Accepted Manuscript Synthesis of novel poly-hydroxyl functionalized acridine derivatives as inhibitors of αGlucosidase and α-Amylase Zahra Toobaei, Reza Yousefi, PhD, Farhad Panahi, Sara Shahidpour, Maryam Nourisefat, Mohammad Mahdi Doroodmand, Ali Khalafi-Nezhad, PhD PII:

S0008-6215(15)00116-0

DOI:

10.1016/j.carres.2015.04.005

Reference:

CAR 6980

To appear in:

Carbohydrate Research

Received Date: 28 February 2015 Revised Date:

7 April 2015

Accepted Date: 9 April 2015

Please cite this article as: Toobaei Z, Yousefi R, Panahi F, Shahidpour S, Nourisefat M, Doroodmand MM, Khalafi-Nezhad A, Synthesis of novel poly-hydroxyl functionalized acridine derivatives as inhibitors of α-Glucosidase and α-Amylase, Carbohydrate Research (2015), doi: 10.1016/j.carres.2015.04.005. 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.

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ACCEPTED MANUSCRIPT Synthesis of novel poly-hydroxyl functionalized acridine derivatives as inhibitors of αGlucosidase and α-Amylase. Zahra Toobaei1, Reza Yousefi,*1,2 Farhad Panahi,3 Sara Shahidpour,1 Maryam Nourisefat,3

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Mohammad Mahdi Doroodmand3, Ali Khalafi-Nezhad*3 Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz

University, Shiraz, Iran Institute of Biotechnology, Shiraz University, Shiraz, Iran

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Department of Chemistry, College of Sciences, Shiraz University, Shiraz, Iran

Corresponding Authors: Reza

Yousefi

(PhD),

email:

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[email protected],

Phone:

++987116137617,

++987116137665, Fax: ++987112280916

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Ali Khalafi-Nezhad (PhD), e-mail: [email protected], Phone: ++987112282380,

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++987116137665, Fax: +++987112280926

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ACCEPTED MANUSCRIPT Abstract In this study a novel series of poly-hydroxyl functionalized acridine derivatives (L1L9) was synthesized and their inhibitory activities against α-Glucosidase (α-Gls) and αAmylase (α-Amy) were evaluated, spectroscopically. The synthetic compounds consist of

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three different substructures, including a 4-(4-aminophenoxy) phenyl group (R3), a pyrimidine fused heterocycle moiety (R2) and a poly-hydroxy chain (R1). The results indicate that among the synthetic compounds, L5 with a chromeno[3',4':5,6]pyrido[2,3-d]pyrimidine

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moiety demonstrates the highest inhibitory activity against both yeast and rat α-Gls enzymes.

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Also, L2 with the thioxo-pyrido[2,3-d:6,5-d'] dipyrimidine moiety plays an important role in the inhibition of yeast α-Gls. In addition, the results may suggest a significant role for the nature of sugar moiety of the synthetic compounds in their inhibitory action against α-Gls. Moreover, in comparison with Acarbose which is a widely used anti-diabetic drug, these compounds show negligible inhibitory activity against pancreatic α-Amy which is important

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in the term of their reduced susceptibility for possible development of the intestinal disturbance side effects. Results of this study may suggest these synthetic compounds as

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novel molecular templates for construction of potentially anti-diabetic drugs with the ability for more convenient management of postprandial hyperglycemia.

