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of some C-nucleosides such as BCX4430 (Fig.1), an imino-C-nucleoside as a potential therapeutic for the treatment of Ebola virus (EBOV) infections. [4]. , and.
Accepted Manuscript Synthesis of novel sugar or azasugar modified anthra[1,2-d] imidazole-6,11-dione derivatives and biological evaluation Qiwei Wang, Chen Ma, Xueyan Li, Xiaofen Wang, Ruixue Rong, Chao Wei, Pingzhu Zhang, Xiaoliu Li PII:

S0008-6215(17)30886-8

DOI:

10.1016/j.carres.2018.02.012

Reference:

CAR 7528

To appear in:

Carbohydrate Research

Received Date: 11 December 2017 Revised Date:

9 February 2018

Accepted Date: 18 February 2018

Please cite this article as: Q. Wang, C. Ma, X. Li, X. Wang, R. Rong, C. Wei, P. Zhang, X. Li, Synthesis of novel sugar or azasugar modified anthra[1,2-d] imidazole-6,11-dione derivatives and biological evaluation, Carbohydrate Research (2018), doi: 10.1016/j.carres.2018.02.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Graphical Abstract O

O glyco--CHO N NH

NH2 O

n=2,3,4,6,8

glyco

N O

O

O

NH2

O

HO

OH HO

ClCH2COOH

OH

N NH

O

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H2N

N NH

NH2 O

O

HO

n

HN

H2N

N

Cl HO

OH

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HO

n=2,3,4,6,8

ACCEPTED MANUSCRIPT

Synthesis of Novel Sugar or Azasugar Modified Anthra[1,2-d] imidazole-6,11-dione Derivatives and Biological Evaluation Qiwei Wanga, Chen Maa, Xueyan Lia, Xiaofen Wanga, Ruixue Ronga, Chao Weia,b,

a

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Pingzhu Zhanga,b*, Xiaoliu Lia,b* Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and

Environmental Science, Hebei University, Baoding 071002, China

Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of

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Education, Hebei University, Baoding 071002, China

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b

Abstract

A series of novel, sugar or azasugar modified anthra[1,2-d] imidazole-6,11-dione derivatives, with different side chain were synthesized, using the synthetic route of

(azasugar)-derived

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imidazole cyclization reaction of 1,2-diaminoanthraquinone with various sugar aldehydes,

and

imidazole

cyclization

reaction

of

1,2-diaminoanthraquinone with chloroacetic acid and then followed by the

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nucleophilic substitution of N-alkylamino azasugar. Their biological activities against HIV-RT and cytotoxic activities against A549, Hela and MCF-7 cells were preliminary evaluated, most compounds showed similar HIV-RT inhibition to the

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control drug (AZT).

Key words: C-nucleoside; sugar; azasugar; anthra[1,2-d]imidazole-6,11-dione; HIV-RT inhibition; cytotoxicity.

*

Corresponding author Tel. & Fax: +86-312-5971116; e-mail: [email protected]

*

Corresponding author Tel. & Fax: +86-312-5971116; e-mail: [email protected] 1

ACCEPTED MANUSCRIPT 1. Introduction Nucleosides (N-nucleosides) have been widely used in the clinic for decades to treat both viral pathogens and neoplasms

[1]

. C-nucleosides

[2,3]

, the analogues of

nucleosides, in which the nucleosidic C-N bond was substituted by C-C bond, is

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expected to be inert towards acidic or enzymatic hydrolysis while still possess strong biological activity of the parent N-nucleosides, while natural and synthetic N-nucleosides are unstable to enzymatic and acid-catalyzed hydrolysis of the nucleosidic bond. Several C-nucleosides are naturally occurring compounds, e.g.,

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pseudouridine (Fig.1) and showdomycin (Fig.1). Especially in recent year, the advent of some C-nucleosides such as BCX4430 (Fig.1), an imino-C-nucleoside as a

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potential therapeutic for the treatment of Ebola virus (EBOV) infections

[4]

, and

GS-6620 (Fig.1), a potential therapeutic agent of hepatitis C virus (HCV) infections [5], making the development of novel synthetic methodologies to prepare and found novel biologically active C-nucleoside analogues are very attractive for chemist and

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pharmacist.

