key: cord-0829197-e0cxrma6
authors: Parmenopoulou, Vanessa; Manta, Stella; Dimopoulou, Athina; Kollatos, Nikolaos; Schols, Dominique; Komiotis, Dimitri
title: Synthesis of novel N-acyl-β-d-glucopyranosylamines and ureas as potential lead cytostatic agents
date: 2016-02-29
journal: Med Chem Res
DOI: 10.1007/s00044-016-1539-5
sha: a001b063da19c74f5a335ea321684b70e00b9e23
doc_id: 829197
cord_uid: e0cxrma6

Novel classes of acetylated and fully deprotected N-acyl-β-d-glucopyranosylamines and ureas have been synthesized and biologically evaluated. Acylation of the per-O-acetylated β-d-glucopyranosylurea (5), easily prepared via its corresponding phosphinimine derivative, by zinc chloride catalyzed reaction of the corresponding acyl chlorides RCOCl (a–f) gave the protected N-acyl-β-d-glucopyranosylureas (6a–f), in acceptable-to-moderate yields. Subsequent deacetylation of analogues 6a–f under Zemplén conditions afforded the fully deprotected derivatives 7a,b,d,e,f, while the desired urea 7c was formed after treatment of 6c with dibutyltin oxide. All protected and unprotected compounds were examined for their cytotoxic activity in different L1210, CEM and HeLa tumor cell lines and were also evaluated against a broad panel of DΝΑ and RNA viruses. Derivative 7c exhibited cytostatic activity against the three evaluated tumor cell lines (IC(50) 9–24 μΜ) and might be the basis for the synthesis of structure-related derivatives with improved cytostatic potential. Only analogue 6f weakly but significantly inhibited the replication of parainfluenza-3 virus, Sindbis virus and Coxsackie virus B4 in cell cultures at concentrations of 45–58 μM.

Cancer figures among the major concerns of modern healthcare due to its rapidly increasing rates and associated mortality. In light of the growing prevalence of malignant carcinomas and their implications, equal impetus is being paid by the scientific community to research on the disease (Ferlay et al., 2015) . Although significant effort has been made to improve the current preventive and/or therapeutic strategies against carcinogenesis, the development of new selective agents capable of suppressing tumor growth and metastasis would contribute greatly to a better prognosis and current therapy (Sethi and Kang, 2011) .

In the beginning of 1990s (Lu et al., 2013) , small molecule kinase inhibitors have shown great potential as novel therapeutics for the treatment of cancer (Sausville, 2000) because of their intimate involvement in oncogenic signal transduction pathways that present multiple physiological responses, tumor cell proliferation and cell survival (Blanc et al., 2013) . Among them, sorafenib (Nevaxar), a diaryl urea analogue, is a small molecular inhibitor of several tyrosine protein kinases for the treatment of advanced renal cell carcinoma (RCC) and advanced hepatocellular carcinoma (HCC) (Wilhelm et al., 2006; Montagut and Settleman, 2009 ). In the same manner, PAC-1 bearing the N-acylhydrazone pharmacophore is promising as a new antitumor drug that can directly influence the apoptotic machinery or suicide of cells and has shown good results in mouse models (Zhang et al., 2012; Peterson et al., 2009; Putt et al., 2006) . Finally, carboxamide analogue sunitinib (Sutent) has proven to be successful in the treatment of renal cancer and pancreatic neuroendocrine tumors, while thiazole carboxamide analogue dasatinib is used for the treatment of several types of leukemia (Jänne et al., 2009) . Probably, the use of amide, urea or heteroatom linkers such as nitrogen or oxygen in the structure of the aforementioned inhibitors are important features for their selectivity that allow the formation of one or two hydrogen bonds with residues of the specific kinase (Davis et al., 2011) .

