We find that all of the monosaccharidedelivery agents display a significantly improved boron delivery capacityover the delivery agents approved for clinical use in vitro, thusproviding a sound foundation for advancing toward in vivo preclinicalassessment studies.Boron neutron capture therapy (BNCT) is a cancer therapyin whichboron delivery agents play a crucial role. In theory, delivery agentswith high tumor targeting capabilities can lead to selective eradicationof tumor cells without causing harmful side effects. We have beenworking on a GLUT1-targeting strategy to BNCT for a number of yearsand found multiple promising hit compounds which outperform the clinicallyemployed boron delivery agents in vitro. Herein, we continue our workin the field by further diversification of the carbohydrate scaffoldin order to map the optimal stereochemistry of the carbohydrate core.In the sweet battle of the epimers, carborane-bearing  In BNCT, the goal is to deliver a sufficient amount of 10B atoms selectively to tumor cells and irradiate the tumorregion with thermal neutrons . Thus, ahighly selective tumor-targeting agent provides the best foundationfor reaching a high 10B-distribution gradient between tumorand healthy tissue, a pre-requisite for a successful treatment outcome.Previous medicinal chemistry campaigns, aimed at improved deliveryagents, have indicated the challenges involved and supplied a substantiallist of criteria that ideal 10B-delivery agents need tofulfill in order to become suitable for BNCT.11 The most important ones relate to selectivity, i.e., high tumor-to-blood(T/B) and tumor-to-normal tissue ratios (T/N) , low systemic toxicity (otherwise other emerging cancertherapies may be better), boron delivery capacity in the range of20−35 μg/1 g of tumor tissue,5 reasonable production costs, well-proven mechanism of action, andknown metabolic fate. Despite the significant efforts invested inthe development of improved delivery agents by a large number of researchteams,11 only three delivery agents  have been approved for clinical use with none displaying optimalproperties. Therefore, there is a need for new and improved deliveryagents.Over the past 70 years,boron neutron capture therapy (BNCT) hassteadily been developing as a promising cancer therapy.neutrons 1.4−6 The17 many attemptshave been made to capitalize on their significant potential.19 Our team has in recent yearscontributed to the exploration of a GLUT1-targeting strategy to BNCT.21 The GLUT1-targeting approachis appealing as it is already a clinically validated tumor-targetingstrategy applied in the diagnosis and staging of many types of cancers[most prominent example is the use of 2-deoxy-2-[fluorine-18]fluoro-d-glucose (18F-FDG) in positron emission tomography(PET) imaging].24 Adequate tumor-to-healthy tissue selectivity canin theory be reached through the “Warburg effect”,25 which describes the inefficient d-glucosemetabolism in cancer cells leading to an overexpression of GLUT1 transporterson the cell membrane and an increased d-glucose uptake. Ourprevious work has showcased that carefully designed d-glucose-baseddelivery agents for BNCT are able to outperform the delivery agentsin current clinical use in vitro.21 In fact, our prime candidate (6-oCb-Glc) was foundto have a 40-fold boron delivery capacity compared to BPA and BSH.16 Nevertheless, the synthesis and in vitro assessmentof positional isomers and different connecting atoms in the carboranecluster revealed that structurally similar glucose-based deliveryagents may not be equally promising.21Since the initial work by Hawthorne and co-workersin 1988 on glycoconjugatedelivery agents for BNCT,27 In addition, these monosaccharides may display altered uptake profilesthrough other potential hexose transporters, thus affecting theirboron delivery capacity. Therefore, we set out to synthesize and assessthe potential of carboranylmethyl-bearing epimers of d-glucose(Glc), i.e., d-mannose (Man), d-galactose ,and d-allose (All) was performed in the CAL 27 oral adenosquamous carcinoma cell line28 in order to be able to compare the results to those obtained earlierin the Glc series.20In ourcontinued work to map the boundaries of the GLUT1-targetingapproach and optimize the delivery agents, we herein focus on assessmentof the stereochemistry in the carbohydrate core. This is a valid approachas the GLUT family is known to accept a variety of monosaccharides.All) see 2. We lim22.11H:499.83 MHz, 13C: 125.69 MHz, 11B: 160.36 MHz).All products were characterized with 1D: 1H, 13C{1H}, 11B{1H}, and 1D-TOCSY and2D-techniques: ed-HSQC, HMBC, and COSY by using pulse sequences providedby the instrument’s manufacturer. Chemical shifts are expressedon the δ scale (in ppm) using TMS, residual chloroform, methanol,or 15% BF3 in CDCl3 (11B NMR) asinternal standards. Coupling constants were obtained through spectralsimulations with the ChemAdder spectral simulation software. Theyare given in Hz and provided only once, when first encountered. Couplingpatterns are given as s (singlet), d (doublet), dd (doublet of a doublet),etc. HRMS spectra were recorded using a Bruker Micro Q-TOF with ESI(electrospray ionization) operated in positive mode. The purity ofglycoconjugates 1–3 was determined by qNMR studiesusing maleic acid as an internal standard. TLC was performed on aluminumsheets precoated with silica gel 60F254 (Merck), and spots were visualizedby spraying with conc. H2SO4:MeOH (1:5) andheating. Flash chromatography was carried out on silica gel 40.All reaction protocols requiring inert reaction conditions wereperformed under an argon atmosphere using dry solvents . Reagents and commercially available starting materialwere purchased from commercial sources. All NMR spectra were recordedwith a 500 MHz Bruker Avance III NMR spectrometer (2.1.12.1.1.1tert-Butyldimethylsilyl chloride (1.1 equiv) was slowlyadded to the reaction mixture. After the addition of the silylatingagent, the reaction mixture was brought to r.t. and stirred for 2–4h. Upon completion of the reaction, the solvent was removed underreduced pressure, and the crude product was purified by column chromatography(DCM:MeOH 8:1).The correspondingmonosaccharide (1.0 equiv) was dissolved in dry pyridine  and brought to 0 °C using an ice bath. 2.1.1.23 . The aqueous phase was extracted with DCM . The combined organic phases werewashed with brine , dried over anhydrousMgSO4, filtered, and concentrated. The crude product waspurified by column chromatography (EtOAc:Hex 1:3).The corresponding carbohydrate was dissolvedin dry DMF  and brought to 0 °Cusing an ice bath. NaH  wasadded slowly to the reaction mixture and left to stir for 15 min.The reaction mixture was brought to r.t., and the corresponding alkylbromide (1.5 equiv/OH group) was added. The mixture was left to stirfor 2–3 h. After the reaction was completed, the reaction mixturewas quenched by the addition of MeOH ,diluted with DCM , and washed witha saturated aqueous solution of NaHCO2.1.1.33 . The aqueous phase was extractedwith DCM . Combined organicphases were washed with brine .The combined organic phases were dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified by columnchromatography (EtOAc:Hex 1:2).The silylated glycoside(1.0 equiv) was dissolved in dry THF at 0 °C, and HF·pyridine  was added to the reaction mixture. The resulting mixturewas slowly brought to r.t. and stirred for 18 h. The reaction mixturewas diluted with DCM  and quenchedwith the addition of saturated aqueous solution of NaHCO2.1.1.410H14 (1.6 equiv)was dissolved in dry MeCN  and heatedto 60 °C. After 1 h, the propargylated glycoside (1.0 equiv)was dissolved in dry toluene  andadded to the reaction mixture. Upon the addition, the temperaturewas increased to 80 °C, and the reaction mixture was left tostir for 16 h. Upon reaction completion, the reaction mixture wasquenched with dry MeOH  and stirredfor an additional 30 min at 80 °C. The solvents were removedunder reduced pressure, and the crude product was purified by columnchromatography (EtOAc:Hex 1:3).B2.1.1.52 three times and then filled with H2 (4 bar). Thereaction was left to stir for 4–6 h. Upon reaction completion,the resulting mixture was filtered through celite, washed with EtOAc:MeOH, and concentrated under reduced pressure. Thecrude product was purified by column chromatography (DCM:MeOH 7:1).The benzyl-protected glycoside was dissolvedin EtOAc:MeOH . 10% Pd/C(1 weight equiv) was added to the reaction mixture, and the reactionvessel was placed in an autoclave. The reactor was flushed with N2.1.1.62O. TFA and H2O wereadded to the reaction mixture  and left to stir at r.t.for 24 h. The resulting mixture was concentrated under reduced pressure,and the crude product was purified by column chromatography (DCM:MeOH7:1).The corresponding protected glycoside (1.0equiv) was dissolved in Et2.1.22.1.2.1d-mannose and TBDMSCl  according to the general procedurefor silylation of the primary 6-OH group of monosaccharides. Thisreaction gave the title compound as a white solid . TLC: Rf: 0.61 (DCM:MeOH 5:1).Thiscompound was synthesized from 1HNMR : δ = 5.05 , 3.94 , 3.83 , 3.77 ,3.75 , 3.74, 3.59 , 0.92 2C(C3H)3) and 0.10 and 0.09 2C(CH3)3) ppm.13C{1H} NMR :δ = 95.7 (C-1), 74.3 (C-3), 72.8 (C-2), 72.4 (C-5), 69.0 (C-4),64.9 (C-6), 26.5 (6-OSi(CH3)2C(CH3)3), 19.3 (6-OSi-(CH3)2C(CH3)3) and −5.10 (6-OSi(CH3)2C(CH3)3)ppm.m/z calcd for C12H26O6SiNa [M + Na]+ 317.1397;found317.1302.HRMS: 2.1.2.2O-(tert-butyldimethylsilyl)-β-d-mannopyranose, NaH , and BnBr  according to the general procedure for alkylation of a freeOH group. This reaction gave the title compound as a colorless oil. TLC: Rf: 0.8 (EtOAc:Hex 1:2).This compound was synthesized from 6-1HNMR : δ = 7.46–7.23, 4.92 and 4.63 , 4.91 , 4.71 and 4.65 , 4.68 and 4.42 , 4.64 and 4.55 , 3.95 , 3.94 , 3.86 , 3.85 , 3.79 , 3.67 , 0.90 2C(C3H)3) and0.09 and 0.07 2C(CH3)3) ppm.α anomer: 13C{1H} NMR : δ= 139.0–127.2 (arom. C), 96.9 (C-1),80.3 (C-3), 75.2 (C-2), 75.1 (4-OCH2Ph and3-OCH2Ph), 75.0 (C-4), 73.5 (C-5), 72.5 (2-OCH2Ph), 68.6 (1-OCH2Ph),62.8 (C-6), 26.0 (6-OSi(CH3)2C(CH3)3), 18.3 (6-OSi(CH3)2C(CH3)3) and −5.1 and −5.2(6-OSi(CH3)2C(CH3)3 ppm.1H NMR : δ = 7.46–7.23 , 4.97and 4.86 , 4.94 and 4.58 ,4.92 and 4.64 , 4.53 and 4.46 , 4.39 ,3.92 , 3.89 , 3.87 , 3.86 ,3.47 , 3.26 , 0.90 2C(C3H)3) and0.09 and 0.07 2C(CH3)3) ppm.