Anthraquinones exist extensively in the nature and perform many biochemical and physiological processes in living organisms, natural and synthetic anthraquinones are very important biology and pharmaceutical chemistry [6]. Because of the extensive

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biological activity, such as antiviral agents against human immunodeficiency virus [7], anti-inflammatory [8], anticancer

[9]

, antioxidant and many others through different

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mechanisms, anthraquinones keep the powerful attraction for chemist and pharmacist to develop novel anthraquinones drugs. Imidazole ring, a very important pharmacophore, exists extensively in various

types of synthetic and naturally occurring pharmaceutical agents, displays diversity of pharmacological activities

[10-16]

, such as anti-tumor, antibacterial, anti-fungal,

anti-virus, anti-parasitic, dopamine receptor, histamine receptor antagonizing actions

2

ACCEPTED MANUSCRIPT and so on. In particular, as the analogue of the base of nucleosides, it also appears

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widely in synthetic and naturally occurring imidazole nucleosides, nucleotides.

Fig.1. The structures of some C-nucleosides

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Combining the excellent bioactivity of anthraquinones and imidazoles, and fusing the anthraquinone which has two hydrogen-bonding accept groups and imidazole ring the base analogue, Here in, we would like to report the design and synthesis of a series of novel C-nucleoside analogues, sugar and azasugar modified anthrax [1,2-d] imidazole-6,11-dione derivatives via the imidazole cyclization

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reaction of 1,2-diaminoanthraquinone with various sugar (azasugar)-derived aldehydes, or with chloroacetic acid, and then followed by the nucleophilic

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substitution of N-alkylamino azasugar.

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2. Results and discussion 2.1. Chemistry

The sugar-, azasugar-derived aldehydes 1a~1c were prepared according to the

corresponding procedure in the literature (1a [17], 1b [18], 1c [19]), using glucose, ribose and mannose as the starting material, respectively. Condensation and cyclization of 1,2-diaminoanthraquinone (2) with 1a~1c under concentrated sulfuric acid as the catalyst in DMF solvent yielded the corresponding targeted compounds 3a~3c (scheme1) in yield of 60%~80%, respectively. The 3

ACCEPTED MANUSCRIPT deprotection of compound 3c in the mixed solution of 1,4-dioxane and hydrochloric

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acid obtained the compound 4 in yield of 92%.

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Scheme1. Synthesis of compounds 3 and 4. Reagents and conditions: (i) H2SO4, DMF, r.t. 5 h, 60%~80%; (ii) HCl, 1,4-dioxane, 40oC, 5 h, 92%.

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Condensation and cyclization of 1,2-diaminoanthraquinone (2) with chloroacetic acid yielded the 2-chloromethyl anthra [1,2-d] imidazole-6,11-dione (5). Compounds N-alkylamino azasugars 6a~6e were synthesized through benzylidenation of D-mannitol, then bistrifluoromethanesulphonation, nucleophilic substitution and deportation [20]. Then through the nucleophilic substitution reaction of N-alkylamino

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azasugar 6a~6e with 5 in the solution of DMF, in which diisopropylethylamine (DIPEA) as the acid-binding agent, and sodium iodide as the catalyzer, obtained the corresponding side chain targeted compounds 7a~7e (scheme2), respectively, in yield

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of 40%~48%. The reaction of 1,2-diaminoanthraquinone (2) with chloroacetic acid could not yielded the compound 5 when contains solvent, such as DMF, THF etc.,

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whether or not contain acid as the catalyzer. Heated the mixture of 1,2-diaminoanthraquinone (2) and chloroacetic acid directly, the compound 5 can be obtained, when the temperature raised to 90oC, the reaction can be completed in 1h, and the compound 5 can be obtained, in yielded of 65%, higher temperature than 90oC, the yielded was reduced because of the side reactions.

4

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Scheme2 The synthesis of compounds 7a~7e. Reagents and conditions: (i) ClCH2COOH, 90oC, 1 h, 60%; (ii) DMF, DIPEA, NaI, 70oC, 3 h, 40%~48%.