In view of the above observations and as a continuation of our long-term interest in glucopyranosyl analogues as antitumor/antiviral agents and potent enzyme inhibitors (Parmenopoulou et al., 2014; Dimopoulou et al., 2013; Manta et al., 2012; Kantsadi et al., 2012) , it was envisaged that compounds bearing a glucopyranosyl moiety linked with various aralkyl and aralkenyl groups (Somsák et al., 2008a, b) via the pharmacophore linkers NHCO and NHCONHCO would be endowed with pronounced antitumor activity. We hereby report the facile synthesis and biological properties of novel acetylated as well as fully deprotected N-acyl-b-D-glucopyranosylamines (3a-f, 4af) and ureas (6a-f, 7a-f), respectively.

Chemistry For the synthesis of the target N-acyl-N 0 -2,3,4,6-tetra-Oacetyl-b-D-glucopyranosylureas (6a-f), the per-O-acetylated b-D-glucopyranosylurea (5), easily prepared via its corresponding phosphinimine derivative (Pinter et al., 1995) , seemed a suitable precursor. Therefore, acylation of compound 5 (Pinter et al., 1995) by zinc chloride (ZnCl 2 ) catalyzed reaction of the corresponding acyl chlorides RCOCl (a-f) gave derivatives 6a-f, in acceptable-tomoderate yields (35-67 %). Although the Lewis acidic zinc chloride not only activates acid chlorides but also catalyze iminium ion formation (Paulsen and Pflufhaupt, 1980; Isbell and Frush, 1958) and, thereby, anomerization (Somsák et al., 2008a, b) , in our case, the b-anomers 6af were solely obtained. 5 Hz, J 3,4 C 8.9 Hz and J 4,5 C 9.4 Hz) arising from the trans-diaxial orientation of these consecutive protons, indicating the b-configuration of the sugar moiety and equatorially oriented acetyl groups.

Subsequent deacetylation of analogues 6a-f under Zemplén conditions (Agoston et al., 2001) at 0°C for 20 min afforded the fully deprotected derivatives 7a,b,d,e,f after flash chromatography, in good yields (72-82 %). Deprotection of acyl urea 6c either with basic or acidic transesterification conditions (sodium methoxide, methanolic ammonia, methanolic hydrogen chloride) was unsuccessful, due to faster cleavage of the N-acyl moiety than removal of the O-protecting groups. In order to overcome this difficulty and to successfully synthesize compound 7c, we sought to couple the free glucosyl urea (McKay and Nguyen, 2014; Helm and Kakhesy, 1989) with the desired acyl chloride RCOCl (c) in the presence of ZnCl 2 , but unfortunately only the starting materials were recovered. To our delight, when the acyl urea 6c was stirred in methanol in the presence of 0.5 eq dibutyltin oxide (Bu 2 SnO) at reflux (Liu et al., 2002) , the acetyl groups were smoothly deprotected and derivative 7c was isolated after flash chromatography, in 83 % yield (Scheme 1).

All compounds were well characterized by 1 H and 13 C NMR, mass spectrometry and elemental analysis and gave satisfactory analytical and spectroscopic data, which were in full accordance with their depicted structure. During the spectroscopic characterization, compound 6f appeared to consist of two rotamers, judged from their 1 H NMR spectra, which is probably due to hindered rotation of the amide bond.

The cytostatic activity of 3a-f, 4a-f, 6a-f and 7a-f was determined against murine leukemia (L1210), human lymphocyte (CEM) and human cervix carcinoma (HeLa) cell cultures, and the results are summarized in Table 1 .

From the overall results obtained, it was clear that the acetylated N-acyl-b-D-glucopyranosylamines (3b-f) and ureas (6a, b, e, f) showed a better cytostatic profile than their corresponding unprotected derivatives 4b-f and 7a, b, e, f, respectively (Table 1) . Exceptions were analogues 4a and 7c, which proved to be more cytostatic than their corresponding acetylated congeners 3a and 6c; in particular derivative 7c proved to be the most cytotoxic for all three tumor cell lines (IC 50 of 9, 24 and 19 lL, respectively). The cytostatic potential of 7c was only 5-to 15-fold lower than the established 6-mercaptopurine anticancer drug. Finally, the acetylated analogue 6d and the unprotected 7d exhibited a comparable degree of cellular cytotoxicity (IC 50 of 16-45 lL). Also, the tested compounds were not cytotoxic in normal (primary) confluent human lung fibroblast (HEL) cell cultures (MCC 50 [ 100 lL).