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Keywords: α-Glucosidase, inhibition, anti-diabetic compounds, postprandial hyperglycemia, α-Amylase

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ACCEPTED MANUSCRIPT 1. Introduction Diabetes mellitus is the most common global endocrine disease and its incidence is growing at an alarming rate [1]. This progressive metabolic disorder is characterized by the chronic elevation of serum glucose level which ultimately leads to micro- and macro-vascular

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changes, causing damages in several tissues including retina, kidney, nerves and blood vessels [2]. The prolonged elevation of blood glucose in diabetic patients accelerates the rate of non-enzymatic reaction between sugar and long-lived proteins which subsequently results

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in formation of protein-bound advanced glycation end products (AGEs). [3]. Moreover,

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chronic hyperglycemia in diabetes mellitus results in the excessive flux of glucose into sorbitol-aldose reductase (SAR) pathway, leading to production and accumulation of intracellular sorbitol in kidney, lens, retina and nerves which eventually cause the cellular damages [4]. Both of these abnormal metabolic processes have been reported to contribute in the pathological events, leading to development of various diabetic secondary complications

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[5]. Therefore, the inhibition of aldose reductase which is the first enzyme in SAR pathway and pharmacological inhibition of AGE formation or disruption of pre-existed AGE-protein

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cross-links provide significant potential therapeutic approaches towards reducing the severity of diabetes associated complications [6]. However, the incidence of secondary complications

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in type-II diabetes is known to tightly correlate with postprandial hyperglycemia which is an indicator of the total glycemic uptake [7]. Therefore, the inhibition of intestinal absorption of sugar which is not interfering with their metabolism can help to control postprandial hyperglycemia in a non-invasive manner [8, 9]. As shown in Fig.1, the digestion of starch mostly occurs in the lumen of mammalian small intestine by α-Amylase (α-Amy) to yield both linear maltose and branched oligosaccharides such as isomaltose. Further digestion of carbohydrates by α-Glucosidase (α-Gls) (EC 3.2.1.20) which is existed in the epithelial mucosa of small intestine releases the absorbable monosaccharides [10]. Therefore, the 3

ACCEPTED MANUSCRIPT inhibition of α-Gls is an effective approach in both preventing and treating diabetes through improvement of postprandial hyperglycaemia [11-13] ►Figure 1◄ The commercial inhibitors of α-Gls (i.e. acarbose, voglibose, miglitol) are currently in

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use for the treatment of diabetes mellitus. The application of these anti-diabetic drugs is associated with the substantial adverse gastrointestinal side effects due to their non-specific inhibition of α-Amy, causing excessive accumulation of undigested carbohydrates in the

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large intestine [14-16]. Therefore, it is necessary to develop more tolerable α-Gls inhibitors with no or less unfavorable side effects. In this regard, it is desirable to search for compounds

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with moderate α-Amy inhibitory properties, displaying potent α-Gls inhibition. In this study, the research objective was to synthesis novel poly-hydroxy functionalized acridine (PHFA) derivatives and to assess their inhibitory properties against both α-Gls and α-Amy. The findings in this study may offer strong foundation to develop new and more specific α-Gls

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inhibitors with potentially therapeutic values for reducing the severity of those secondary complications which are normally associated with type-II diabetes mellitus.

2.1. Materials

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2. Material and Methods

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Yeast α-Gls (EC.3.2.1.20), porcine pancreatic α-Amy (EC 3.2.1.1) and paranitrophenyl-α-D-Glucopyranoside (pNPG) were purchased from Sigma Aldrich (Gillingham, Dorset, UK) Chemical Company. Other chemicals were purchased from Fluka and Aldrich chemical companies and used without further purification. 1H and

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C-NMR spectra were

recorded on a Bruker Advance 250 MHz spectrometer in DMSO solution with TMS as an internal standard. Shimadzu GC/MS-QP 1000-EX apparatus; in m/z (rel%) was used for mass analysis of products. FTIR spectroscopy (Shimadzu FT-IR 8300 spectrophotometer) was employed for the compound characterization. Melting points were determined in open

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ACCEPTED MANUSCRIPT capillary tubes in a Barnstead Electrothermal 9100 BZ, circulating oil melting point apparatus. The reaction monitoring was accomplished by TLC on silica gel PolyGram SILG/UV254 plates. 2.2. Methods

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2.2.1. General procedure for the synthesis of compounds L1-L9

A mixture of sugar (1.0 mmol), diketone (2.0 mmol for identical diketones and 1 mmole for different diketones) 4,4'-oxydianiline (1.0 mmol), and PTSA (0.05g, 30 mol%) in EtOH (5