β anomer: 13C{1H} NMR : δ= 139.0–127.2 (arom. C), 99.9 (C-1),82.3 (C-3), 77.1 (C-5), 75.1 (4-OCH2Ph), 74.9(C-4), 74.1 (C-2), 73.7 (2-OCH2Ph), 71.4 (3-OCH2Ph), 70.4 (1-OCH2Ph),62.9 (C-6), 25.9 (6-OSi(CH3)2C(CH3)3), 18.4 (6-OSi(CH3)2C(CH3)3) and −5.1 and −5.2(6-OSi(CH3)2C(CH3)3 ppm.m/z calcdfor C40H50O6SiNa [M + Na]+ 677.3275; found677.3036.HRMS: 2.1.2.3O-benzyl-6-O-(tert-butyldimethylsilyl)-d-mannopyranoside according to the general procedure for silyl groupremoval using HF·pyridine. This reaction gave the title compoundas a white solid . TLC: Rf: 0.34 (EtOAc:Hex 1:2).This compound wassynthesized from 1,2,3,4-tetra-1H NMR : δ= 7.46–7.22 , 4.94 and 4.62 , 4.90 ,4.88 and 4.64 , 4.75 and 4.66 , 4.65 and 4.43 , 4.00 , 3.97 ,3.92 , 3.82 , 3.78 , 3.70  and 2.02  ppm.α anomer: 13C{1H} NMR : δ = 138.7–127.6 (arom. C), 97.7 (C-1),80.3 (C-3), 75.4 (4-OCH2Ph), 75.0 (C-4), 74.9(C-2), 73.0 (2-OCH2Ph), 72.5 (C-5), 72.4 (3-OCH2Ph), 69.2 (1-OCH2Ph)and 62.5 (C-6) ppm.1H NMR : δ = 7.46–7.22 , 4.98and 4.88 , 4.95 and 4.60 ,4.94 and 4.64 , 4.52 and 4.47 , 4.48 ,3.94 , 3.93 , 3.91 , 3.78 , 3.51 , 3.32  and 2.15 ppm.β anomer: 13C{1H} NMR : δ = 138.7–127.6 (arom.C), 100.6 (C-1),82.5 (C-3), 76.0 (C-5), 75.4 (4-OCH2Ph), 75.0(C-4), 74.2 (C-2 and 2-OCH2Ph), 71.7 (3-OCH2Ph), 71.3 (1-OCH2Ph)and 62.7 (C-6) ppm.m/z calcd for C34H36O6Na [M + Na]+ 563.2410; found563.3879.HRMS: 2.1.2.44 , NaH , and propargyl bromide  according to the general procedure for alkylation of a freeOH group. This reaction gave the title compound as a colorless oil. TLC: Rf: 0.71 (EtOAc:Hex 1:2).This compound was synthesized from 1H NMR : δ = 7.46–7.22, 4.94 , 4.92 and 4.67 , 4.71 and 4.70, 4.69 and 4.44 ,4.62 and 4.60 , 4.28 ,4.21 , 4.01 , 3.94 , 3.89 , 3.82 , 3.80 , 3.75 and 2.38  ppm.α anomer: 13C{1H} NMR : δ = 138.9–127.5 (arom.C), 97.5 (C-1),80.3 (6-OCH2C≡CH), 80.0 (C-3), 75.3(4-OCH2Ph), 74.9 (C-4), 74.7 (C-2), 74.6 (6-OCH2C≡CH), 72.7 (2-OCH2Ph), 72.3 (3-OCH2Ph), 71.9 (C-5), 69.2 (1-OCH2Ph), 68.9 (C-6) and 58.7 (6-OCH2C≡CH) ppm.1H NMR: δ = 7.46–7.24, 5.00and 4.88 , 4.99 and 4.58 ,4.94 and 4.67 , 4.50 and 4.44 , 4.42 ,4.27 , 4.24 ,3.93 , 3.92 , 3.87 ,3.86 , 3.48, 3.42  and 2.38  ppm.β anomer: 13C{1H}NMR : δ = 138.9–127.5(arom. C), 100.5 (C-1),82.4 (C-3), 80.3 (6-OCH2C≡CH), 75.8(C-5), 75.3 (4-OCH2Ph), 74.8 (C-4), 74.6 (6-OCH2C≡CH), 74.0 (2-OCH2Ph), 73.9 (C-2), 71.6 (3-OCH2Ph), 71.0 (1-OCH2Ph), 69.2 (C-6) and 58.8 (6-OCH2C≡CH) ppm.m/z calcd For C37H38O6Na [M + Na]+ 601.2566; found601.2229.HRMS: 2.1.2.55  and B10H14  according to the general procedure for installationof the carboranyl moiety. This reaction gave the title compound asa white solid . TLC: Rf:0.70 (EtOAc:Hex 1:2).This compound was synthesizedfrom 1H NMR : δ = 7.44–7.23 ,4.94 and 4.84 , 4.94 and 4.59 , 4.90 and 4.57 , 4.51 and 4.44 , 4.41 ,4.06 , 4.01 and 3.94 , 3.91 , 3.85 , 3.81 , 3.64 , 3.46 , 3.30  and 2.98–1.36 ppm.13C{1H} NMR : δ= 138.6–127.7 (arom. C), 100.5 (C-1),82.3 (C-3), 75.8 (C-5), 75.3 (2-OCH2Ph), 74.2(4-OCH2Ph), 74.1 (C-4), 74.0 (C-2), 73.2 (carborane C), 73.1 (6-OCH2-carborane), 71.6(3-OCH2Ph), 71.1 (1-OCH2Ph), 71.0 (C-6) and 58.0 (carborane CH) ppm.11B{1H} NMR :δ = −2.4, −4.3, −8.7, −11.2,and −12.7 ppm.m/z calcd for C37H48B10O6Na [M + Na]+ 721.4279;found 721.4298.HRMS: 2.1.2.66 and 10% Pd/C (0.22 g) according to the general procedure for deprotectionof benzyl groups. This reaction gave the title compound as a whitesolid . TLC: Rf: 0.70 (DCM:MeOH 5:1).This compound was synthesized from 1H NMR : δ = 5.03 , 4.68 , 4.06 and 4.02 , 3.783, 3.781 , 3.77 , 3.73 ,3.71 , 3.62  and 2.99–1.36  ppm.α anomer: 13C{1H} NMR :δ = 95.9 (C-1), 75.2 (carborane C), 73.9 (6-OCH2-carborane), 73.3 (C-2), 72.7 (C-6), 72.6 (C-2),72.2 (C-5), 68.4 (C-4), and 60.4 (carborane CH) ppm.11B{1H} NMR : δ = −2.8, −4.6, −9.0, −11.2,and −12.7 ppm.All forms: 1H NMR :δ = 4.71 , 4.68 , 4.07 and 4.03 , 3.80 , 3.78 ,3.73 , 3.54, 3.43 , 3.28  and 2.99–1.36  ppm.β anomer: 13C{1H} NMR :δ = 95.6 (C-1), 77.2 (C-5), 75.3 (C-3), 75.2 (carborane C), 73.9 (6-OCH2-carborane), 73.0(C-6), 72.4 (C-2), 68.1 (C-4) and 60.6 (carborane CH)ppm.m/z calcd for C9H24B10O6Na [M + Na]+ 361.2401;found 361.2375.HRMS: 2.1.2.7O-benzyl-6-O-(tert-butyldimethylsilyl)-d-allopyranoside according to the general procedure for silyl groupremoval using HF-pyridine. This reaction gave the title compound asa white solid . TLC: Rf:0.32 (EtOAc:Hex 1:2).This compound wassynthesized from 1,2,3,4-tetra-1H NMR : δ = 7.45–7.22 ,5.02 and 4.86 , 4.97 , 4.80 and 4.57 ,4.58 and 4.42 , 4.56 and 4.54 , 4.24 , 4.19 , 3.75 , 3.73 , 3.42  and3.40  ppm.13C{1H} NMR : δ = 139.75–127.2(arom. C), 96.4 (C-1),77.0 (C-2), 74.9 (C-4), 73.7 (3-OCH2Ph), 72.5(C-3), 71.4 (2-OCH2Ph), 71.0 (4-OCH2Ph), 69.8 (1-OCH2Ph), 67.1 (C-5)and 62.2 (C-6) ppm.m/z calcd for C34H36O6Na [M + Na]+ 563.2410; found563.2381.HRMS: 2.1.2.87 , NaH , and propargyl bromide  according to the general procedure for alkylation ofa free OH group. This reaction gave the title compound as a whitesolid . TLC: Rf: 0.73 (EtOAc:Hex1:2).This compound was synthesized from 1H NMR :δ = 7.45–7.20 , 5.00 , 4.99 and 4.87 , 4.81 and 4.57 , 4.57 and 4.50 , 4.528 and 4.529 , 4.28 , 4.19 , 4.10 , 4.10 ,3.87 , 3.58 , 3.53 , 3.43 and2.35  ppm.13C{1H} NMR : δ = 139.6–127.1 (arom. C), 96.7 (C-1),79.7 (6-OCH2C≡CH), 76.8 (C-2), 74.9(6-OCH2C≡CH), 74.5 (C-4), 73.8 (3-OCH2Ph), 73.0 (C-3), 71.4 and 71.3 (2-OCH2Ph and 4-OCH2Ph), 69.8 (1-OCH2Ph), 68.4 (C-6), 66.4 (C-5) and 58.7 (6-OCH2C≡CH) ppm.m/z calcd. For C37H38O6Na [M + Na]+ 601.2566; found601.3194.HRMS: 2.1.2.98  and B10H14  according to the general procedure for installationof the carboranyl moiety. This reaction gave rise to the title compoundas a white solid . TLC: Rf: 0.72 (EtOAc:Hex 1:2).This compound was synthesizedfrom 1H NMR : δ = 7.44–7.21 ,5.03 and 4.83 , 4.97 , 4.78 and 4.56 ,4.58 and 4.57 , 4.56 and 4.32 , 4.27 , 4.24 , 3.89 and 3.80 , 3.75 , 3.67,3.54 , 3.41 , 3.32  and 2.99–0.92 ppm.13C{1H} NMR : δ= 139.3–127.3 (arom. C), 96.3 (C-1),77.4 (C-2), 74.6 (C-4), 73.8 (3-OCH2Ph), 73.0(carborane C), 72.9 (6-OCH2-carborane),72.2 (C-3), 71.5 (2-OCH2Ph), 71.4 (C-6), 70.5(4-OCH2Ph), 69.8 (1-OCH2Ph), 66.8 (C-5) and 57.6 (carborane CH) ppm.11B{1H} NMR :δ = −3.2, −5.0, −9.3, −11.9,and −13.4 ppm.m/z calcd for C37H48B10O6Na [M + Na]+ 721.4279;found 721.4239.HRMS: 2.1.2.109  and 10% Pd/C (0.28 g) according to the general procedure fordeprotection of benzyl groups. This reaction gave the title compoundas a white solid . TLC: Rf: 0.69 (DCM:MeOH 5:1). Product distribution: α-pyranose:β-pyranose:α-furanose:β-furanose28:63:3:6.This compound was synthesized from Pyranose Forms1H NMR :δ = 5.01 , 4.60 , 4.06 , 4.03 and 4.02 , 3.95 ,3.77 , 3.76 , 3.50 , 3.46 and 2.99–1.36  ppm.α anomer: 13C{1H} NMR :δ = 95.2 (C-1), 75.3 (carborane C), 74.1 (C-3),73.8 (6-OCH2-carborane), 72.7 (C-6), 69.0(C-2), 68.1 (C-4), 68.0 (C-5) and 60.6 (carborane CH)ppm.1H NMR :δ = 4.79 , 4.63 , 4.04 and 4.01 , 4.04 , 3.77 ,3.76 , 3.68 , 3.47 , 3.22 and 2.99–1.36  ppm.β anomer: 13C{1H} NMR :δ = 95.4 (C-1), 75.3 (carborane C), 75.4 (C-5),73.9 (6-OCH2-carborane), 73.4 (C-2), 72.8(C-6 and C-3), 68.7 (C-4), and 60.6 (carborane CH) ppm.Furanose Forms1H NMR :δ = 5.20 , 4.68 , 4.08 , 4.04 and 4.02 , 3.96 , 3.94 , 3.71 , 3.62 , 3.55  and2.99–1.36  ppm.α anomer: 1H NMR :δ = 5.11 , 4.68 , 4.29 , 4.04 and 4.01 , 3.821, 3.820 , 3.78 , 3.67 , 3.58  and2.99–1.36  ppm.β anomer: 11B{1H} NMR : δ = −2.7, −4.6, −8.9, −11.1,−12.2, and −12.6 ppm.All forms: m/z calcd for C9H24B10O6Na [M + Na]+ 361.2401;found 361.2440.HRMS: 2.1.2.11O-(isopropylidene)-α-d-galactopyranoside, NaH , and propargyl bromide according to the general procedure for alkylationof a free OH group. This reaction gave the title compound as a whitesolid . TLC: Rf: 0.34 (EtOAc:Hex1:4).This compound was synthesized from 1,2:3,4-bis-1H NMR :δ = 5.54 , 4.61 , 4.32 , 4.26, 4.25 , 4.20 , 4.00, 3.77 , 3.67 , 2.43 , 1.54, 1.45, 1.34and 1.33 2 and 3,4-C(CH3)2) ppm.13C{1H} NMR: δ = 109.5 , 108.8 , 96.5 (C-1), 79.8 (6-OCH2C≡CH),74.7 (6-OCH2C≡CH), 71.3 (C-4), 70.8(C-3), 70.6 (C-2), 68.9 (C-6), 66.9 (C-5), 58.7 (6-OCH2C≡CH), 26.2, 26.1, 25.1 and 24.6 2 and 3,4-C(CH3)2) ppm.m/z calcd for C15H22O6Na[M + Na]+ 298.1416; found298.1458.HRMS: 2.1.2.1210  and B10H14  according to the general procedure for installation ofthe carboranyl moiety. This reaction gave the title compound as awhite solid . TLC: Rf: 0.43(EtOAc:Hex 1:4).This compound was synthesized from 1H NMR : δ = 5.50 , 4.60 , 4.31 , 4.16 ,4.10 , 4.04 and 3.93 , 3.91 , 3.70 , 3.61, 3.16–1.10 , 1.53, 1.43, 1.33 and 1.32 2 and 3,4-C(CH3)2) ppm.13C{1H} NMR :δ = 109.7 , 108.9 , 96.4(C-1), 74.7 (carborane-C), 72.7 (6-OCH2-carborane), 71.2 (C-6), 71.1 (C-4), 70.8 (C-3),70.6 (C-2), 67.4 (C-5), 57.8 (carborane-CH), 26.2, 26.1,25.0, and 24.5 2 and3,4-C(CH3)2) ppm.11B{1H} NMR :δ = −3.1, −5.0, −9.3, −11.8,and −13.4 ppm.m/z calcd for C15H32B10O6Na [M + Na]+ 441.3027;found 441.3055.HRMS: 2.1.2.1311  accordingto the general procedure for removal of acetal protecting groups.This reaction gave the title compound as a white solid .TLC: Rf: 0.64 (EtOAc:MeOH 5:1). Productdistribution: α-pyranose:β-pyranose:α-furanose:β-furanose40:47:8:5.This compoundwas synthesized from Pyranose Forms1H NMR :δ = 5.14 , 4.60 , 4.15 , 4.04 and4.00 , 3.84 , 3.78 , 3.73 , 3.727 , 3.63 , and 3.01–1.43 ppm.α anomer: 13C{1H} NMR :δ = 94.2 (C-1), 75.1 (carborane C), 73.7 (6-OCH2-carborane), 72.6 (C-6), 71.1 (C-3), 71.0 (C-4),70.3 (C-2), 70.0 (C-5) and 60.7 (carborane CH) ppm.1H NMR :δ = 4.44 , 4.63 , 4.05 and 4.01 , 3.78 , 3.74 , 3.69 , 3.68 , 3.48 , 3.46  and 3.01–1.43  ppm.β anomer: 13C{1H} NMR :δ = 98.7 (C-1), 75.1 (carborane C), 74.8 (C-3 andC-5), 73.7 (C-2), 73.66 (6-OCH2-carborane),72.3 (C-6), 70.4 (C-4) and 60.8 (carborane CH) ppm.Furanose Forms1H NMR :δ = 5.12 , 4.66 , 4.03 , 4.03 and 4.00 , 4.00 , 3.90 , 3.82 , 3.63 , 3.58  and3.01–1.43  ppm.α anomer: 1H NMR :δ = 5.17 , 4.66 , 4.14 , 4.03 and4.01 , 3.91 , 3.77 , 3.76 , 3.62 , 3.54  and 3.01–1.43 ppm.