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2.2. Biological activities

The inhibitions against HIV-1 reverse transcriptase (HIV-RT) and the

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cytotoxicity of some of compounds 3a, 3b, 4 and 7 were preliminarily evaluated. The HIV-RT inhibitory activities of compounds were measured using colorimetric reverse transcriptase assay by comparison with AZT. As shown in table 1, most of the tested compounds exhibited good inhibitions against HIV-RT, better or similar to the positive control AZT. Xylosyl-C-α-nucleoside analogue 3a showed nice inhibitions

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against HIV-RT with the IC50 values of 8.51 µM, better than AZT (31.24 µM). For compounds 7, the length of the chain between anthra[1,2-d] imidazole-6,11-dione and azasugar, had certain influence to the activities against HIV-RT, the azasugar modified anthra[1,2-d] imidazole-6,11-dione derivatives, which contained 2CH2,

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3CH2 and 6CH2 chain long between azasugar and amino methyl anthra[1,2-d] imidazole-6,11-dione exhibited good inhibitions with the IC50 values of 24.54 µM,

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27.78 µM and 31.12 µM. The cytotoxicity in vitro of the compounds 3a, 3b, 4 and 7 against HeLa cell

lines, breast cancer cell line Michigan Cancer Foundation-7 (MCF-7) and human lung adenocarcinoma epithelial cell line (A-549) was examined by the modified Mosmann’s protocol with comparison to the control drug (Cisplatin) [21]. As shown in table2, most of the compounds have not shown obvious activities against all of the tested cell lines, but compound 3b and 7e, the ribosyl-C-β-nucleoside analogue which 5

ACCEPTED MANUSCRIPT have a CH2 between the ribosyl and anthra[1,2-d] imidazole-6,11-dione, and the azasugar modified anthra[1,2-d] imidazole-6,11-dione derivatives, which has 8CH2 chain long between azasugar and amino methyl anthra[1,2-d] imidazole-6,11-dione showed good inhibitions to HeLa with the IC50 values of 8.23µM and 6.80µM, similar

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to that of the positive control Cisplatin (6.09µM). For compounds 7a-7d, the lengths of the chain between anthra [1,2-d] imidazole-6,11-dione and azasugar influenced the cytotoxic activities, the cytotoxicity to all of the tested cell lines, were increased with

imidazole-6,11-dione.

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the CH2 decrease of chain long between azasugar and amino methyl anthra [1,2-d]



In summary, a series of novel, sugar and azasugar modified anthra[1,2-d] imidazole-6,11-dione derivatives with different side chain were synthesized, via the

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key step of imidazole cyclization reaction of 1,2-diaminoanthraquinone with various sugar(azasugar)-derived aldehydes, or with chloroacetic acid and then followed by the nucleophilic substitution of N-alkylamino azasugar. Their biological activities against HIV-RT and cytotoxic activities against A549, HeLa and MCF-7 cells were

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preliminarily evaluated, most compounds showed similar HIV-RT inhibition to the control drug. Xylosyl-C-α-nucleoside analogue 3a showed nice inhibitions against

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HIV-RT. Some of the compounds showed a certain cytotoxic activity, compound 3b and 7e showed good inhibitions to HeLa, similar to that of the positive control cisplatin.

3. Experimental 3.1. General methods

6

ACCEPTED MANUSCRIPT Melting points were measured on an SGW®X-4 micro melting point apparatus and are uncorrected. 1H NMR, 13C NMR spectra were measured on Bruker AVANCE 600 (600 MHz) spectrometer using tetramethylsilane (Me4Si) as the internal standard. High-resolution mass spectra (HRMS) were carried out on a FTICR-MS (Ionspec

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7.0T) mass spectrometer in the electrospray ionization (ESI) mode. The microwave assisted reactions were performed on a DISCOVER S-Class Auto Focused Microwave Synthesis System (CEM Corporation, USA).

