-(4-methylphenyl)urea (193). The product was obtained as a light yellow solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (162 mg; 62% yield), mp 214–215 ºC. 1H NMR (500 MHz, DMSO-d6)  8.45 (s, 1H, NHAr), 8.38–8.33 (m, 4H, Ar), 8.24 (d, J = 8.4 Hz, 2H, Ar), 8.19 (d, J = 8.3 Hz, 2H, Ar), 7.26 (d, J = 6.0 Hz, 2H, Ar), 7.03 (d, J = 7.0 Hz, 2H, Ar), 6.45 (t, J = 5 Hz, 1H, CH2NH) 5.56–5.52 (m, 1H, CH), 4.75 (dd, J = 3,5 Hz, J = 8,6 Hz, 1H, CH2O), 4.62 (dd, J = 6,8 Hz, J = 5,2 Hz, 1H, CH2O), 3.71–3.61 (m, 2H, CH2NH), 2.24 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  164.0, 163.9, 155.3, 150.3, 137.6, 134.9, 134.6, 130.7, 130.6, 129.9, 128.9, 123.8, 117.8, 72.5, 64.4, 39.5, 20.2. HRMS (m/z): calcd for C25H22N4O9Na 545.1279 M+Na+; found 545.1274. Anal. Calcd for C25H22N4O9: C, 57.47; H, 4.24; N, 10.72. Found: C, 56.99; H, 4.20; N, 10.68. 139 CHAPTER 6: EXPERIMENTAL SECTION N-{2,3-Bis[4-(trifluoromethyl)benzoyloxy]propyl}-N’-(4-methylphenyl)urea (194). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (179 mg; 63% yield), mp 137–138 ºC. 1H NMR (500 MHz, DMSO-d6)  8.43 (s, 1H, NHAr), 8.20 (d, J = 8.2 Hz, 2H, Ar), 8.15 (d, J = 8.2 Hz, 2H, Ar), 7.92 (d, J = 7.0 Hz, 4H, Ar), 7.26 (d, J = 8.4 Hz, 2H, Ar), 7.04 (d, J = 8.3 Hz, 2H, Ar), 6.43 (t, J = 6.1 Hz, 1H, CH2NH), 5.55–5.51 (m, 1H, CH), 4.74 (dd, J = 3,4 Hz, J = 8,7 Hz, 1H, CH2O), 4.60 (dd, J = 6,7 Hz, J = 5,4 Hz, 1H, CH2O), 3.70–3.60 (m, 2H, CH2NH), 2.24 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  164.4, 164.3, 155.4, 137.6, 133.3, 133.1, 133.0, 132.8, 132.6, 130.2, 130.0, 129.9, 129.0, 125.9, 125.8, 125.7, 125.6, 124.7, 122.6, 117.9, 72.3, 64.2, 20.2. HRMS (m/z): calcd for C27H22F6N2O5Na 681.4719 M+Na+; found 681.4715. Anal. Calcd for C27H22F6N2O5: C, 57.05; H, 3.90; N, 4.93. Found: C, 56.80; H, 3.88; N, 4.90. N-[2,3-Bis(2,4-dimethoxybenzoyloxy)propyl]-N’-(4-methylphenyl)urea (195). The product was obtained as a white resin and purified by column chromatography using hexane-ethyl acetate (1:1.5) as eluent (169 mg; 61% yield). 1H NMR (500 MHz, DMSO-d6)  8.45 (s, 1H, NHAr), 7.75 (q, J = 8.6 Hz, 2H, Ar), 7.29 (d, J = 8.0 Hz, 2H, Ar), 7.04 (d, J = 8.1 Hz, 2H, Ar), 6.64 (s, 2H, Ar), 6.59 (t, J = 8.5 Hz, 2H, Ar), 6.32–6.28 (m, 1H, CH2NH), 5.36–5.29 (m, 1H, CH), 4.51–4.43 (m, 1H, CH2O), 4.37 (dd, J = 6.5 Hz, J = 9.5 Hz, 1H, CH2O), 3.84-3.80 (m, 12H, OCH3), 3.55–3.50 (m, 2H, CH2NH), 2.24 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  164.1, 164.0, 163.9, 160.9, 155.3, 137.8, 133.2, 133.1, 129.9, 129.0, 117.8, 111.7, 111.4, 105.3, 105.2, 98.9, 98.8, 70.6, 63.0, 55.5, 55.4, 20.2. HRMS (m/z): calcd for C29H32N2O9Na 575.2000 M+Na+; found 575.1993. Anal. Calcd for C29H32N2O9: C, 63.04; H, 5.84; N, 5.07. Found: C, 62.97; H, 5.81; N, 4.99. N-[2,3-Bis(3,4,5-trimethoxybenzoyloxy)propyl]-N’-(4-methylphenyl)urea (196). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:1.5) as eluent (267 mg; 87% yield), mp 151–152 ºC. 1H NMR (500 MHz, DMSO-d6)  8.43 (s, 1H, NHAr), 7.26–7.24 (m, 4H, Ar), 7.22 (s, 2H, Ar), 7.02 (d, J = 8.1 Hz, 2H, Ar), 6.38 (t, J = 8.5Hz 1H, CH2NH), 5.47–5.42 (m, 1H, CH), 4.65 (dd, J = 3.6 Hz, J = 8.5Hz, 1H, CH2O), 4.45 (dd, J = 3.5 Hz, J = 8.4 Hz, 1H, CH2O), 3.82-3.72 (m, 18H, OCH3) 3.63–3.55 (m, 2H, CH2NH), 2.22 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  165.0, 164.9, 155.4, 152.7, 152.6, 142.0, 141.9, 137.7, 130.0, 128.9, 124.7, 124.4, 117.9, 106.8, 106.6, 71.8, 63.7, 60.1, 55.9, 55.8, 39.5, 20.2. HRMS (m/z): calcd for C31H36N2O11Na 635.2211 M+Na+; found 635.2202. Anal. Calcd for C25H22N4O9: C, 60.78; H, 5.92; N, 4.57. Found: C, 60.90; H, 5.94; N, 4.58. 140 CHAPTER 6: EXPERIMENTAL SECTION N-[2,3-Bis(4-methylbenzoyloxy)propyl]-N’-[4-(trifluoromethyl)phenyl]urea (197). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (3:1) as eluent (217 mg; 84%yield), mp 111–112 ºC. 1H NMR (500 MHz, DMSO-d6)  9.00, (s, 1H, NHAr), 7.89 (d, J = 8.2 Hz, 2H, Ar), 7.83 (d, J = 8.2 Hz, 2H, Ar), 7.61–7.55 (m, 4H, Ar), 7.32–7.29 (m, 4H, Ar), 6.59 (t, J = 5.9 Hz, 1H, CH2NH), 5.48–5.44 (m, 1H, CH), 4.63 (dd, J = 3.3 Hz, J = 12.1 Hz, 1H, CH2O), 4.48 (dd, J = 6.9 Hz, J = 12.1 Hz, 1H, CH2O), 3.67–3.58 (m, 2H, CH2NH), 2.37, 2.36 (2s, 6H, CH3). 13C NMR (125 MHz, DMSO-d6)  165.4, 165.2, 154.9, 144.0, 143.8, 143.7, 129.4, 129.3, 129.2, 129.1, 129.0, 126.8, 126.6 125.8, 125.7, 117.3, 71.3, 63.5, 21.1, 21.0. HRMS (m/z): calcd for C27H25F3N2O5Na: 537.1608 M+Na+; found 537.1600 Anal. Calcd for C27H25F3N2O5: C, 63.03; H, 4.90; N, 5.44. Found: C, 62.88; H, 4.87; N, 5,38. N-[2,3-Bis(2-methylbenzoyloxy)propyl]-N’-[4-(trifluoromethyl)phenyl]urea (198). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2.5:1) as eluent (188 mg; 73% yield), mp 112–113 ºC. 1H NMR (500 MHz, DMSO-d6)  9.05 (s, 1H, NHAr), 7.90 (d, J = 8.2 Hz, 1H, Ar), 7.85 (d, J = 8.2 Hz, 1H, Ar), 7.61 (q, J = 8.7 Hz, 4H, Ar), 7.52–7.47 (m, 2H, Ar), 7.35–7.28 (m, 4H, Ar), 6.63 (t, J = 6.1 Hz, 1H, CH2NH), 5.54-5.50 (m, 1H, CH), 4.67 (dd, J = 3,5 Hz, J = 11,8 Hz, 1H, CH2O), 4.52 (dd, J = 6,8 Hz, J = 11,9 Hz, 1H, CH2O), 3.68–3.61 (m, 2H, CH2NH), 2.51 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6)  166.5, 166.3, 154.9, 143.9, 139.3, 139.2, 132.3, 132.2, 131.6, 131.5, 130.2, 130.1, 129.3, 128.9, 125.9, 123.5, 121.3, 117.4, 71.2, 63.5, 39.5, 21.0, 20.9. HRMS (m/z): calcd for C27H25F3N2O5Na 515.1788 M+Na+; found 515.1782. Anal. Calcd for C27H25 F3N2O5: C, 63.03; H, 4.90; N, 5.44. Found: C, 62.52; H, 4.92; N, 5.49. N-[2,3-Bis(4-methoxybenzoyloxy)propyl]-N’-[4-(trifluoromethyl)phenyl]urea (199). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (195 mg; 71% yield), mp 128–129 ºC. 1H NMR (500 MHz, DMSO-d6)  9.01 (s, 1H, NHAr), 7.98–7.96 (m, 2H, Ar), 7.94–7.89 (m, 2H, Ar) 7.60 (q, J = 8.0 Hz, 4H, Ar), 7.07–7.03 (m, 4H, Ar), 6.60 (t, J = 6.9 Hz, 1H, CH2NH), 5.47–5.43 (m, 1H, CH), 4.61 (dd, J = 3.6 Hz, J = 11.8 Hz, 1H, CH2O), 4.48 (dd, J = 7.0 Hz, J = 11.9 Hz, 1H, CH2O), 3.85 (s, 6H, OCH3), 3.65–3.60 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  165.1, 164.9, 163.3, 163.2, 154.9, 143.9, 132.6, 131.4, 131.2, 123.5, 121.7, 121.5, 117.3, 114.1, 114.0, 113.9, 113.8, 71.1, 63.4, 55.5, 39.5. HRMS (m/z): calcd for C27H25F3N2O7Na 569.1506 M+Na+; found 569.1497. Anal. Calcd for C27H25F3N2O7: C, 59.34; H, 4.61; N, 5.13. Found: C, 59.18; H,4.57; N, 5.51. 