β anomer: 11B{1H} NMR : δ = −2.6, −4.5, −8.9, −11.1,−12.2, and −12.7 ppm.All forms: m/z calcd for C9H24B10O6Na [M + Na]+ 361.2401;found 361.2462.HRMS: 2.2l-glutamine(2.0 mM), heat-inactivated fetal bovine serum (10%), and penicillin(50 U/mL)–streptomycin (50 μg/mL) at 37 °C with5% CO2 and 95% relative humidity. The GLUT1 affinity studies,cellular uptake, as well as the cytotoxicity studies followed theprotocols reported in our earlier work and will only be briefly describedhere.20CAL27 (ATCC CRL-2095) cells used in in vitro experiments were acquiredfrom ATCC  and cultured in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 2.2.11–3 (0.1–1800 μM) containing [14C]-d-glucose  in glucose-free HBSS . The reaction was quenched with ice-cold buffer,followed by two times washing of the cells, lysis with 250 μLof 0.1 M NaOH, and mixing with 1.0 mL of emulsifier safe cocktail. The IC50 values were obtainedby nonlinear regression analysis.The CAL27 cells were incubated at r.t. for 5 min with the glycoconjugates 2.2.21–3 were added in a concentration range of 10–400 μM in250 μL of glucose-free HBSS. They were further incubated for5, 30, and 120 min at r.t. The reaction was quenched with adding ice-coldbuffer followed by washing of the cells. The cell lysate from fourwells was combined and centrifuged at 4 °C. Supernatant (800μL) from each sample was collected and digested in 1.0 mL ofHNO3 for 24 h. Milli-Q water was added to obtain 10 mLof the sample, and the boron amount was measured using ICP-MS. Themeasurements were performed in triplicate. The data analysis was performedwith PerkinElmer Syngistix Data Analysis Software, and the statisticalanalyses was done using GraphPad Prism v.5.03.CAL 27cells were pre-incubated after which the glycoconjugates 2.2.31–3 at concentrations of 5, 25, 50, 125, and 250 μM for 24 h at37 °C, 5% CO2 atmosphere, and 95% relative humidity.Each concentration was done in triplicate. After 24 h, media was removed,and the cells were washed twice with 1 × DPBS  (pH 7.4). To assess cell viability, anequal volume of 1 × HBSS and CellTiter-Glo reagent (50 μLeach) was added to all the wells, and they were left at r.t. in thedark for 10 min. The luminescent signal from viable cells was measuredusing the Synergy H1 Hybrid multimode microplate reader . After luminescence reading, the total protein content ineach sample was quantified using the Pierce colorimetric bicinchoninicacid (BCA) protein assay .Briefly, a sample of 25 μL from each well was transferred toa separate transparent 96-well plate. BCA working reagent (200 μL)was then added to each well, and the plates were wrapped with aluminumfoil and incubated for another 30 min at 37 °C. The absorbancewas recorded at 562 nm using the microplate reader. The protein contentwas calculated using the BSA standard curve (concentration range:0–2000 μg/mL) and used to normalize cell viability data.CAL 27cells were seeded at a density of 5000 cells per well in a white-walled96-well plate and incubated overnight. The cells were incubated incell culture media containing glycoconjugates 2.331 with the doubly polarized triple-ζbasis set def2-TZVPP.32 Partial atomiccharges were computed using the restrained electrostatic potential(RESP) protocol.33 For the RESP chargecalculation, the molecule was divided into two parts, with one partconsisting of the carborane and a linking carbon atom and the otherpart comprising the sugar. Partial charges of hydrogen atoms bondedto the same carbon atom were constrained to be equal. The geometryoptimizations were performed with Turbomole 7.635 and the RESP calculations with NWChem 6.8.1.36The initial geometriesof the ligands were optimized to a local minimum at the DFT levelusing the dispersion-corrected hybrid Tao–Perdew–Scuseria–Staroverovfunctional TPSSh-D3(BJ),38 All rotatable bonds in the carborane partwere set to nonrotatable (inactive). For docking, the number of torsionaldegrees of freedom was set to 8 (torsdof 8) for the pyranoses andto 9 (torsdof 9) for the furanoses. The docking studies were performedusing the XylE inward-open 4QIQ(6N3I(4QIQ), adding hydrogens,merging them, and then computing Gasteiger partial charges. For allproteins, a grid of size 46 × 56 × 60 was used, with a gridspacing value of 0.375. The grid center was in the middle of the proteincavity for the grid box to cover the binding site. During docking,the protein was kept rigid and only ligand torsional angles changed.For each ligand, 2000 independent search runs, each with max 2.5 millionenergy evaluations and a population size of 150 with max 27,000 generations,were performed using the default settings of the Lamarckian geneticalgorithm (LGA), that is, a mutation rate of 0.02 and a crossoverrate of 0.8, with one top individual surviving to the next generation.Conformations were clustered (ranked) with a cluster RMS of 2.0 Å.Molecular docking studies were performed using AutoDock4.2.6.open 4QIQ and outw4QIQ)moiety.43 First, our selected conjugationreaction between the decaborane complex [B10H12(MeCN)2] and a terminal alkyne does not tolerate the presenceof free hydroxyl groups.44 Second, the ortho-carborane cluster is known to be base-labile and harshbasic conditions need to be avoided during deprotection sequences.44 With these factors in mind, two synthetic pathwayswere devised. Glycoconjugates 1 and 2 weresynthesized in a similar fashion to that previously reported for the d-Glc analogue,16 while for glycoconjugate 3, a shorter synthetic sequence employing isopropylidene protectinggroups was devised in accordance with a previous report in the field.46 A summary of the synthetic routes is supplied in As mentioned above, our goal was to incorporate an 1 and 2 was initiated by the selective protection of the 6-OH group.This can be achieved with a bulky substituent, here a tert-butyldimethylsilyl ether was installed in yields ranging from 68to 78%.47 The remaining hydroxyl groupswere benzylated in good yields,48 followedby cleaving of the temporary protected 6-OTBDMS group with a HF·pyridinecomplex.49 The newly demasked 6-OH groupwas deprotonated with NaH, and the corresponding alkoxide ion wasreacted with propargyl bromide to introduce the conjugation site forthe [B10H12(MeCN)2] complex. The[B10H12(MeCN)2] complex was generatedby gentle heating of decaborane in dry acetonitrile, and the carboranylinstallation was successfully completed by the addition of the protectedcarbohydrate derivative (5 or 8) bearingthe terminal alkyne handle in dry toluene. The 53–66% yieldsobserved in the carboranyl installation are typical for reactionsof this type.44 The final hydrogenolysisof benzyl groups was performed in an autoclave using 10% Pd/C as thecatalyst.50 This led to the formation of 1 and 2 in yields ranging from 80 to 83%. Theoverall efficiencies of these synthetic routes were thus 10–20%over six steps.In short, the synthesis of glycoconjugates 3 can be accessedthrough a shortersynthetic route starting from commercially available 1,2:3,4-bis-O-isopropylidene-α-d-galactopyranose. Similarstrategies as those earlier reported for the synthesis of glycoconjugate 3 were employed.46 Propargylation andcarboranyl installation with B10H12(MeCN)2 proceeded in similar yields to those observed in the syntheticroutes leading to 1 and 2. The deprotection,performed under acidic conditions, gave glycoconjugate 3 in an excellent yield, improving upon the deprotection yield reportedin the literature for 3.46 The overall efficiency of this synthetic route was 42% over threesteps. All three synthetic routes were relatively short and displayedoverall yields in the 10–42% range. They employed commerciallyavailable and affordable reagents and can therefore be consideredto be cost-effective. Further optimization of work processes wouldhowever be required in order to supply materials for clinical studies.Glycoconjugate 3.21–3 was confirmed by qNMR-techniques employing maleicacid as an internal standard. Apart from the purity assessment, specialemphasis was placed on characterization of NMR spectra. This was consideredimportant because mutarotation is a naturally occurring phenomenonin carbohydrates leading to the possible formation of furanose andpyranose forms as both α- and β-anomers. These differentforms and anomeric ratios may impact how the substrates interact withthe transporters in a biological environment, and thus, having informationon the distribution between these can be useful. To this end, we initiallyemployed the following 1D-experiments: 1H, 1D-TOCSY, 13C{1H} and 11B{H}, and 2D-experiments:COSY, ed-HSQC, and HMBC. Especially the use of 1D-TOCSY proved criticalin the current study.51 With the aid of 1D-TOCSY, wewere able to analyze the different spin systems overlapping in the 1H NMR spectra. This provided us with a good base for a detailedanalysis of the spectra with the ChemAdder program.53 Through the use of ChemAdder, the spectra were simulated and fittedwith the experimentally derived ones. The solid match between thesimulated and experimental 1H NMR spectra resulted in accessto information such as accurate chemical shifts, coupling constants,and distribution between the forms/anomers present in solution. Anexample of the NMR spectroscopic characterization studies of glycoconjugate 3 is provided in All key intermediates and final compounds wereinitially characterized by a conventional set of 1D- and 2D-NMR experimentsand high-resolution mass spectrometry (HRMS). The purity of glycoconjugates 1, only the pyranoseforms werevisible in the spectra, whereas for glycoconjugates 2 and 3, small amounts of signals resembling furanosescould likewise be found. The ratio between α- and β-anomerswas also largely affected by the stereochemistry of the carbohydratecore. In the d-Glc analogue, 6-oCb-Glc, andfor glycoconjugate 1, the α-pyranose form dominates. In d-Gal, an equal distribution between the anomers is observed,and in d-All, the β-pyranose form dominates (α:βapprox. 1:2).For glycoconjugate 3.3d-Glc. In ourearlier work, we have proven the competitive advantage of glucoconjugatesthrough an experimental cis-inhibition assay andanalyzed the interactions taking place between the ligands and theGLUT1 transporter through molecular docking studies.21 Here, similar assessment protocolswere employed.For GLUT1-targeting agents to be viablein BNCT, they need to be able to compete for the hexose transporterseven with the high blood concentrations of 1–3 and the carbohydrate recognition domain of GLUT1 were assessed throughdocking studies employing our existing GLUT1 model.21 Our GLUT1 model is derived from the insideopen (PDB ID 4QIQ)39 and outside open (PDB ID 6N3I)40 crystal structures reported for a d-xylose-protonsymporter 54 through virtual mutation of GLN415 in the symporterin order to match the ASN found in the carbohydrate recognition domainof GLUT1.56 Glycoconjugates 1–3 werefound to interact more strongly with the transporter than d-Glc which indicates that their targeting capabilities may be superiorcompared to the natural substrate . The mean bindingenergies for the three glycoconjugates were all within the 1 kcal/molerror limit  of the computational methodsemployed, and therefore, details on which of these would be the bestcould not be deduced based on the computational modeling data. Thecomputational model did however suggest that the pyranose forms bindmore strongly to the carbohydrate recognition domain of GLUT1 thanthe furanose forms . Therefore, monosaccharides in which the furanoseform dominates might be inferior to ones in which the pyranose