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mL) was stirred at 50 °C for 12 h. In the case of compounds L4 and L5 one mmole of each diketone was used. After cooling down to room temperature of the reaction mixture, the

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precipitate was filtered and washed with ethanol (15 mL) to afford the pure product. 2.2.2. Spectral data for the synthesized compounds

10-(4-(4-aminophenoxy)phenyl)-5-((1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl)-5,10dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L1)

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Yield: 82% (0.48 g); brown solid, m.p. 139-141 ºC. 1H-NMR (250 MHz, DMSO-d6/TMS): 3.27-3.43 (m, 6H), 3.56-3.64 (m, 4H), 3.86 (s, 1H, CH), 4.16 (s, 1H, CH), 4.77 (m, 2H), 6.796.91 (m, 8H, Ar), 11.07-11.21 (m, 4H, NH).13C-NMR (62.5 MHz, DMSO-d6/TMS): 18.4,

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43.4, 55.9, 63.1, 65.3, 70.5, 72.0, 76.2, 118.1, 119.0, 138.2, 150.6, 212.6.. MS: 582 (17.5%,

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M+). Anal. Calcd for C26H26N6O10 (582.53): C, 53.61; H, 4.50; N, 14.43. Found: C, 53.45; H, 4.39; N, 14.32.

10-(4-(4-aminophenoxy)phenyl)-5-((1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl)-2,8dithioxo-2,3,5,8,9,10-hexahydropyrido[2,3-d:6,5-d']dipyrimidine-4,6(1H,7H)-dione (L2) Yield: 79% (0.48 g), brown solid, m.p. 150-152 ºC, 1H-NMR (250 MHz, DMSO-d6/TMS): 3.13–3.48 (m, 3H), 3.52–3.74 (m, 3H), 4.24 (d, J = 3.2 Hz, 1H), 6.42 (d, J = 7.7 Hz, 2H), 6.76-6.80 (m, 6H), 8.81 (brs, 2H), 11.35 (brs, 2H).

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C-NMR (62.5 MHz, DMSO-d6/TMS):

22.1, 65.1, 66.6, 71.7, 72.5, 73.8, 87.4, 117.8, 122.6, 122.5, 135.1, 143.3, 144.8, 157.5, 163.5,

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ACCEPTED MANUSCRIPT 176.2. MS: 614 (17.5%, M+). Anal. Calcd for C26H26N6O8S2 (614.65): C, 50.81; H, 4.26; N, 13.67. Found: C, 50.72; H, 4.17; N, 13.58. 10-(4-(4-aminophenoxy)phenyl)-3,3,6,6-tetramethyl-9-((1S,2R,3R,4R)-1,2,3,4,5pentahydroxypentyl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (L3)

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Yield: 67% (0.41 g), yellow solid, m.p. 161-163 ºC. 1H-NMR (250 MHz, DMSO-d6/TMS): 0.96 (s, 6H), 0.99 (s, 6H), 2.17-2.28 (Distorted AB system, 4H), 2.47 (s, 4H), -3.15–3.50 (m, 3H), 3.53–3.75 (m, 3H), 4.23 (d, J = 2.9 Hz, 1H), 6.42 (d, J = 6.9 Hz, 2H), 6.75-6.79 (m, 6H). 13

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C-NMR (62.5 MHz, DMSO-d6/TMS): 23.1, 27.3, 29.4, 31.9, 32.3, 40.8, 50.8, 65.0, 66.9,

71.7, 72.8, 116.7, 121.9, 122.5, 122.8, 134.7, 141.8, 144.9, 151.8, 196.4. MS: 606 (22.1%,

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M+). Anal. Calcd for C34H42N2O8 (606.72): C, 67.31; H, 6.98; N, 4.62. Found: C, 67.23; H, 6.90; N, 4.54.