141 CHAPTER 6: EXPERIMENTAL SECTION N-[2,3-Bis(4-cyanobenzoyloxy)propyl]-N’-[4-(trifluoromethyl)phenyl]urea (200). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (205 mg; 76% yield), mp 170–171 ºC. 1H NMR (500 MHz, DMSO-d6)  9.00, (s, 1H, NHAr), 8.14 (d, J = 8.2 Hz, 2H, Ar), 8.08 (d, J = 8.2 Hz, 2H, Ar), 8.02-7.99 (m, 4H, Ar), 7.56 (m, 4H, Ar), 6.63 (t, J = 5.9 Hz, 1H, CH2NH), 5.53–5.49 (m, 1H, CH), 4.71 (dd, J = 3.5 Hz, J = 12.1 Hz, 1H, CH2O), 4.57 (dd, J = 6.7 Hz, J = 12.1 Hz, 1H, CH2O), 3.69–3.62 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  164.3, 164.2, 155.0, 143.9, 133.4, 133.2, 132.8, 132.7, 130.0, 129.8, 125.9, 117.9, 117.4, 115.6, 72.2, 64.3. HRMS (m/z): calcd for C27H19F3N4O5Na: 559.1200 M+Na+; found 559.1192 Anal. Calcd for C27H19F3N4O5: C, 60.45; H, 3.57; N, 10.44. Found: C, 59.85; H, 3.56; N, 10.42. N-[2,3-Bis(4-nitrobenzoyloxy)propyl]-N’-[4-(trifluoromethyl)phenyl]urea (201). The product was obtained as a yellow solid and purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (195 mg; 68% yield), mp 186–187 ºC. 1H NMR (500 MHz, DMSO-d6)  9.00, (s, 1H, NHAr), 8.35–8.32 (m, 4H, Ar), 8.23 (d, J = 8.7 Hz, 2H, Ar), 8.17 (d, J = 8.6 Hz, 2H, Ar), 7.56 (m, 4H, Ar), 6.65 (t, J = 5.4 Hz, 1H, CH2NH), 5.54 (m, 1H, CH), 4.74 (dd, J = 2.8 Hz, J = 12.1 Hz, 1H, CH2O), 4.61 (dd, J = 6.4 Hz, J = 12.1 Hz, 1H, CH2O), 3.71–3.63 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  164.0, 163.9, 155.0, 150.4, 150.3, 134.9, 134.7, 130.8, 130.6, 125.9, 123.9, 123.8, 117.3, 129.1, 129.0, 126.8, 126.6 125.8, 125.7, 117.3, 72.4, 64.4. HRMS (m/z): calcd for C25H19F3N4O9Na: 599.0996 M+Na+; found 599.0988. Anal. Calcd for C25H19F3N4O9: C, 52.09; H, 3.32; N, 9.72. Found: C, 51.63; H, 3.22; N, 9.78. N-{2,3-Bis[4-(trifluoromethxyl)benzoyloxy]propyl}-N’-[4-(trifluoromethyl)phenyl]urea (202). The product was obtained as a white solid and purified by column chromatography using hexaneethyl acetate (2.5:1) as eluent (236 mg; 76% yield), mp 135–136 ºC. 1H NMR (500 MHz, DMSO-d6)  9.01 (s, 1H, NHAr), 8.17 (d, J = 8.2 Hz, 2H, Ar), 8.11 (d, J = 8.2 Hz, 2H, Ar), 7.90-7.86 (m, 4H, Ar), 7.58-7.52 (m, 4H, Ar), 6.63 (t, J = 6.0 Hz, 1H, CH2NH), 5.55–5.50 (m, 1H, CH), 4.52 (dd, J = 3.4 Hz, J = 12.1 Hz, 1H, CH2O), 4.58 (dd, J = 6.6 Hz, J = 12.2 Hz, 1H, CH2O), 3.70–3.61 (m, 2H, CH2NH). 13C NMR (125 MHz, DMSO-d6)  164.9, 164.7, 155.5, 144.4, 133.8, 133.6, 133.3, 130.7, 130.5, 127.4, 126.4, 126.3, 126.2, 125.2, 123.9, 117.8, 72.6, 64.7. HRMS (m/z): calcd. for C27H19F9N2O5Na 645.1042 M+Na+; found 645.1035. N-[2,3-Bis(2,4-dimethoxybenzoyloxy)propyl]-N’-[4-(trifluoromethyl)phenyl]urea (203). The product was obtained as a white solid and purified by column chromatography using hexane-ethyl