formdominates, although without experimental evidence to support the modelingdata definite conclusions cannot be drawn. Nevertheless, in glycoconjugates 2 and 3, the furanose forms are present in minoramounts (9–13%) and would not be expected to cause seriousconcerns in this regard.The interactions between cis-inhibitionassay employed, glycoconjugates 1–3 were forcedto compete for the transporter againstradiolabeled d-Glc. This assay provides essential informationon the targeting capabilities of the delivery agents on a molecularbiology level. In more detail, the inhibition of d-Glc uptakeat low concentrations was taken as a sign of promising transportertargeting capabilities. In this regard, the IC50 valuesreported represent the concentrations at which the glycoconjugatesinhibit 50% of the radiolabeled d-Glc uptake. These studieswere performed on the CAL 27 cell line, which is an oral adenosquamouscarcinoma cell line28 of human origin and thus relevantfrom the BNCT treatment perspective. In addition, we have previouslydetermined the GLUT1-expression levels, verified the GLUT1 function,and used it as a model for comparison of GLUT1-targeting agents.16 Our current hit molecule, 6-oCb-Glc, has an IC50 value of 43.96 μM, while the nonradiolabeledcontrol d-Glc has an IC50 value exceeding 1 mMin the CAL 27 cell line.16 The IC50 values of the glycoconjugates 1–3 are summarizedin 1–3 are able to match theGLUT1 affinity observed for 6-oCb-Glc. It is importantto not over-interpret these results as they reflect upon the competitionfor transporters targeted by d-Glc. If glycoconjugates 1–3 display different hexose transporter targetingprofiles than d-Glc, information obtained through the employedassay does not necessarily account for such factors. Nevertheless,the results still indicate that the glucose transporter targetingcapabilities are superior to that of the natural substrate.In the 3.457 While the amounts are bound to be different depending on the efficiencyof delivery agents and strategies employed, it nevertheless underlineswhy a low systemic toxicity is highly beneficial for delivery agentsin BNCT.In order to determine whether delivery agents displaysufficient potential to warrant future in vivo studies, we have studiedtheir cytotoxicity and boron delivery capacity. Both are importantfactors for eventual translation into more detailed preclinical andclinical assessment studies. One of the important prospects of BNCTis that nontoxic delivery agents can be employed. Thereby, harmfulside effects can be limited compared to conventional chemotherapywhich employs toxic cancer therapeutics. In order to reach the requiredcellular boron concentrations, considerable amounts of delivery agentsare used in clinical treatments. For example, the most commonly employeddelivery agent BPA is administered intravenously at a dose of roughly250–400 mg/kg of patient weight.1–3 were assessed over the 5–250μM concentration range in order to be able to compare the resultsto our earlier work. The preclinical and clinical administration doseswould be expected to fall within this concentration range. While wehave earlier studied the toxicity of delivery agents at multiple timepoints, we have concluded that the 24 h time point is sufficient forpreliminary evaluation. The results from the cytotoxicity assay aresummarized in 1–3, a completegrowth medium was used as a negative control (for cytotoxicity), 1%v/v Triton X-100 as a positive control (for cytotoxicity), and BSHas the reference of a clinically employed boron delivery agent.21 Overall, glycoconjugates 1–3 were found to be less toxic than BSH across the concentration range,indicating that their in vitro toxicity profiles are more amenable,and their use in BNCT is not precluded.Here, we performed a cytotoxicity evaluation in thesquamous cell carcinoma CAL 27 cell line. The toxicity profiles ofglycoconjugates 1–3. This is one of the keyaspects of the development of improved delivery agents for BNCT. Therefore,we considered it important to include clinically employed deliveryagents (BPA and BSH) in the study to have a solid reference to comparethe results to. It should be noted that these two agents enter thecells via different mechanisms than the hexose transporters targetedin our approach. BPA is a LAT1-targeting delivery agent, and BSH entersthe cells by passive diffusion through the cell membrane.58 Therefore, in addition to comparing the deliverycapacity of glycoconjugates, our experimental design allows a crudecomparison of these three distinct targeting strategies. We evaluatedthe boron delivery capacity over three time points  and a concentration range of 10–400 μM. In moredetail, the CAL 27 cells were first incubated with the delivery agentsat set concentrations and time points. The cells were then washedand lyzed, and the supernatant from four wells was combined beforeacidic digestion in order to prepare samples for boron quantificationby inductively coupled plasma-mass spectrometry (ICP-MS). The resultsare summarized in 1–3 is superior to that of the clinically employed agents BPA and BSH.While the significant potential embedded in targeting hexose transporteris undeniable, there are a few important findings that emerge whenthe results are compared to those obtained in our earlier work.21 First, modification of the position6 seems to be a promising strategy regardless of the stereochemicalconfiguration in the carbohydrate core. Second, the stereochemicalconfiguration does not seem to be as crucial to the functioning principleof the carbohydrate delivery agents as having the appropriate interconnectingatoms in the boron cluster and linker.21 Altogether, all three glycoconjugates were found to display similarboron delivery capacities as our previous hit compound 6-oCb-Glc. Based on the preliminary in vitro assessment reported herein, theyshould all therefore be viewed as promising delivery agents for BNCT,and future in vivo studies will be necessary to further explore theirpotential.As a last measure, we sought to determine the borondelivery capacityof glycoconjugates 421 We have previously synthesized and studied a numberof glucoconjugates and found delivery agents that outperform the onesin current clinical use in key in vitro preclinical assessment studies.One of the hit molecules was 6-oCb-Glc in which the position6 of d-Glc bears a carboranylmethyl substituent. Since thehexose transporters, including the glucose transporters, also recognizeother monosaccharides than d-Glc, we here sought to identifywhich stereochemical configuration in the carbohydrate core wouldbe optimal for the design of the next generation of carbohydrate deliveryagents. To this end, we synthesized the corresponding carboranylmethylderivatives of d-Man, d-All, and d-Gal(epimers of d-Glc) and studied their GLUT affinity, cytotoxicity,and boron delivery capacity as part of a preclinical assessment campaign.All three glycoconjugates were prepared from affordable starting materialsvia short synthetic routes and carefully characterized by NMR andHRMS. To our surprise, all three delivery agents displayed equallypromising biological profiles. They were found to be capable of targetingthe glucose transporters in the presence of d-Glc. In addition,they display acceptable in vitro cytotoxicity profiles and, most importantly,significantly boosted boron delivery capacity compared to the deliveryagents in current clinical use. While the new glycoconjugates didnot outperform the previous hit compound in the in vitro assessmentstudies, we consider that there is a possibility that more strikingdifferences may yet be uncovered in vivo. In our opinion, the currentresults are supportive of advancing these molecules to the next stageof the development pipeline.The increasing emergenceof in-hospital BNCT treatment facilitiesis having a revolutionizing effect on the entire BNCT field. The lastbottleneck of this therapeutic modality are the suboptimal propertiesdisplayed by the boron delivery agents in clinical use. Therefore,the development of improved delivery agents is important. In our team,we have focused on the development of carbohydrate-based deliveryagents targeting hexose transporters.


Z)-3-benzylideneisobenzofuran-1(3H)-ones in one-pot fashion under ultrasound irradiation. The one-pot reaction was scalable and efficiently produced the desired products (1a–m) in high yields in a short reaction time. Evaluation of their in vitro antiplasmodium assay against the 3D7 (chloroquine-sensitive) and FCR3 (chloroquine-resistant) strains of Plasmodium falciparum demonstrated that they displayed moderate to strong antiplasmodium activities  and low resistance indices. The in silico prediction of ADME and physicochemical properties showed that the synthesized compounds met the drug-likeliness requirements and featured low toxicity effects. Based on the evaluation of the antiplasmodium profiles, 3-substituted-isoindolinone derivatives of 1a, 1d, 1h, and 1l may become potential antiplasmodium candidates.As the attempts to control malaria through chemotherapy strategies are restricted, we have prepared a small library of 3-substituted-isoindolinones from ( A library of 3-substituted-isoindolin-1-ones has been synthesized from 3-alkylidenephtalides and primary amines through one-pot reaction under ultrasonic irradiation. Four isoindolin-1-ones have great potential to be antiplasmodium candidates. Anopheles mosquito where the protozoan parasite of Plasmodium falciparum is the most predominant species which is responsible for the mortality.1 The efforts to control malaria through chemotherapy are restricted since Plasmodium falciparum has developed resistance to artemisinin combination therapies (ACT) and other antimalaria drugs such as chloroquine, quinine, mefloquine and halofantrine.2,3 Therefore, the development of more effective antimalaria agents with good resistance profile is urgently required.Malaria is one of severe infectious diseases which threaten the global population both in tropical and sub-tropical countries. This disease is transmitted by the female 4 such as entonalactam A5 and (−)-goniolanceolactam.6 The synthetic γ-lactams have been reported to exhibit various pharmaceutical applications including N-p-methoxy-1,α-dihydroaristoyagonine7 as anticancer and N-benzylisoindolin-1-one8 as antibacterial. Moreover, isoindolin-1-one derivatives as exemplified by pazinaclone and pagoclone are commercial anxiolytic drugs.9 Due to their interesting applications in medicinal chemistry, a considerable attention has been devoted to produce 3-substituted-isoindolin-1-ones through