(R)-10-(4-(4-aminophenoxy)phenyl)-8,8-dimethyl-5-((1S,2R,3R,4R)-1,2,3,4,5pentahydroxypentyl)-5,8,9,10-tetrahydropyrimido[4,5-b]quinoline-2,4,6(1H,3H,7H)-trione

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(L4)

Yield: 68% (0.40 g), yellow solid, m.p. 173-175 ºC. 1H-NMR (250 MHz, DMSO-d6/TMS): 0.94 (s, 6H), 2.19-2.29 (m, 4H), 3.19–3.53 (m, 3H), 3.55–3.71 (m, 3H), 4.29 (d, J = 3.2 Hz, 13

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1H), 6.45 (d, J = 7.1 Hz, 2H), 6.73-6.78 (m, 6H), 10.59 (s, 1H), 11.08 (s, 1H).

C-NMR

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(62.5 MHz, DMSO-d6/TMS): 22.9, 27.1, 33.6, 31.9, 43.5, 51.5, 65.1, 66.8, 72.2, 72.9, 88.1, 116.3, 116.5, 116.9, 122.5, 135.1, 142.4, 145.1, 150.2, 152.1, 162.5, 163.2, 197.1. MS: 594 (19.5%, M+). Anal. Calcd for C30H34N4O9 (594.62): C, 60.60; H, 5.76; N, 9.42. Found: C, 60.51; H, 5.68; N, 9.34.

(S)-12-(4-(4-aminophenoxy)phenyl)-7-((1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl)-7,12dihydro-6H-chromeno[3',4':5,6]pyrido[2,3-d]pyrimidine-6,8,10(9H,11H)-trione (L5) Yield: 74% (0.45 g), yellow solid, m.p. 179-181 ºC. 1H-NMR (250 MHz, DMSO-d6/TMS): 3.23–3.72 (m, 6H), 5.91 (s, 1H), 6.44 (d, J = 7.4 Hz, 2H), 6.72-6.79 (m, 6H), 7.38 (d, J = 8.3

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ACCEPTED MANUSCRIPT Hz, 2H), 7.64 (t, J = 8.1 Hz, 1H), 7.98-8.03(m, 1H), 10.68 (s, 1H), 11.04 (s, 1H). 13C-NMR (62.5 MHz, DMSO-d6/TMS): 22.8, 65.2, 67.0, 72.3, 73.90, 88.0, 104.5, 115.1, 116.7, 117.1, 122.1, 122.8, 123.2, 126.2, 129.4, 134.9, 142.3, 145.4, 149.8, 150.7, 152.5, 161.9, 162.5, 165.8. MS: 616 (11.0%, M+). Anal. Calcd for C31H28N4O10 (616.58): C, 60.39; H, 4.58; N,

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9.09. Found: C, 60.28; H, 4.49; N, 8.97.

10-(4-(4-aminophenoxy)phenyl)-5-((1S,2R,3S,4R)-1,2,3,4,5-pentahydroxypentyl)-5,10dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L6)

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Yield: 76% (0.44g); brown solid, m.p. 238-240 ºC.. 1H-NMR (250 MHz, DMSO-d6/TMS) δ (ppm): 3.21 (s, 1H), 3.24 (s, 1H), 3.28-3.33 (m, 2H), 3.39-3.52 (m, 5H), 3.58 (s, 1H), 3.62 (s,

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1H), 3.90 (t, 1H, J = 9.5 Hz), 4.90 (brs, 2H), 6.76-6.84 (m, 8H), 11.04 (s, 1H), 11.10 (s, 1H), 11.16-11.20 (m, 2H). 13C-NMR (62.5 MHz, DMSO-d6/TMS) δ (ppm): 18.4, 43.5, 48.3, 55.9, 63.1, 70.5, 72.0, 76.2, 78.1, 80.9, 118.1, 119.0, 138.2, 150.0, 212.6. MS: 582 (8%, M+). Anal. Calcd for C26H26N6O10 (582.53): C, 53.61; H, 4.50; N, 14.43. Found: C, 53.53; H, 4.46;

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N, 14.32.