The optical densities for examining the inhibitory activities against HIV-RT and

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anti-tumor activity were measured on a TU-1901 UV–vis spectrophotometer and a Bio-Rad Model 3550 microplate spectrophotometer, respectively. Thin-layer

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chromatography (TLC) was performed on precoated plates (Qingdao GF254) with detection by UV light or with phosphomolybdic acid in EtOH–H2O followed by heating. Column chromatography was performed using SiO2 (Qingdao 300–400 mesh).

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3.2. General procedure for the synthesis of compounds 3

238.0 mg (1.0 mmol) 1,2-diaminoanthraquinone was dissolved in 5.0 mL DMF, 0.1 mL concentrated sulfuric acid and the solution of 160.0 mg compound 1a in 3.0

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mL DMF, were added dropwise successively at 0oC with stirring under nitrogen atmosphere, the mixture was stirred at room temperature for 5h, then sodium

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carbonate was added to adjust the pH of the reaction solution to 7. The solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using dichloromethane/methanol (v/v = 5:1) as the eluent to obtain light yellow solid 3a 250.0 mg, yield 65%. Under the similar condition, compounds 3b and 3c were obtained in yields of 80% and 60%, respectively. 3.2.1.

2-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)

-1H-anthra[1,2-d]imidazole-6,11-dione (3a) Yield 65%; light yellow solid, mp 218.4-219.8oC; [α]20D: +32.6 (c 0.1, MeOH); 7

ACCEPTED MANUSCRIPT 1

H NMR (600 MHz, DMSO-d6): δ 4.55 (s, 2H, CH2), 5.49 (brs, 1H, H-2’), 6.59 (d,

1H, J = 3.0 Hz, H-3’), 7.98 (brs, 1H, H-4’), 7.91-7.93 (m, 2H, H-5, H-8), 8.03-8.06 (m, 2H, H-6, H-7), 8.18-8.21 (m, 2H, H-1, H-2); 13C NMR (150 MHz, DMSO-d6): δ 50.29, 109.99, 115.81, 118.82, 121.62, 125.10, 126.65, 127.24, 128.40, 132.75, 133.43, 133.59, 134.69, 134.90, 134.96, 143.63, 149.85, 159.27, 182.67, 183.60;

3.2.2.

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HRMS: Calcd. For C20H16N2O6+: 380.1008; found: 380.1012. 2-(((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)

methyl)-1H-anthra[1,2-d]imidazole-6,11-dione (3b)

1

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Yield 80%; light yellow solid, mp 198.0-199.2oC; [α]20D: +21.3 (c 0.1, MeOH); H NMR (600 MHz, DMSO-d6): δ 3.53-3.62 (m, 2H, CH2), 4.03 (q, 1H, J = 4.8 Hz,

CH2), 4.09 (q, 1H, J = 4.8 Hz, CH2), 4.52 (q, 1H, J = 4.8 Hz), 4.87 (q, 1H, J = 4.8 Hz),

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5.12 (d, 1H, J = 4.8 Hz, H-1’), 5.67 (d, 1H, J = 4.8 Hz, H-3’), 5.75 (d, 1H, J = 4.8 Hz), 7.92 (brs, 2H, H-5, H-8), 8.04-8.13 (m, 2H, H-6, H-7), 8.19-8.22 (m, 2H, H-1, H-2); 13

C NMR (150 MHz, DMSO-d6): δ 62.13, 78.11, 80.19, 81.29, 86.61, 118.93, 121.00,

125.75, 126.71, 127.31, 128.54, 132.25, 133.45, 133.66, 134.75, 134.99, 148.89, 160.97, 182.90, 183.90; HRMS: Calcd. for C21H18N2O6+: 394.1165; found: 394.1161. (3aS,4R,6aR)-tert-butyl-(6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imida

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3.2.3.

zol-2-yl)-2,2-dimethyldihydro-3aH-[1,3]dioxolo[4,5-c]pyrrole-5(4H)-carboxylate (3c)

1

H NMR (600 MHz,

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Yield 60%; light yellow solid, mp 162.0-163.1oC;