simple and facile synthetic strategies -ones with primary amines.11,12 Other groups have carried out the ortho-alkenylation of substituted benzamides to generate ortho-vinybenzamides in the presence of transition metal catalysts such as Co,13,14 Ru15 and Rh.16 Then, the in situ generated synthetic intermediates may undergo subsequent intramolecular amidation to furnish lactam 1. Another application of transition metal catalyst in the synthesis of isoindolin-1-ones is Rh-catalyzed-three component reaction using benzamides, ketones and hydrazines.17 The formation of isoindolin-1-ones 1 has been achieved through the addition of Grignard reagent to 2-(N-sulfinylimines)benzoate esters followed by the intramolecular lactamization.18–20 Other strategies involved the modification of other isoindolin-1-one moieties such as alkylation of isoindolin-1-ones at C3 position,21,22 hydrogenation of 3-alkylideneisoindolin-1-ones,23,24 or reduction of 3-hydroxyisoindolin-1-ones.25,26 In spite of these robust methodologies, a facile, rapid and straightforward approach to efficiently generate biologically active isoindolin-1-ones 1 should be still developed.Isoindolin-1-ones 1 have been synthesized through intramolecular hydroamidation of Z)-3-benzylideneisobenzofuran-1(3H)-ones 2. We demonstrated that the reactivity of 3-hydroxyisoindolin-1-ones 7 can be further extended to give various motifs of isoindolin-1-ones. On reaction of 3-benzyl-3-hydroxyisoindolin-1-one with hydride source (NaBH3CN) under acidic condition, 3-benzylisoindolin-1-one was generated through the formation of N-acyliminium intermediate (NAI). We also found that ultrasound irradiation may accelerate the rate of those reactions.27Our recent study reported an efficient synthesis of 3-hydroxyisoindolin-1-ones 7 through nucleophilic addition reaction of primary amines 3 into  and 3D7 (chloroquine-sensitive) strains as well as prediction of their physicochemical and pharmacokinetic properties.We present, herein, one-pot synthesis of 3-substituted-isoindolin-1-ones 1 under ultrasound irradiation, evaluation of their cytotoxicity against Z)-3-benzylideneisobenzofuran-1(3H)-one 2a  and butylamine 3a in iso-propanol at 50 °C under ultrasound irradiation. This reaction in situ produced the synthetic intermediate of 3-hydroxyisoindolin-1-ones 7. After completion of the reaction, p-TSA as the acid catalyst (10 equiv.) and NaBH3CN (3 equiv.) as the hydride source were introduced to the subsequent NAI reaction in the same flask without isolating the intermediates 7.The one-pot synthesis of 3-substituted-isoindolin-1-ones 1 was initially investigated by performing the nucleophilic addition reaction between (p-TSA was required to completely convert 3-hydroxyisoindolin-1-ones 7 into the corresponding NAI intermediates 8. The reaction was further irradiated at 50 °C for more 1 h (3CN (entry 3). Further survey of solvents (entries 5–7) and catalysts (entries 8–10) demonstrated that the excellent yield of 1a was obtained when the reaction proceeded in acetonitrile in the presence of trifluoroacetic acid (entry 9). The control experiment demonstrated that the lactam 1a was obtained in lower yields when the one-pot reaction was carried out using conventional heating for 10 h (entries 9 vs. 11). The increase of both reaction yields and rates of the ultrasound-assisted-one-pot reaction was because the reaction medium received additional energy which was supplied by the mechanical effect of cavitation phenomenon generated from ultrasound waves.34–36The use of high amount of more 1 h , entry 137 The ultrasound-assisted-one-pot reaction also worked well (1c) when benzylamine was used as substrate.The scopes of one-pot synthesis of 3-substituted-isoindolin-1-ones 1 have been examined under the optimized reaction conditions . The rea38,39 Therefore, we decided to install both electron withdrawing  and donating (Me and OMe) groups at para position of benzylamines. These groups were tolerated to the reaction conditions, delivering the products (1d–h) in high yields. Furthermore, benzylamines with methoxy group at the ortho and meta positions smoothly reacted to give the corresponding isoindolin-1-ones 1i–j. Heterocyclic amines such as furfurylamine and 2-thiophenemethylamine could be subjected to our methodology leading to the direct formation of the products 1k–l in good yields. As exemplified by 1m, we also managed to employ the precursor of (Z)-3-(4-methylbenzylidene)isobenzofuran-1(3H)-one in the developed one-pot reaction.The nature of substituent at benzylamine was then evaluated under the optimal reaction conditions. One of strategies to enhance antiplasmodium activity is by incorporating halogen, trifluoromethyl and methoxy groups to the lead compounds.Z)-3-benzylideneisobenzofuran-1(3H)-one 2a allowed us to obtain the product 1a in 89% yield -3-benzylideneisobenzofuran-1(3H)-ones 2 results in enols 5, which subsequently transformed into the stable keto form 6. Then, the intramolecular nucleophilic addition of ketoamides 6 produces 3-hydroxyisoindolin-1-ones 7. The in situ generated lactams 7 reacts with acid catalyst of trifluoroacetic acid leading to the formation the reactive N-acyliminium ions (NAI) 8.40–42 Then, two possible reaction mechanism can be considered. The first one is the trapping of the reactive ketimines 8 with hydride source (NaBH3CN) to directly furnish the isoindolin-1-ones 1  and -resistant (FCR3) strains of Plasmodium falciparum.43 The antiplasmodium activities were presented as mean of IC50 values.Having prepared a small library of racemic 3-substituted-isoindolin-1-ones, we then turned our attention to the evaluation of their antiplasmodium activities. These products 1a–m were tested N-butylisoindolin-1-one 1a displayed great activities of 8.18 and 5.98 μM against chloroquine-sensitive (3D7) and -resistant (FCR3) strains, respectively. The antiplasmodium activity was reduced when phenylethyl group was installed in the isoindolin-1-one skeleton 1b. Introducing halogen substituents, such as Cl, F and CF3, on the N-benzyl moiety at 4-position gave positive influence on antiplasmodium activity if compared with hydrogen substituent (1cversus1d–f). However, a slight reduction of activity against 3D7 strain was detected for 1d derivative. The presence of halogen substituents on the bioactive compounds may improve their pharmacological properties  and may block metabolic pathway such as cytochrome P450 oxidation.44,45 The effect of electron donating groups, such as Me and OMe, was evaluated and enhanced activities against both strains was observed (1cversus1g–h). Change in the position of methoxy groups to 3- and 2- positions reduced their antiplasmodium activity, signifying the importance of substituent at 4-position (1gversus1h–i). The position of substituent may direct the orientation of molecules and affect the binding ability and energy towards the target proteins of Plasmodium parasites.46 Interestingly, heterocycle-substituted-isoindolin-1-ones 1k–l exhibited high activities against both strains. However, the decreased activities on 3D7 and FCR3 strains were observed when methyl group was installed at C-benzyl moiety (1aversus1m).The results of the antiplasmodium assay are presented in 50 < 1 μM), active (IC50 1–20 μM), moderate (IC50 20–100 μM), low (IC50 100–200 μM), and not active (IC50 > 200 μM). By considering the IC50 values /IC50(3D7)). The high value of RI correlates to the high level of resistance, while low RI value indicates that the compounds have potential as antiplasmodium agents.47,48 It should be noted that the RI values of all products were relatively low and in the range of 0.27–1.73. Despite the antiplasmodium activity of chloroquine against both strains were higher than the tested compounds, the RI values of the isoindolin-1-ones 1a–m were 6–40 times lower than that of chloroquine.For all tested compounds, the corresponding resistance indice (RI) was also calculated  number of hydrogen bond donor (HBD) was lower than 5 and (d) number of hydrogen acceptor was lower than 10. All the tested compounds were compliant to the Lipinski rule, indicating that isoindolin-1-ones 1a–m may be easily absorbed, have good permeability and promising potential as antiplasmodium agents.The physicochemical properties were calculated through ompounds  showed tin silico pharmacokinetic properties of synthesized compounds were also predicted using the online web tools (http:/preadmet.bmdrc.org), while the excretion parameters were calculated using the pkCSM web (https://biosig.lab.uq.edu.au/pkcsm/). The absorption of drugs depends on the human intestinal absorption (HIA), CaCo-2 cell permeability and Madin–Darby canine kidney (MDCK) cell permeability. The results showed that all lactams 1a–m were predicted to have more than 70% HIA values, suggesting that they had good human intestinal absorption. For CaCo-2 cell permeability, all isoindolin-1-ones 1a–m exhibited moderate permeability. On the MDCK cell permeability, five derivatives  were anticipated to show moderate permeability and the other compounds were expected to display low permeability.The eb tools  and 4. T51The blood–brain barrier (BBB) penetration and plasma protein binding (PPB) are used to characterize the distribution of compounds. All compounds 1a–m exhibited higher BBB penetration values in the range 0.21–10.36, indicating that they can cross the BBB. For the plasma protein binding, values of % bound of all isoindolin-1-ones 1a–m were estimated more than 90%, indicating decreased excretion and increased half-life.Cytochrome P450 isoforms  is an important enzyme for drug metabolism in liver. Metabolism is predicted based on the CYP models for substrate or inhibition. This investigation revealed that most of 3-substituted-isoindolin-1-ones were non-inhibitors for CYP2C19 and CYP2C9 and were not substrates for CYP2D6, except 1g. Only a few compounds (1i–k) were not inhibitor for CYP2D6. The compound 1a–m were non-inhibitors and substrate of the CYP3A4 enzyme.52 The prediction showed that the total clearance of 1e was the highest and followed by 1a, 1m, 1b, 1h, 1i, 1k, 1j, 1c, 1g, 1l, 1f and 1d.Excretion is predicted based on the total clearance. The higher the total clearance value of the compound, the faster the excretion process.The toxicity of the compounds 1a–m was predicted by the Ames toxicity (mutagenicity) and carcinogenicity mouse and rat. The toxicity prediction showed that all compounds act as mutagen. Only compound 1e and 1f had positive carcinogenicity in mouse while only compound 1k had positive for rat.Z)-3-benzylideneisobenzofuran-1(3H)-ones 