Compounds Only the acetylated glucopyranosyl urea 6f was found to inhibit the proliferation of parainfluenza-3 virus, Sindbis virus and Coxsackie virus B4 in Vero cells at the concentration of 45 and 58 lM (vide IC 50 ), respectively, whereas the well-described antiviral drug ribavirin showed no activity at all (C250 lM). The polyanionic compound dextran sulfate (DS) (mw 10,000) was also included as a control compound (Table 2 ).

In this study, we report the synthesis of novel acetylated as well as fully deprotected N-acyl-b-D-glucopyranosylamines and ureas as potential cytotoxic agents. Since the N-acyl-b-D-glucopyranosylureas proved to be more cytostatic than the corresponding amines and the final analogues were less potent than their acetylated congeners, it seems that the cytostatic activity of compounds is associated with the type of the linker, while the acetyl moiety may also contribute to the antitumor effect. Derivative 7c exhibited the most enhanced cytostatic activity (IC 50 of 9, 24 and 19 lL, respectively) and might be the basis for preparing structure-related derivatives with improved cytostatic potential, while analogue 6f inhibited the proliferation of parainfluenza-3 virus, Sindbis virus and Coxsackie virus B4 in Vero cell cultures at the concentration of 45, 58 and 58 lM (vide IC 50 ), respectively, as compared to ribavirin ([250 lM).

Thin layer chromatography (TLC) was performed on Merck precoated 60F254 plates. Reactions were monitored by TLC Mass spectra were obtained on a ThermoQuest Finnigan AQA Mass Spectrometer (electrospray ionization). Optical rotations were measured using an Autopol I polarimeter.

All reactions sensitive to oxygen or moisture were carried out under nitrogen atmosphere using oven-dried glassware. Chloroform (CHCl 3 ) was distilled from phosphorus pentoxide and stored over 4E molecular sieves. Methanol (MeOH) was stored over 3E molecular sieves. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl azide (1) (Tropper et al., 1992) , 2,3,4,6-tetra-O-acetyl-b-Dglycopyranosylamine (2), N-acyl-b-D-glucopyranosylamines 3a-f and 4a-f (Parmenopoulou et al., 2014) and 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylurea (5) (Pinter et al., 1995) were prepared according to the procedures described in literature, and their chemical and physical properties were in agreement with previous data.