DMSO-d6): δ 1.13 (s, 5H, CH3), 1.29 (s, 3H, CH3), 1.39 (s, 4H, CH3), 1.46 (d, 3H, J = 14.4 Hz, CH3), 3.67 (dd, 1H, J = 34.8 Hz, 12.6 Hz, H-1’), 3.90-3.94 (m, 1H, H-2’),

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4.83-4.86 (m, 1H, CH2), 4.89-4.92 (m, 1H, CH2), 5.41 (s, 0.5H, , H-3’), 5.52 (s, 0.5H, , H-3’), 7.92-7.94 (m, 2H, H-5, H-8), 8.02-8.09 (m, 2H, H-6, H-7), 8.20-8.25 (m, 2H, H-1, H-2); 13C NMR (150 MHz, DMSO-d6): δ 8.32, 25.21, 27.22, 27.26, 28.26, 28.48, 53.06, 53.41, 58.58, 61.21, 61.79, 78.55, 79.95, 84.66, 84.85, 111.65, 111.70, 120.99, 125.46, 126.75, 127.29, 128.38, 133.41, 133.62, 134.76, 134.99, 154.29, 154.49, 160.86, 182.89, 183.70; HRMS: Calcd. for C27H27N3O6+: 489.1900; found: 489.1906. 3.3. Synthesis of compound 4 8

ACCEPTED MANUSCRIPT Compound 3c (290.0 mg, 0.6 mmol) was dissolved in the mixed solution (5.0 mL 1,4-dioxane and 5.0 mL 6N hydrochloric acid), and the reaction mixture was stirred at 40oC for 5h, the solvents 1,4-dioxane were removed in vacuum, the residue was extracted with dichloromethane (3×10 mL), the combined organic phases were dried over MgSO4, concentrated under reduce pressure, the crude product was =1:1)) to afford the compound 4 in the yield of 92%. 3.3.1.

.

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purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v

2-((2R,3S,4R)-3,4-dihydroxypyrrolidin-2-yl)-1H-anthra[1,2-d]imidazole-

6,11-dione (4)

1

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Yield 92%; light yellow solid, mp 224.2-225.6oC; [α]20D: +9.0 (c 0.1, MeOH); H NMR (600 MHz, DMSO-d6): δ 2.85 (d, 1H, J = 11.4 Hz), 3.31 (dd, 1H, J = 11.4

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Hz, 4.8 Hz, H-2’), 4.08 (s, 1H, H-3’), 4.21(t, 1H, J = 5.4 Hz, ), 4.39 (d, 1H, J = 6.6 Hz, H-4’), 7.92 (brs, 2H, H-5, H-8), 8.04 (brs, 2H, H-6, H-7), 8.20 (brs, 2H, H-1, H-2); 13

C NMR (150 MHz, DMSO-d6): δ 58.14, 65.49, 66.34, 110.16, 118.61, 121.63,

126.83, 126.89, 130.07, 133.51, 133.86, 134.21, 134.84, 135.39, 155.21, 180.61, 183.86; HRMS: Calcd. for C19H15N3O4+: 349.1063; found: 349.1059.

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3.4. General procedure for the synthesis of compounds 7 The mixture of 2.4 g (10.0 mmol) 1,2-diaminoanthraquinone and 18.8 g (0.2 mol)

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chloroacetic acid was stirred at 90oC for 1h under nitrogen atmosphere. Cooled to room temperature and then 100 mL water was added to precipitate solids. After

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filtration, washing with water and ethyl alcohol, the crude compound 5 (1.3 g, 60%) was obtained as a yellow solid. Crude compound 5 1.2 g (4.0 mol) was dissolved in 20.0 mL dimethylformamide

(DMF), 1.4 mL (8.0 mmol) DIPEA and 120.0 mg (0.8 mmol) KI were added, and then the solvent of 1.6 g (7.7 mmol) compound 6a in 10.0 mL DMF was added dropwise, the reaction mixture was heated at 70°C for 3h with stirring, the solution was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane /methanol (v/v =5:1)) to afford the compound 7a in the yield of 43%. Under the similar condition, compounds 7b~7e 9