2 and primary amines 3 thanks to ultrasound-assisted-one-pot reaction strategy. The developed methodology provided the desired compounds 1a–m as a racemic mixture in good to excellent yields and can be scaled up using 10 mmol scale. The results showed that introducing aliphatic, halogen, methyl, methoxy and heterocyclic groups on isoindolin-1-one scaffold could enhance their antiplasmodium activities against chloroquine-sensitive (3D7) and -resistant (FCR3) strains of Plasmodium falciparum. In addition, the presence of substituent at 4-position of N-benzyl moiety played important role in antiplasmodium activity. Based on the in vitro antiplasmodium activity, the resistance indice value, the prediction of physicochemical and pharmacokinetic properties, 4 out of 13 tested compounds, namely 1a, 1d, 1h, and 1l, may become promising antiplasmodium agents.Numbers of substituted 3-substitutedisoindolin-1-ones 1 has been conveniently and directly accessed from various . The solvents for the synthesis were initially distilled from calcium hydride. The chemicals used in this study were purchased from Merck and used without any further purification. The reaction was monitored using thin layer chromatography on Merck Kieselgel 60 F254 plates and was observed under UV light (254 nm). The purification of product was carried out using column chromatography on Merck Kiesgel 60 (0.040–0.063 nm).1H and 125 MHz for 13C). The chemical shifts were reported in parts per million (ppm) relative to the internal solvent signal of CDCl3 (δH 7.26 ppm and δC 77.16 pm). Multiplicity is indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), ddd (doublet of doublet of doublet), dd (doublet of doublet), dt (doublet of triplet), and td (triplet of doublet). The additional NMR analysis was conducted using DEPT, COSY and HMQC. The high-resolution mass spectrometry experiments were conducted using a QSTAR Elite mass spectrometer (Applied Biosystems SCIEX) or a SYNAPT G2 HDMS mass spectrometer (Waters) equipped with an electrospray ionization source operated in the positive ion mode. The IR spectra were recorded from Shimadzu Prestige-21, where the samples were prepared as KBr pellets.The melting point was determined using the electrothermal apparatus . The NMR spectra were recorded on JEOL JNM-ECZ500R (at 500 MHz for Z)-3-benzylideneisobenzofuran-1(3H)-one derivatives 2  and primary amines 3  was dissolved in 1 mL of acetonitrile. The flask was placed in the pre-heated ultrasonic bath (50 °C) and the reaction was carried out for 30 min. The reaction mixture was placed in the ice bath, followed with the addition of NaBH3CN  and trifluoroacetic acid . The reaction was continued under ultrasonic irradiation at 50 °C for 60 min. The reaction was quench with the addition of the saturated aqueous solution of NaHCO3, followed with the extraction with dichloromethane (3 × 5 mL). The combined organic layer was washed with brine, dried over Na2SO4, filtered and removed under vacuum. The crude product was purified by column chromatography using the eluent of n-hexane/ethyl acetate (9 : 1).: δ = 7.78–7.76 , 7.40–7.36 , 7.28–7.22 , 7.09–7.07 , 6.92–6.90 , 4.80 , 4.13–4.07 , 3.40 , 3.22 , 2.84 , 1.71–1.59 , 1.40–1.32 , 0.96 ; 13C-NMR : δ = 168.3  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O), 144.9 (CAr), 136.1 (CAr), 132.5 (CAr), 130.8 (CHAr), 129.6 (2CHAr), 128.6 (2CHAr), 128.2 (CHAr), 127.1 (CHAr), 123.6 (CHAr), 123.0 (CHAr), 60.0 (CH), 40.0 (CH2), 38.5 (CH2), 30.5 (CH2), 20.2 (CH2), 13.9 (CH3); HR-MS (ESI): m/z [M + Na]+ cald for C19H21NONa+: 302.1515, found: 302.1517.Small scale: white solid; yield: 130 mg (93%); mp: 88–91 °C. Scaled up synthesis: white solid; 2.48 g (89%); mp: 89–92 °C. IR (KBr): 3024, 2970, 2924, 2862, 1681, 1604, 1458, 1419, 1273, 1095, 702 cm−1; 1H-NMR : δ = 7.79–7.77 , 7.41–7.35 , 7.30–7.21 , 7.19–7.17 , 6.98–6.96 , 6.86–6.85 , 4.52 , 4.33–4.27 , 3.44–3.34 , 3.25 , 3.01–2.90 3.44–3.34 , 2.76 ; 13C-NMR : δ = 168.4 (CO), 145.1 (CAr), 139.1 (CAr), 136.2 (CAr), 132.3 (CAr), 131.0 (CHAr), 129.5 (2CHAr), 128.9 (2CHAr), 128.8 (2CHAr), 128.6 (2CHAr), 128.2 (CHAr), 127.1 (CHAr), 126.7 (CHAr), 123.6 (CHAr), 122.9 (CHAr), 60.8 (CH), 42.2 (CH2), 38.6 (CH2), 35.0 (CH2); HR-MS (ESI): m/z [M + H]+ cald for C23H22NO+: 328.1696, found: 328.1693.Yellow oil; yield: 130 mg (79%); IR (nujol): 3023, 2970, 2924, 2860, 1681, 1604, 1412, 1080, 756, 702 cm−1; 1H-NMR : δ = 7.85–7.83 , 7.43–7.37 , 7.34–7.28 , 7.25–7.21 , 7.00–6.98 , 6.87 , 5.46 , 4.57 , 4.22 , 3.37 , 2.83 ; 13C-NMR : δ = 168.6 (CO), 145.2 (CAr), 137.2 (CAr), 136.2 (CAr), 132.1 (CAr), 131.2 (CHAr), 129.6 (2CHAr), 128.9 (2CHAr), 128.7 (2CHAr), 128.3 (CHAr), 128.2 (2CHAr), 127.8 (CHAr), 127.1 (CHAr), 123.9 (CHAr), 123.1 (CHAr), 59.7 (CH), 44.4 (CH2), 38.5 (CH2); HR-MS (ESI): m/z [M + H]+ cald for C22H20NO+: 314.1539, found: 314.1539.Light yellow solid; mp: 98–101 °C; yield: 121 mg (77%); IR (KBr): 3024, 2970, 2924, 2860, 1681, 1604, 1435, 1072, 972, 694 cm−1; 1H-NMR : δ = 7.85–7.83 , 7.45–7.39 , 7.31–7.24 , 7.12–7.11 , 7.02–7.00 , 6.91 , 5.36 , 4.57 , 4.18 , 3.30 , 2.86 ; 13C-NMR : δ = 168.6 (CO), 145.2 (CAr), 136.2 (CAr), 135.8 (CAr), 131.9 (CAr), 131.4 (CHAr), 129.7 (CAr), 129.59 (2CHAr), 129.56 (2CHAr), 129.1 (2CHAr), 128.7 (2CHAr), 128.4 (CHAr), 127.2 (CHAr), 124.0 (CHAr), 123.1 (CHAr), 59.8 (CH), 43.8 (CH2), 38.9 (CH2); HR-MS (ESI): m/z [M + H]+ cald for C22H19ClNO+: 348.1150, found: 348.1146.Yellow solid; mp: 108–110 °C; yield: 131 mg (75%); IR (KBr): 3020, 2975, 2925, 2860, 1680, 1603, 1404, 1072, 879, 702 cm−1; 1H-NMR : δ = 7.85–7.83 , 7.44–7.39 , 7.28–7.23 , 7.18–7.15 , 7.02–6.98 , 6.90 , 5.37 , 4.56 , 4.19 , 3.32 , 2.86 ; 13C-NMR : δ = 168.6 (CO), 162.4 , 145.2 (CAr), 136.2 (CAr), 133.0 , 132.0 (CAr), 131.4 (CHAr), 129.9 , 129.6 (2CHAr), 128.7 (2CHAr), 128.4 (CHAr), 127.2 (CHAr), 124.0 (CHAr), 123.1 (CHAr), 115.7 , 59.7 (CH), 43.7 (CH2), 38.8 (CH2); 13F-NMR : δ = −114.7; HR-MS (ESI): m/z [M + H]+ cald for C22H19FNO+: 332.1445, found: 332.1443.Yellow solid; mp: 108–112 °C; yield: 119 mg (72%); IR (KBr): 3024, 2975, 2924, 2865, 1682, 1603, 1404, 1219, 1072, 764, 702 cm−1; 1H-NMR : δ = 7.87–7.85 , 7.55 , 7.46–7.42 , 7.28–7.25 , 7.02–7.00 , 6.95–6.96 , 5.41 , 4.59 , 4.28 , 3.28 , 2.91 ; 13C-NMR : δ = 168.8 (CO), 145.2 (CAr), 141.5 (CAr), 136.1 (CAr), 131.8 (CAr), 131.6 (CHAr), 130.1 , 129.5 (2CHAr), 128.8 (2CHAr), 128.5 (CHAr), 128.4 (2CHAr), 127.4 (CHAr), 125.9 (CHAr), 125.8 (CHAr), 124.1 (CHAr), 123.1 (CHAr), 122.1 , 60.0 (CH), 44.1 (CH2), 39.0 (CH2); 13F-NMR : δ = −62.5; HR-MS (ESI): m/z [M + H]+ cald for C23H19F3NO+: 382.1413, found: 382.1415.Yellow oil; yield: 160 mg (84%); IR (nujol): 3022, 2974, 2924, 2860, 1683, 1404, 1217, 1072, 764, 702 cm−1; 1H-NMR : δ = 7.81 , 7.41–7.34 , 7.24–7.21 , 7.14–7.10 , 7.00–6.97 , 6.83 , 5.42 , 4.54 , 4.16 , 3.36 , 2.80 , 2.32 ; 13C-NMR : δ = 168.5 (CO), 145.2 (CAr), 137.5 (CAr), 136.2 (CAr), 134.3 (CAr), 132.2 (CAr), 131.2 (CHAr), 129.6 (2CHAr), 129.5 (2CHAr), 128.6 (2CHAr), 128.33 (2CHAr), 128.32 (CHAr), 127.1 (CHAr), 123.9 (CHAr), 123.1 (CHAr), 59.6 (CH), 44.1 (CH2), 38.5 (CH2), 21.3 (CH3); HR-MS (ESI): m/z [M + H]+ cald for C23H22NO+: 328.1696, found: 328.1693.White solid; mp: 118–120 °C; yield: 130 mg (79%); IR (KBr): 3025, 2973, 2924, 2865, 1681, 1404, 1072, 702 cm−1; 1H-NMR : δ = 7.82 , 7.41 , 7.37 , 7.25–7.23 , 7.16 , 7.01 , 7.00 , 6.86–6.84 , 6.84 , 5.41 , 4.55 , 4.15 , 3.79  3.37 , 2.82 ; 13C-NMR : δ = 168.5 (CO), 159.2 (CAr), 145.2 (CAr), 136.3 (CAr), 132.3 (CAr), 131.2 (CHAr), 129.6 (4CHAr), 129.3 (CAr), 128.7 (2CHAr), 128.3 (CHAr), 127.1 (CHAr), 123.9 (CHAr), 123.1 (CHAr), 114.3 (2CHAr), 59.6 (CH), 55.4 (CH3), 43.7 (CH2), 38.5 (CH2); HR-MS (ESI): m/z [M + H]+ cald for C23H22NO2+: 344.1645, found: 344.1645.White solid; mp: 92–98 °C; yield: 148 mg (86%); IR (KBr): 3025, 2973, 2924, 2860, 1682, 1512, 1404, 1242, 1180, 1072, 756, 702 cm−1; 1H-NMR : δ = 7.83 , 7.42 , 7.38 , 7.25–7.22 , 7.00 , 6.87 , 6.83 , 6.81 , 6.76 , 5.43 , 4.60 , 4.19 , 3.77 , 3.37 , 2.83 ; 13C-NMR : δ = 168.5 (CO), 160.1 (CAr), 145.2 (CAr), 138.8 (CAr), 136.2 (CAr), 132.1 (CAr), 131.2 (CHAr), 129.9 (CHAr), 129.6 (2CHAr), 128.7 (2CHAr), 128.3 (CHAr), 127.1 (CHAr), 124.0 (CHAr), 123.1 (CHAr), 120.6 (CHAr), 113.8 (CHAr), 113.3 (CHAr), 59.7 (CH), 55.4 (CH3), 44.3 (CH2), 38.6 (CH2); HR-MS (ESI): m/z [M + H]+ cald for C23H22NO2+: 344.1645, found: 344.1646.Yellow oil; yield: 131 mg (76%); IR (nujol): 3025, 2973, 2924, 2860, 1682, 1512, 1411, 1257, 1049, 702 cm−1; 1H-NMR : δ = 7.77 , 7.37–7.32 , 7.28–7.20 , 6.99–6.98 , 6.91–6.90 , 6.78 , 5.23 , 4.59 , 3.87  3.56–3.53 , 2.80–2.75 ; 13C-NMR : δ = 168.6 (CO), 157.5 (CAr), 145.3 (CAr), 136.4 (CAr), 132.4 (CAr), 130.9 (CHAr), 130.5 (CHAr), 129.7 (2CHAr), 129.1 (CHAr), 128.5 (2CHAr), 128.1 (CHAr), 127.0 (CHAr), 125.5 (CAr), 123.7 (CHAr), 123.1 (CHAr), 121.1 (CHAr), 110.6 (CHAr), 60.2 (CH), 55.6 (CH3), 38.6 (CH2), 38.1 (CH2); HR-MS (ESI): m/z [M + H]+ cald for C23H22NO2+: 344.1645, found: 344.1642.Yellow oil; yield: 130 mg (76%); IR (nujol): 3032, 2973, 2924, 2860, 1682, 1412, 1103, 756, 702 cm−1; 1H-NMR : δ = 7.81–7.79 , 7.41–7.35 , 7.28–7.22 , 7.06–7.04 , 6.87–6.85 , 6.33 , 6.27 , 5.33 , 4.67 , 4.30 , 3.47 , 2.82 ; 13C-NMR : δ = 168.2 (CO), 150.6 (CAr), 145.2 (CAr), 142.6 (CHAr), 136.2 (CAr), 132.0 (CAr), 131.3 (CHAr), 129.7 (2CHAr), 128.7 (2CHAr), 128.3 (CHAr), 127.2 (CHAr), 123.9 (CHAr), 123.1 (CHAr), 110.6 (CHAr), 108.8 (CHAr), 60.3 (CH), 38.4 (CH2), 37.2 (CH2); HR-MS (ESI): m/z [M + H]+ cald for C20H18NO2+: 304.1332, found: 304.1330.Orange solid; mp: 98–101 °C; yield: 120 mg (79%); IR (KBr): 3032, 2973, 2932, 2860, 1682, 1466, 1420, 1211, 1103, 756, 710 cm−1; 1H-NMR : δ = 7.82 , 7.43–7.38 , 7.29–7.23 , 7.07 , 6.97–6.95 , 6.89 , 6.27 , 5.53 , 4.71 , 4.42 , 3.41 , 2.88 ; 13C-NMR : δ = 168.3 (CO), 145.3 (CAr), 139.6 (CAr), 136.2 (CAr), 131.9 (CAr), 131.4 (CHAr), 129.6 (2CHAr), 128.7 (2CHAr), 128.4 (CHAr), 127.2 (CHAr), 127.1 (CHAr), 126.9 (CHAr), 125.8 (CHAr), 124.0 (CHAr), 123.1 (CHAr), 59.6 (CH), 38.9 (CH2), 38.7 (CH2); HR-MS (ESI): m/z [M + H]+ cald for C20H18NOS+: 320.1104, found: 320.1102.White solid; mp: 106–108 °C; yield: 120 mg (75%); IR (KBr): 3032, 2973, 2928, 2860, 1682, 1420, 1080, 694, 570 cm−1; 1H-NMR : δ = 7.77–7.76 , 7.39–7.37 , 7.05 , 6.95 , 6.94–6.93 , 4.75 , 4.10–4.04 , 3.34 , 3.20 , 2.77 , 2.31 , 1.69–1.58 , 1.39–1.31 , 0.94 ; 13C-NMR : δ = 168.4 (CO), 145.1 (CAr), 136.7 (CAr), 133.0 (CAr), 132.6 (CAr), 130.8 (CHAr), 129.4 (2CHAr), 129.3 (2CHAr), 128.2 (CHAr), 123.6 (CHAr), 123.0 (CHAr), 60.1 (CH), 40.0 (CH2), 38.0 (CH2), 30.6 (CH2), 21.2 (CH3), 20.3 (CH2), 13.9 (CH3); HR-MS (ESI): m/z [M + H]+ cald for C20H24NO+: 294.1852, found: 