General procedure for preparation of the N-acyl-N 0 -2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylureas (6a-f) To a solution of an acyl chloride (18 mmol) in 20 mL of dry CHCl 3 , anhydrous ZnCl 2 (0.59 mmol) and 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylurea (5) (2.56 mmol) were added with stirring. The reaction mixture was refluxed until TLC showed the complete transformation of the urea. Then, the reaction mixture was poured into ice water and was extracted with chloroform (29). The organic phases were collected and washed with sat. aq. NaHCO 3 solution and water. After drying, the solvent was evaporated under vacuo and the residue was purified by flash chromatography (n-hexane/EtOAc 1:1). 5 Hz, , 4.10 (dd, 1H, J = 1.9, 12.5 Hz, H-6b), 3.83 (ddd, 1H, J = 2.1, 4.1, 10.0 Hz, H-5), 2.06, 2.05, 2.03, 2.01 (4s, 12H, 4OAc); 13 C NMR (75.5 MHz, CDCl 3 ) d 170. 6, 170.1, 169.8, 169.4, 166.4, 154.8 (CO), 145.4, 143.7, 139.9, 132.8, 128.9, 128.8, 128.0, 127.7, 127.0, 79.0, 73.6, 73.0, 70.0, 68.2, 61 .6 (C-1-C-6), 20. 7, 20.6, 20.5 (OCOCH 3 H, 5.41; N, 4.70; Found: C, 60.17; H, 5.59; N, 6, 7, 8, 170.5, 169.9, 169.7, 169.4, 152.9 (CO), 137.7, 137.3, 135.2, 129.3, 129.1, 79.1, 73.7, 73.2, 70.4, 68.4, 36.4, 34.4, 29.4, 29.1, 26.0, 23.3, 23 .2 (3CH 2 and 4CH 2 of tetrahydronaphthalene moiety), 20. 7, 20.5, 20.4 C, 58.97; H, 6.49; N, 4.74; Found: C, 59.31; H, 6.67; N, 7H, ArH), 7.00 (d, 2H, J = 8.6 Hz, ArH), 2H, J = 9.4, 9.2 Hz, 2H, J = 9.7, 9.5 Hz, 4.63, 4.58 (q, 2H, J = 16.3 Hz, CH 2 ), 4.28 (dd, 1H, J = 4.4, 12.5 Hz, 4.13 (dd, 1H, J = 2.1, 12.5 Hz, 3.83 (ddd, 1H, J = 2.2, 4.3, 10.1 Hz, , 2.09, 2.04, 2.03, 2.02 (4s, 12H, 4OAc); 13 C NMR (75.5 MHz, CDCl 3 ) d 170. 7, 170.0, 169.8, 169.5, 169.4, 169.3 (CO), 157.4, 140.6, 134.8, 128.8, 128.4, 128.2, 126.8, 77.9, 75.0, 72.9, 70.1, 68.0, 67.5, 20.7, 20.6, 20.5 (OCOCH 3 , 5.37; N, 4.66; Found: C, 57.88; H, 5.73; N, 5. (100, 20, etc., lM) of the test compounds. The viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. The antiviral activity was expressed as the EC 50 or the compound concentration required to reduce virus-induced cytopathogenicity or viral plaque (VZV) plaque formation by 50 %. The minimal cytotoxic concentration (MCC) of the compounds was defined as the compound concentration that caused a microscopically visible alteration of cell morphology. Alternatively, the cytostatic activity of the test compounds was measured based on the inhibition of cell growth. HEL cells were seeded at a rate of 5 9 103 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, the medium containing different concentrations of the test compounds was added. After three days of incubation at 37°C, the cell number was determined with a Coulter counter. The cytostatic concentration was calculated as the CC 50 , or the compound concentration required to reduce cell proliferation by 50 % relative to the number of cells in the untreated controls. The methodology of the anti-HIV assays was as follows: human CEM (*39105 cells/cm 3 ) cells were infected with 100 CCID 50 of HIV-1IIIB or HIV-2ROD and seeded in 200-lL wells of a microtiter plate containing appropriate dilutions of the test compounds. After 4 days of incubation at 37°C, the HIVinduced CEM giant cell formation was examined light microscopically.

Antiproliferative assays Novikov et al., 2013) The cytostatic effects of the test compounds on murine leukemia cells (L1210), human T-lymphocyte cells (CEM), and human cervix carcinoma cells (HeLa) were evaluated as follows: an appropriate number of cells suspended in growth medium were allowed to proliferate in 200-lLwells of 96-well microtiter plates in the presence of variable amounts of test compounds at 37°C in a humidified CO 2 -controlled atmosphere. After 48 h (L1210), 72 h (CEM) or 96 h (HeLa), the number of cells was determined in a Coulter counter. The IC 50 value was defined as the compound concentration required to inhibit cell proliferation by 50 %.

Cytotoxic activity assay Novikov et al., 2013) Confluent human lung fibroblast (HEL) cultures in 96-well microtiter plate were exposed to serial dilutions of the test compounds (i.e., 100, 20, 4, 0.8 lL) . After 3 days of incubation at 37°C, microscopical detectable alterations of cell morphology were examined.