ACCEPTED MANUSCRIPT were obtained in yields of 40%~48%, respectively. 3.4.1. 2-(((2-((2S,3R,4R,5S)-3,4-dihydroxy-2,5-bis(hydroxymethyl)pyrrolidin-1-yl) ethyl)amino)methyl)-1H-anthra[1,2-d]imidazole-6,11-dione (7a) Yield 43%; yellow solid, mp 140.6-142.4oC; [α]20D: -39.8 (c 0.1, MeOH); 1H

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NMR (600 MHz, DMSO-d6): δ 1.91 (s, 2H, CH2), 2.95 (m, 1H, H-1’), 3.19 (m, 1H, H-4’), 3.35 (m, 1H, H-2’) 3.48 (m, 1H, H-4’), 3.61-3.81 (m, 2H, CH2), 4.00-4.09 (m, 2H, CH2), 4.24 (m, 2H, CH2 ), 4.80 (s, 2H, CH2), 7.92 (m, 2H, H-5, H-8), 8.04 (m, 2H, H-3, H-4), 8.19 (m, 2H, H-2, H-1); 13C NMR (150 MHz, DMSO-d6): δ 14.55, 21.23,

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21.53, 56.76, 57.61, 60.23, 60.71, 72.60, 72.73, 72.80, 74.47, 75.22, 75.72, 81.77, 120.99, 126.73, 127.28, 128.32, 133.42, 133.59, 134.76, 134.99, 161.89, 172.47,

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182.90, 183.84; HRMS: Calcd. for C24H26N4O6+: 466.1852; found: 466.1848. 3.4.2. 2-(((3-((2S,3R,4R,5S)-3,4-dihydroxy-2,5-bis(hydroxymethyl)pyrrolidin-1-yl) propyl)amino)methyl)-1H-anthra[1,2-d]imidazole-6,11-dione (7b) Yield 40%; yellow solid, mp 147.8-148.5℃; [α]20D: +12.4 (c 0.1, MeOH); 1H NMR (600 MHz, DMSO-d6): δ 1.14-1.20 (m, 2H, CH2), 1.81 (m, 1H, CH), 2.82 (s,

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1H, CH), 3.04-3.08 (m, 2H, CH2), 3.40-3.42 (t, J = 7.2 Hz, 2H, CH2), 3.52-3.61 (m, 4H, 2CH2), 3.67-3.08 (m, 1H, CH), 4.01 (s, 1H, CH), 7.86-7.91 (m, 2H, Ar-H), 8.12-8.13 (m, 4H, Ar-H);

13

C NMR (150 MHz, DMSO-d6) δ (ppm): 24.97, 43.84,

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44.10, 47.56, 48.32, 58.69, 59.23, 63.29, 76.31, 76.58, 76.23, 116.47, 124.49, 127.18, 128.78, 128.92, 133.33, 134.45, 134.82, 135.12, 144.09, 166.09, 181.85, 182.78;

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HRMS: Calcd. for C25H28N4O6+: 480.2009; found: 480.2002. 3.4.3. 2-(((4-((2S,3R,4R,5S)-3,4-dihydroxy-2,5-bis(hydroxymethyl)pyrrolidin-1-yl) butyl)amino)methyl)-1H-anthra[1,2-d]imidazole-6,11-dione (7c) Yield 40%; yellow solid, mp 143.6-145.4oC; [α]20D: +44.2 (c 0.1, MeOH); 1H

NMR (600 MHz, DMSO-d6): δ 1.26-1.37 (m, 4H, 2CH2), 3.10 (m, 1H, H-1’), 3.15 (s, 2H, CH2), 3.26 (m, 1H, H-4’) 3.51 (m, 2H, CH2’), 3.56 (m, 1H, H-3’), 3.71 (m, 1H, H-2’), 3.37 (m, 2H, CH2), 4.12-4.20 (m, 2H, CH2), 7.81-7.88 (m, 2H, H-5, H-8), 8.01-8.06 (m, 2H, H-3, H-4), 8.12 (s, 2H, H-2, H-1); 13C NMR (150 MHz, DMSO-d6): 10

ACCEPTED MANUSCRIPT δ 12.50, 22.59, 23.21, 24.58, 41.94, 49.01, 52.06, 52.17, 52.14, 53.64, 54.33, 54.96, 63.26, 6.35, 66.81, 70.14, 71.77, 118.24, 118.27, 120.58, 121.35, 125.21, 126.49, 126.69, 127.12, 127.2, 127.88, 132.11, 133.20, 133.36, 134.56, 134.76, 134.84, 134.98, 135.07, 149.04, 149.92, 160.44, 182.61, 183.51; HRMS: Calcd. for C26H30N4O6+: 494.2165; found: 494.2170.