294.1854.White solid; mp: 96–101 °C; yield: 121 mg (82%); IR (KBr): 3031, 2973, 2925, 2865, 1682, 1404, 1072, 702 cmPlasmodium falciparum strains of 3D7 (chloroquine-sensitive) and FCR3 (chloroquine-resistant). The antiplasmodium assay was conducted using candle jar method.The cytotoxicity of products was evaluated using in vitro assay, each synthesized compound was dissolved in DMSO and was prepared in a series of concentration, i.e. 10, 5, 2.5, 1.25 and 0.625 μg mL−1 in RPMI medium. A total of 100 μL of each series of concentrations was put into the 96-well microplate with three repetition and then 100 μL Plasmodium suspension was added. The culture was incubated at 37 °C for 72 h, a thin blood smear was made and treated with 20% Giemsa dyes. The percentage of parasitemia was determined by calculating the number of the infected erythrocytes for minimum 1000 erythrocytes and then used to calculate the inhibition percentage of P. falciparum growth. The antiplasmodium activities were presented as mean of IC50 values. The IC50 value was calculated by probit analysis using SPSS software.For the http:/preadmet.bmdrc.org). Moreover, the excretion parameters were determined using the pkCSM web (https://biosig.lab.uq.edu.au/pkcsm/).The pharmacokinetic properties including absorption, distribution, metabolism, and toxicity parameters were screened through the PreADMET web (Conceptualization, M. I. D. M. and L. C.; synthesis and analysis, M. F. H., H. S. S. and M. I. D. M.; antiplasmodium assay, C. R. M.; supervision, M. I. D. M., E. S. K. and B. P.; writing-draft preparation, M. I. D. M. and L. C.; writing-review and editing, M. I. D. M. and L. C. All authors have read the manuscript.There are no conflicts to declare.RA-013-D3RA02829A-s001


More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [18F]fluororhamnose. The overall radiochemical yield was 7–27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[18F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[ It has recently been approved for the imaging of infection and inflammation fluoride in the labeled intermediate and the final product (The collected fraction was diluted with water and trapped on a solid phase extraction (SPE) cartridge. The trapped intermediate was eluted with ethanol through a potassium carbonate cartridge into vial containing a stir bar. The eluted mixture was stirred for 10 min and neutralized. The final product was retained on an FDG purification cartridge and eluted with normal saline. The radiochemical yields (RCYs) for  product .18F]fluoride, drying, and fluorination reaction. Vials 13 and 14 were used for the trap and release of the purified intermediate. The valve 15 normal close was connected to the crude product vial through an inline K2CO3 cartridge for hydrolysis. The crude product was formulated by trapping on an FDG cartridge and eluting with normal saline.After successful manual standardization of the procedure, an automated synthesis was performed in a GE Tracerlab FX-N module . The syn5 was stable at 37 °C in whole human serum and compound 7 was slowly decomposed  at room temperature for at least 4 h. Compound composed . Howeverfter 4 h .18F]fluoro-L-rhamnose (5) and the averaged time activity curves are shown in 5 is not metabolized by mammalian cells, as expected. Additionally, it was not detected within the skeletal system at any point and thus the ligand is stable for at least 60 min in vivo. The time activity curves showed that 5 has a short biological half-life of 3.4 ± 1.7 min. The in vivo biodistribution of the other two radiolabeled derivatives of rhamnose (6 and 7) were also obtained using dynamic PET imaging. Both 6 and 7 showed rapid defluorination upon injection in mice, as evidenced by the increasing skeletal uptake in the representative PET images . An FDG cartridge was purchased from NucMedCor . Mass spectrometry (MS) was performed on a 6130 Quadrupole LC/MS, Agilent Technologies instrument equipped with a diode array detector. 1H, 13C, and 19F NMR spectra were recorded on a Varian spectrometer (400 MHz). Chemical shifts (ppm) were reported relative to the solvent residual peaks. HPLC for purification and analytical analysis were performed on an Agilent 1200 Series instrument equipped with multi-wavelength detectors along with a flow count radiodetector . LC-MS/MS analysis was performed on an Agilent 6460C triple quadrupole mass spectrometer with an ESI source and a flow count radiodetector . The LC unit was an Agilent 1200 series chromatographic system equipped with a 1260 binary pump, 1290 thermostatted column compartment and 1260 high-performance autosampler. Instrument control and data processing were performed using Aglient’s MassHunter Software.Methyl-rhamnopyranoside was obtained from Combi-Blocks Inc. . All other chemicals and solvents were received from Sigma Aldrich  and used without further purification. Fluorine-18 was received from the National Institutes of Health cyclotron facility . A Chromafix PS-HCO3 cartridge was purchased from Synthra GmbH (Hamburg Germany). Oasis HLB plus and K-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (8). The synthetic process followed the literature , found 355.00 [M + Na].terature . L-Rhamn-2-bromo-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (9). To a solution of compound 8  in glacial acetic acid (20 mL) and acetic anhydride (1.6 mL), HBr in acetic acid  was added dropwise in an ice-water bath and vigorous stirring. The resulting mixture was stirred at rt overnight and slowly quenched with a pre-cooled saturated NaHCO3 solution (500 mL). The brominated intermediate was extracted with CHCl3 (200 mL × 2). The organic layer was combined and dried over anhydrous Na2SO4. The bromide intermediate 9 was obtained by removing the volatiles under reduced pressure as a yellow oil (17.8 g).-2-ethoxy-2,5-dimethyltetrahydro-5H-dioxolo pyran-6,7-diyl diacetate (10). The oily bromide intermediate 9 was dissolved in a mixture of anhydrous acetonitrile (8 mL), and 2,4,6-collidine (11 mL) and ethanol  was added. The resulting mixture was stirred at rt overnight, diluted with CH2Cl2 (300 mL), and washed with water (300 mL × 2) and brine (200 mL). The organic layer was dried over anhydrous Na2SO4. Crude product was obtained by removing the volatiles under reduced pressure. Product 10 was purified using flash column chromatography with hexane/ethyl acetate 4/1 to 2/1 gradient . 1H NMR  δ 5.41 , 5.16–5.02 , 4.59 , 3.65–3.47 , 2.12 , 2.07 , 1.75 , 1.30–1.14 . MS (ESI) calculated mass for the parent C14H22O8 318.13 [M], found 341.00 [M + Na].-3-hydroxy-6-methyltetrahydro-2H-pyran-2,4,5-triyl triacetate (11). Hydrochloric acid  was added to a solution of the orthoester 10  and acetone (15 mL). The mixture was stirred at rt for 10 min and volatiles were removed under reduced pressure. The resulting crude product was dissolved in CH2Cl2 (150 mL) and washed with water (150 mL × 2). The organic layer was dried over anhydrous Na2SO4. Crude product was obtained by removing the volatiles under reduced pressure. Product 11 was purified using flash column chromatography with hexane/ethyl acetate 4/1 to 1/1 gradient . 1H NMR  δ 5.76 , 5.15 , 4.99 , 4.22–4.15 , 3.65 , 2.5–2.25 , 2.17 , 2.11 , 2.06 , 1.27 . MS (ESI) calculated mass for the parent C12H18O8 290.10 [M], found 313.00 [M + Na].-3-hydroxy-6-methyltetrahydro-2H-pyran-2,4,5-triyl triacetate (13). The triacetate 11  was dissolved in anhydrous CH2Cl2 (20 mL) and anhydrous pyridine (3.5 mL) and cooled with an ice-salt bath. Trifluoromethanesulfonic anhydride  in CH2Cl2 (10 mL) was added dropwise. The mixture was stirred at rt for 20 min, and then sequentially washed with HCl , saturated NaHCO3 (30 mL), and brine (30 mL). The organic layer was dried over anhydrous Na2SO4. The crude triflate was obtained by removing the volatiles under reduced pressure.12, 1.35 g) was stirred with acetonitrile (30 mL) and tetrabutylammonium nitrate  at rt for 1 h. Crude product was obtained by removing the volatiles under reduced pressure. Product 13 was purified using flash column chromatography with hexane/ethyl acetate 3/1 to 1/1 gradient . 1H NMR  δ 5.58 , 5.07 , 4.77 , 3.76–3.60 , 3.05 , 2.15 , 2.07 , 2.04 , 1.20 . MS (ESI) calculated mass for the parent C12H18O8 290.10 [M], found 313.00 [M + Na].The crude triflate -6-methyl-3-(((trifluoromethyl)sulfonyl)oxy)tetrahydro-2H-pyran-2,4,5-triyl triacetate (14). The triacetate 13  was dissolved in anhydrous CH2Cl2 (20 mL) and anhydrous pyridine (1.2 mL) and cooled with an ice-salt bath. Trifluoromethanesulfonic anhydride  in CH2Cl2 (10 mL) was added dropwise. The mixture was stirred at rt for 20 min, and then sequentially washed with HCl , saturated NaHCO3 (30 mL), and brine (30 mL). The organic layer was dried over anhydrous Na2SO4. Crude product was obtained by removing the volatiles under reduced pressure. Flash column chromatography was used to purify product 14 with hexane/ethyl acetate 3/1 to 1/1 gradient . 1H NMR  δ 5.80 , 5.38 , 4.89–4.76 , 3.78 , 2.16 , 2.07 , 2.05 , 1.25 . 13C NMR  δ 169.53, 169.51, 168.44, 118.21 , 90.21, 80.92, 77.35, 77.23, 77.03, 76.71, 73.02, 71.27, 71.24, 20.49, 20.37, 20.26, 17.02. MS (ESI) calculated mass for the parent C13H17F3O10S 422.05 [M], found 362.90 [M-OAc].-3-fluoro-6-methyltetrahydro-2H-pyran-2,4,5-triol . To a solution of 2-hydroxyl analogue 14  in anhydrous acetonitrile (2 mL), TBAF in THF  was added. The solution was stirred at 65 °C overnight. The volatiles were removed under reduced pressure. Flash column chromatography was used to purify product 7 with hexane/ethyl acetate 5/1 to 2/1 gradient . 1H NMR  δ 6.02 , 5.78 , 5.35–5.30 , 3.30–5.25 , 5.18–5.10 , 5.08–4.95 , 4.88 , 4.00–3.80 , 3.73–3.65 , 2.20 , 2.09 , 2.08 , 2.12 , 2.08 , 2.02 , 1.30 , 1.25 . MS (ESI) calculated mass for the parent C12H17FO7 292.10 [M], found 273.10 [M-F]. Triacetate  was dissolved in TFA (1.0 mL) and stirred at 50 °C for 1 h. The volatiles were removed under a reduced pressure to yield 2-deoxy-2-fluoro-L-rhamnose (25) as a yellow oil . The 19F NMR chromatogram was compared with the literature which found an identical result , found 268 [M].-4-(benzyloxy)-5-hydroxy-6-methyltetrahydro-2H-pyran-2,3-diyldiacetate (18). Compound 16  was dissolved in anhydrous pyridine (12 mL) and Ac2O (7 mL). The solution was stirred at room temperature for 15 h. Solvents were evaporated and the residue was dissolved in EtOAc (300 mL), washed with sat. NaHCO3, 1 N HCl, H2O, and brine, and dried over Na2SO4. After the evaporation of solvents, the crude product 17 was used for next step. H2SO4 (0.6 mL) was added dropwise to a solution of 17  in Ac2O (20 mL) and the solution was stirred at room temperature for 5 h. The reaction mixture was poured into a stirred mixture of ethyl acetate (150 mL) and sat. NaHCO3 (80 mL). The organic phase was separated and washed with sat. NaHCO3 and brine and dried over Na2SO4. After the evaporation of solvents, the residue was purified using silica gel flash chromatography to afford the product 18 as a colorless oil . 