E)-3-(4-Isopropylphenyl)acryloyl-N 0 -(2,3,4,6-tetra-Oacetyl-b-D-glucopyranosyl)-urea (6d)

White foam

ArH cumenyl), 7.28 (d, 2H, J = 10.1 Hz, ArH cumenyl), 6.39 (d, 1H, CH=CH)

,3,4,6-tetra-Oacetyl-b-D-glucopyranosyl)-urea (6e) Yellow foam

CHCl 3 ); 1 H NMR (300 MHz

Anal

,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-urea (6f) Yellow foam

The 1 H NMR spectrum showed hindered rotation around the amide bond. 1 H NMR (300 MHz, CDCl 3 ): d 9.11 (t, 1H, J = 10.1 Hz, NH), 8.10 (2br s, 1H, NH), 7.17-7.05 (m, 4H, ArH cumenyl), 5.32-5.18 (2pseudo t, 2H, J = 9

ESI-MS: m/z, 579.25 [M ? H] ? . Anal

18 mmol) were dissolved in dry MeOH (1 mL), 1-2 drops of 1 M methanolic sodium methoxide (NaOMe) solution were added and the reaction mixture was kept at 0°C until completion of the transformation (20 min, TLC, EtOAc/MeOH 4:1)

E)-3-(Biphenyl-4-yl)acryloyl-N 0 -(b-D-glucopyranosyl)-urea (7a)

White syrup

CD 3 OD): d 7.83 (d, 1H, J = 15.7 Hz, CH=CH), 7.71-7.65 (m, 6H, ArH), 7.51-7.36 (m, 3H, ArH)

ESI-MS: m/z, 429.17 [M ? H] ? . Anal. calcd for C 22 H 24 N 2 O

8-Tetrahydronaphthalen-2-yl)butanoyl-N 0 -(b-Dglucopyranosyl)-urea (7b) Yellow syrup

MeOH); 1 H NMR (300 MHz

m, 4H, H-2-H-5), 2.71 (m, 4H, tetrahydronaphthalene moiety), 2.56 (t

ESI-MS: m/z, 423.18 [M ? H] ? . Anal. calcd for C 21 H 30 N 2 O

Biphenyl-4-yloxy)acetyl-N 0 -(b-D-glucopyranosyl)-/MeOH 4:1 as eluting solvents

MeOH); 1 H NMR (300 MHz

E)-3-(4-Isopropylphenyl)acryloyl-N 0 -(b-Dglucopyranosyl)-urea (7d)

White syrup

Hz ArH cumenyl), 6.64 (d, 1H, CH=CH), 4.94 (d, 1H, J = 9.0 Hz, H-1), 3.85 (dd, 1H, J = 1.8, 11.6 Hz, H-6b), 3.67 (dd, 1H, J = 4.4, 11.6 Hz, H-6a

3-(4-Ethylphenyl)butanoyl-N 0 -(b-D-glucopyranosyl)-urea (7e) Yellow syrup

H-1), 3.83 (dd, 1H, J = 4.5, 11.8 Hz, H-6a), 3.70-3.62 (m, 1H, H-6b

ESI-MS: m/z, 397.16 [M ? H] ? . Anal

3-(4-Isopropylphenyl)-2-methylpropanoyl-N 0 -(b-D-glucopyranosyl)-urea (7f) Yellow syrup

68 (dd, 1H, J = 4.3, 12.0 Hz, H-6a), 3.46-3.35 (m, 4H, H-2-H-5), 3.00-2.83 and 2.81-2.57 (2 m, 4H, CH 2 , CH, CH 3 -CH-CH 3 ), 1.22 (d, 6H, J = 6

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Acknowledgments This work was supported in part by the Postgraduate Programmes ''Biotechnology-Quality assessment in Nutrition and the Environment,'' ''Application of Molecular Biology-Molecular Genetics-Molecular Markers,'' Department of Biochemistry and Biotechnology, University of Thessaly, and the KU Leuven (GOA 10/14). We thank Lizette van Berckelaer, Leen Ingels, Leentje Persoons and Frieda De Meyer for excellent technical assistance in the evaluation of these compounds in the cellular assays.