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3.4.4. 2-(((6-((2S,3R,4R,5S)-3,4-dihydroxy-2,5-bis(hydroxymethyl)pyrrolidin-1-yl) hexyl)amino)methyl)-1H-anthra[1,2-d]imidazole-6,11-dione (7d)

Yield 45%; yellow solid, mp 149.6-150.5oC; [α]20D: +22.1 (c 0.1, MeOH); 1H

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NMR (600 MHz, DMSO-d6): δ 0.85-0.91 (m, 4H, CH2), 1.17-1.21 (m, 2H, CH2), 1.52-1.56 (m, 2H, CH2), 2.43 (t, J = 6.6 Hz, 2H, CH2), 2.91-2.99 (m, 1H, CH),

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3.17-3.20 (m, 2H, CH2), 3.47 (m, 2H, CH2), 3.52-3.53 (d, J = 8.4 Hz, 1H, CH), 3.58 (s, 2H, CH2), 3.61-3.63 (m, 1H, CH), 4.00 (m, 1H, CH), 7.87 (m, 2H, Ar-H), 8.13-8.14 (m, 4H, Ar-H), 8.47 (s, 1H, Ar-H);

13

C NMR (150 MHz, DMSO-d6) δ

(ppm): 25.13, 55.95, 59.56, 79.17, 79.40, 79.61, 116.58, 124.46, 127.13, 128.54, 128.85, 133.54, 134.58, 134.91, 144.56, 171.45, 181.72, 182.80; HRMS: Calcd. for

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C28H34N4O6+: 522.2478; found: 522.2474.

3.4.5. 2-(((8-((2S,3R,4R,5S)-3,4-dihydroxy-2,5-bis(hydroxymethyl)pyrrolidin-1-yl) octyl)amino)methyl)-1H-anthra[1,2-d]imidazole-6,11-dione (7e) Yield 48%; yellow solid, mp 149.8-151.2oC; [α]20D: -56.5 (c 0.1, MeOH); 1H

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NMR (600 MHz, DMSO-d6): δ 1.40 (m, 12H, 6CH2), 3.37 (s, 1H, H-1’), 3.47 (m, 2H, CH2), 3.58 (m, 1H, H-4’) 3.89 (m, 2H, CH2), 3.99 (m, 1H, H-2’), 4.05 (m, 2H, CH2),

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4.08 (m, 2H, CH2), 4.10 (m, H, H-3’), 4.30 (d, 2H, J = 3.0 Hz, CH2), 7.81 (m, 2H, H-5, H-8), 7.98 (m, 1H, H-1), 8.08 (m, 1H, H-2), 8.17 (m, 2H, CH2); 13C NMR (150 MHz, DMSO-d6): δ 7.08, 37.24, 37.79, 36.64, 40.09, 41.79, 44.42, 44.45, 52.26, 56.01, 56.14, 58.77, 71.79, 74.57, 80.12, 82.64, 96.69, 118.22, 118.83, 112.14, 123.49, 125.18, 128.21, 130.13, 151.63, 162.04, 162.09, 162.52, 164.97, 172.14; HRMS: Calcd. for C30H38N4O6+: 550.2791; found: 550.2785. 3.5. In vitro colorimetric reverse transcriptase assay The HIV-RT inhibition assay was performed by using a colorimetric reverse 11