1H NMR : δ 7.37–7.26 , 6.12 , 6.03 , 5.34 , 5.23 , 5.16 , 5.07 , 4.72–4.43 , 3.94–3.79 , 2.16 , 2.12 , 2.11 , 2.10 , 2.05 , 2.04 , 1.23 , 1.21. MS (ESI) calculated mass for the parent C19H24O8 380 [M], found 403 [M + Na].-4-hydroxy-6-methyltetrahydro-2H-pyran-2,3,5-triyl triacetate (19). 10% Pd/C (1.5 g) was added to 18  in EtOAc (200 mL). The mixture was stirred at room temperature under a H2 atmosphere for 2 h and filtered through a celite pad. The filtrate was evaporated and the residue was purified using silica gel flash chromatography to afford 19 as a white solid . 1H NMR : δ 6.10 , 6.06 , 5.25 , 5.17 , 5.09 , 4.90 , 4.10–4.00 , 3.97–3.84 , 2.16 , 2.12 , 2.11 , 2.10 , 2.05 , 2.04 , 1.23 , 1.21. MS (ESI) calculated mass for the parent C12H18O8 290 [M], found 313 [M + Na].-6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)tetrahydro-2H-pyran-2,3,5-triyl triacetate (20). Trifluoromethanesulfonic anhydride  was added to a mixture of compound 19  and pyridine (0.22 mL) in dichloromethane (18 mL) at −18 °C. After stirring for 0.5 h, the mixture was warmed up to 0 °C and stirred for an additional 0.5 h. Water (50 mL) was added and the organic layer was separated. The aqueous layer was extracted with dichloromethane (3 × 50 mL). The combined organic layers were washed with 10% H2SO4, sat. NaHCO3, and brine and dried over MgSO4. After the evaporation of solvents, the residue was purified using silica gel flash chromatography to afford product 20 as a colorless oil . 1H NMR : δ 6.06 , 5.38 , 5.28 , 5.18 , 3.92 , 2.21 , 2.18 , 2.15 , 1.27 . 19F NMR : δ −75.0. MS (ESI) calculated mass for the parent C13H17F3O10S 422 [M], found 445 [M + Na].-4-hydroxy-6-methyltetrahydro-2H-pyran-2,3,5-triyl triacetate (21). Compound 20  was dissolved in dry CH3CN (2 mL) and solid tetrabutylammonium nitrite  was added. After stirring for 1 h at rt, the reaction mixture was evaporated. The residue was dissolved in CH2Cl2, washed with brine, and dried over MgSO4. After the evaporation of solvents, the residue was purified using silica gel flash chromatography to afford product 21 as a white solid . 1H NMR : δ 5.95 , 5.91 , 5.10 , 5.02 , 5.00 , 4.89 , 4.27 , 4.12–4.06 , 3.74 , 2.15 , 2.13 , 2.12 , 2.11 , 2.10 , 1.33 , 1.25. MS (ESI) calculated mass for the parent C12H18O8 290 [M], found 313 [M + Na].-4-fluoro-6-methyltetrahydro-2H-pyran-2,3,5-triyl triacetate (22). DAST  was slowly added to a solution of 21  in anhydrous CH2Cl2 (1 mL) at −40 °C. The reaction was stirred at room temperature for 24 h. After being cooled down to −20 °C, MeOH (0.2 mL) was added and the solvent was removed under reduced pressure. The residue was diluted with CH2Cl2 (30 mL), washed with water, and dried over MgSO4. After the evaporation of solvents, the residue was purified using silica gel flash chromatography to afford product 22 as a colorless oil . 1H NMR : δ 6.05 , 5.19 , 4.87 , 4.38 , 4.26 , 2.13 , 2.12 , 2.11 , 1.36 . 19F NMR : δ −205.1. MS (ESI) calculated mass for the parent C12H17FO7 292 [M], found 292 [M].-4-fluoro-6-methyltetrahydro-2H-pyran-2,3,5-triol (23). NaOMe  was added to a suspension of 22  in dry MeOH (1.7 mL). The mixture was stirred at room temperature for 15 h. Then, the reaction mixture was neutralized with Dowex (H+) resin, filtrated, concentrated, and purified using silica gel flash column chromatography to afford 23 as a white solid . 1H NMR : δ 4.93 , 4.90 , 4.22 , 4.28 , 4.10–4.03 , 3.97 , 3.80 , 3.51 , 3.38 , 1.30 ; 1.25 ; 19F NMR : δ −201.6, −206.1; HRMS (ESI) calculated mass for the parent C6H11FO4 166.0641 [M], found 165.0565 [M − H].-6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)tetrahydro-2H-pyran-2,3,5-triyl triacetate (24). Compound 24  was prepared using the same preparation procedure as compound 12. 1H NMR : δ 5.94 , 5.35 , 5.09 , 4.89 , 4.42 , 2.16 , 2.15 , 2.12 , 1.46 ; 19F NMR : δ −74.9. HRMS (ESI) calculated mass for the parent C13H17F3O10S 422.0495 [M], found 445.0377 [M + Na].-6-((trityloxy)methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (26). Triphenylmethyl chloride  was added to L-Mannose 25  in anhydrous pyridine (10 mL). The mixture was stirred at room temperature for 15 h. A total of 6 mL of Ac2O was added afterwards and the solution was stirred for another 15 h. The mixture was poured into ice-cold water and extracted with EtOAc (3 × 100 mL). The combined organic layer was washed with brine and dried over Na2SO4. After the evaporation of solvents, the residue was purified using silica gel flash chromatography to afford product 26 as a white solid . 1H NMR : δ 7.46–7.22 , 6.10 , 5.85 , 5.52 , 5.43–5.52 , 3.91 , 3.64 , 3.34 , 3.18 (0.3H), 3.07 , 2.24 , 2.23 , 2.17 , 2.14 , 2.00 , 1.98 , 1.76 , 1.75 . MS (ESI) calculated mass for the parent C33H34O10 590 [M], found 613 [M + Na].-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (27). 33% HBr in HOAc (1.6 mL) was added to the solution of compound 26  in glacial acetic acid (16 mL) at 10 °C. The mixture was stirred for 10 min. The formed triphenylmethyl bromide was immediately removed via filtration. The filtrate was diluted with cold water and extracted with EtOAc (3 × 100 mL). The combined organic layer was washed with water and brine and dried over Na2SO4. After the evaporation of solvents, the residue was purified using silica gel flash chromatography to afford product 27 as a white solid . 1H NMR : δ 6.09 , 5.87 , 5.49 , 5.40 , 5.33 , 5.27 , 5.17 , 3.85 , 3.73 , 3.66–3.58 , 2.21 , 2.17 , 2.16 , 2.10 , 2.08 , 2.04 , 2.02 , 2.01 . MS (ESI) calculated mass for the parent C14H20O10 348 [M], found 371 [M + Na].-6-(fluoromethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (28). Compound 28  was prepared using the same preparation procedure as compound 22. 1H NMR : δ 6.11 , 5.88 , 5.49 , 5.38–5.35 , 5.31 , 5.26 , 5.15 , 4.56 , 4.44 , 4.02 , 3.80 ; 2.21 , 2.17 , 2.16  2.11 , 2.07 , 2.01 ; 19F NMR : δ −231.9, −232.4. MS (ESI) calculated mass for the parent C14H19FO9 350 [M], found 350 [M].-6-(fluoromethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol (29). Compound 29  was prepared using the same preparation procedure as compound 23. 1H NMR : δ 5.18 , 4.92 , 4.78–4.57 , 3.93 , 3.87 , 3.77 , 3.68 . HRMS (ESI) calculated mass for the parent C6H11FO5 182.0591 [M], found 181.0521 [M − H].3S,5S,6R)-6-((((trifluoromethyl)sulfonyl)oxy)methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (30). Trifluoromethanesulfonic anhydride  was added to a mixture of compound 27  and pyridine (0.25 mL) in dichloromethane (20 mL) at −10 °C. After stirring for 2 h, water (50 mL) was added. The organic layer was separated and the aqueous layer was extracted with dichloromethane (3 × 50 mL). The organic layers were combined, washed with 10% H2SO4, sat. NaHCO3, and brine, and dried over MgSO4. After the evaporation of solvents, the residue was purified using silica gel flash chromatography to afford product 30 as a white solid . 1H NMR : δ 6.12 , 5.89 , 5.49 , 5.39 , 5.33 , 5.30 , 5.26 , 5.16 , 4.58–4.54 , 4.14 , 3.92 , 2.22 , 2.19 , 2.12 , 2.10 , 2.05 , 2.03 , 2.02 . 19F NMR : δ −74.3, −74.4. HRMS (ESI) calculated mass for the parent C15H19F3O12S 480.0549 [M], found 503.0433 [M + Na].All three tracers were prepared following the general procedure described below.2CO3 cartridge was incorporated between valve 15 and the product vial. Specifically, the reagent vials contained the following: Vial 1: tetrabutylammonium bicarbonate solution , 50 µL water, and MeOH (1 mL); Vial 2: ACN (1 mL); Vial 3: triflate precursor (5 mg) in ACN (0.6 mL); Vial 4: water (1 mL); Vial 5: HPLC solvent (2.0 mL); Vial 13: EtOH (2 mL); Vial 14: water (6 mL); HPLC dilution flask: water (30 mL). The vial 11 inlet port was connected to the V15 right port to transfer the intermediate to reactor 2 (R2).Radiosyntheses were performed on a GE Tracerlab FX-N2 synthesizer. The synthesis consisted of 7 reagent vials on the GE synthesizer. Vials 1–5 were used for the elution, the drying of fluorine-18, and the fluorination reaction. Vials 13–14 were used for the formulation of the purified intermediate. An inline K18F]fluoride in 2.5 mL of water was passed through a Chromafix PS-HCO3 cartridge, which was rinsed with 1 mL of acetonitrile. The retained [18F]fluoride was eluted from the cartridge into reactor 1 (R1) with the eluent in Vial 1 and dried under a N2/vacuum at 75 °C for 4 min. R1 was cooled to 50 °C, acetonitrile in Vial 2 was added and the activity was azeotropically dried at 55 °C for 3 min and at 95 °C for an additional 3 min under a N2/vacuum. The activity was further dried using a vacuum for 3 min. The [18F]fluoride drying cycle took about 20 min.Typically, 7.4 GBq (200 mCi) -fluoro-L-rhamnose, a Mediso PET/CT scanner was used. The imaging methodology and the dose administered did not differ from the previous scans conducted on the Inveon imager. The images were analyzed using Fusion software .A preclinical Inveon PET/CT scanner  was used. Each animal was first secured onto the scanner bed and placed symmetrically within the center FOV. A CT scan was then performed for attenuation correction and anatomic guidance when placing the volumes of interest for the calculation of radioactivity concentrations (VOIs). Immediately after the PET acquisition was started, the tracer was injected via the tail vein (~9 MBq) as a bolus (18F]fluoro-L-rhamnose and 6-[18F]fluoro-L-rhamnose showed rapid defluorination after in vivo injection (18F]fluoro-L-rhamnose was found to be very stable upon injection in healthy mice. This ligand rapidly cleared and there was minimal uptake in various organs indicating lack of uptake and/or metabolism by mammalian cells. There was also no liver metabolism detected and excretion was completely via the renal route (Three fluorine-18-labeled L-rhamnose derivatives have been developed for PET imaging studies with the ultimate goal of using those compounds to image various infectious diseases. Using triflate precursors, these new radioligands were prepared with medium to high RCY. The radiolabeling methods were successfully automated with the intention of large-scale production. All three derivatives were used for PET/CT imaging in mice. Of these, 3-deoxy-3-[njection . On the al route . Further