ACCEPTED MANUSCRIPT transcriptase assay (Roche), and the procedure for assaying reverse transcriptase (RT) inhibition was performed as described in the kit protocol. Briefly, the reaction mixture consists of template/primer complex, 2’-deoxy-nucleotide-5’-triphosphates (dNTPs) and reverse transcriptase (RT) enzyme in the lysis buffer with or without inhibitors. 1h

incubation

at

37°C

the

reaction

mixture

was

transferred

to

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After

streptavidine-coated microtitre plate (MTP). The biotins labeled dNTPs that are incorporated in the template due to activity of RT were bound to streptavidine. The unbound dNTPs were washed using wash buffer and antidigoxigenin-peroxidase

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(DIG-POD) was added in MTP. The DIG-labeled dNTPs incorporated in the template was bound to anti-DIG-POD antibody. The unbound anti-DIG-POD was washed and

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the peroxide substrate (ABST) was added to the MTP. A colored reaction product was produced during the cleavage of the substrate catalyses by a peroxide enzyme. The absorbance of the sample was determined at OD (optical density) 405 nM using microtiter plate ELISA reader. The resulting color intensity is directly proportional to the actual RT activity. The percentage inhibitory activity of RT inhibitors was

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calculated by comparing to a sample that does not contain an inhibitor. The percentage inhibition was calculated by formula as given below: % Inhibition = 100[(OD 405 nm with inhibitor/OD 405 nm without inhibitor) ×100].

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3.6. Cytotoxicity analysis

The cytotoxicity of the compounds against HeLa cell lines (human cervical

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cancer cells) and human lung adenocarcinoma epithelial cell line (A-549) was examined by the modified Mosmann’s protocol as follows: Briefly, cells (104 cells per well) were plated in 96-well culture plates and cultured overnight at 37°C in a 5% CO2 humidified incubator. Compounds were added to the wells at final concentrations of 1, 10 and 100 µmol/L. Control wells were prepared by addition of DMEM. Wells containing DMEM without cells were used as blanks. The plates were incubated at 37°C in a 5% CO2 incubator for 48 h. Upon completion of the incubation, stock MTT dye solution (10 µL, 5 mg/mL) was added to each well. After 4 h of incubation, the 12

ACCEPTED MANUSCRIPT supernatant was removed and dimethyl sulfoxide (DMSO) (100 µL) was added to dissolve the MTT. The optical density of each well was measured on a microplate spectrophotometer at a wavelength of 570 nm. The cytotoxicity effect was calculated according to the formula: (ODcontrol _ ODtreated)/ODcontrol ×100%.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 21572044, 21372060 and 21172051), the The Colleges and Universities

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Science Technology Research Project of Hebei Province (QN2017015), the Hebei Natural Science Foundation (B2016201031) and Program for Changjiang Scholars

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and Innovative Research Team in University (IRT-15R43) References

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ACCEPTED MANUSCRIPT 1057-1059. [21] T. J. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, Immunol. Meth. 65 (1983)

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Table1 The inhibitions against HIV-1 reverse transcriptase of compounds 3a, 4 and 7. compound

4 40.86 ± 3.29

7a 24.54 ± 1.56

7b 27.78 ± 3.65

7c 33.13 ± 1.43

7d 31.12 ± 1.45

7e 36.31 ± 1.29

AZT 31.24 ±2.87

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IC50 (µM)

3a 8.51 ±0.86

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Table2 The cytotoxic activities of compounds 3, 4 and 7. Cytoxicity (IC50, µM) Compound A549

3a

12.32±0.07

>100

-a

3b

8.23±0.09

36.12±0.05

4

20.34±0.06

>100

7a

44.23±0.06

25.17±0.04

7b

80.39±0.07

49.48±0.05

7c

123.64±0.09

50.00±0.03

7d

191.80±0.03

97.77±0.05

84.81±0.03

7e

6.80±0.08

53.64±0.03

23.16±0.04

Cisplatin

6.09±0.02

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-a -a

28.72±0.02

47.24±0.01

73.49±0.01

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: no test

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MCF-7

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a

Hela

6.59±0.05

8.93±0.01

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Highlights > Novel C-nucleoside analogues > Sugar and azasugar modified anthra[1,2-d]

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imidazole-6,11-dione derivatives.