10-(4-(4-aminophenoxy)phenyl)-5-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-5,10dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L7)

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Yield: 70% (0.38g); brown solid, m.p. 168-172 ºC. 1H-NMR (250 MHz, DMSO-d6/TMS) δ

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(ppm): 3.15-3.44 (m, 7H), 3.59 (t, 1H, J = 5.0 Hz), 3.82-3.91 (m, 2H), 4.67 (brs, 2H), 6.736.81 (m, 8H), 11.08 (s, 2H), 11.18 (s, 2H).

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C-NMR (62.5 MHz, DMSO-d6/TMS) δ (ppm):

22.0, 24.1, 61.7, 73.2, 77.4, 119.0, 119.1, 119.2, 120.2, 120.5, 125.4, 128.1, 136.7, 150.9, 151.2, 151.5, 167.6, and 210.8. MS: 552 (14%, M+). Anal. Calcd for C25H24N6O9 (552.50): C, 54.35; H, 4.38; N, 15.21. Found: C, 54.28; H, 4.32; N, 15.14. 10-(4-(4-aminophenoxy)phenyl)-5-((1S,2S,3R,4R)-1,2,4,5-tetrahydroxy-3(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-

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ACCEPTED MANUSCRIPT yl)oxy)pentyl)-5,10-dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L8) Yield: 63% (0.47g); pale yellow crystal, m.p. 150-151 ºC. 1H-NMR (250 MHz, DMSOd6/TMS/D2O) δ (ppm): 3.24-3.27 (m, 6H), 3.40 (s, 1H), 3.43 (s, 1H), 3.45-3.49 (m, 6H), 3.59 13

C-NMR

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(s, 4H), 3.71-3.72 (m, 2H), 4.44 (brs, 2H), 6.73 (m, 8H), 11.09-11.27 (m, 4H).

(62.5 MHz, DMSO-d6/TMS) δ (ppm): 18.4, 48.11, 56.0, 60.3, 60.4, 68.0, 69.3, 69.7, 70.4, 71.2, 71.9, 73.1, 75.1, 75.3, 80.5, 81.1, 91.9, 103.6, 103.7, 117.8, 119.0, 138.6, 150.5, 150.9,

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151.5, 167.8. MS: 744 (6%, M+). Anal. Calcd for C32H36N6O15 (744.67): C, 51.61; H, 4.87; N, 11.29. Found: C, 51.52; H, 4.80; N, 11.21.

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10-(4-(4-aminophenoxy)phenyl)-5-((1S,2S,3R,4R)-1,2,4,5-tetrahydroxy-3-

(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)pentyl)-5,10-dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L9)

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Yield: 65% (0.48g), Brown crystal, m.p. 178-180 ºC. 1H-NMR (250 MHz, DMSO-d6/TMS) δ (ppm): 3.32-3.43 (m, 14H), 3.58-3.63 (m, 6H), 3.71-3.72 (m, 2H), 4.67 (brs, 2H), 6.75 (s, 8H), 11.07-11.08 (s, 4H).

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C-NMR (62.5 MHz, DMSO-d6/TMS) δ (ppm): 18.4, 55.9, 60.6,

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60.7, 69.7, 73.2, 118.9, 119.0, 136.8, 151.2, 151.5, 167.6, 167.7. MS: 744 (9%, M+). Anal.

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Calcd for C32H36N6O15 (744.67): C, 51.61; H, 4.87; N, 11.29. Found: C, 51.53; H, 4.82; N, 11.22.

2.2.3. Preparation of the acetone powder of rat intestine The acetone powder of rat intestine was prepared, according to the method reported

previously [17, 18]. 2.2.4. α-Gls inhibition assay The inhibition assay of yeast and rat α-Gls was performed according to our previous publications with minor changes [19]. The inhibition of yeast α-Gls (0.2 U/mL) was

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ACCEPTED MANUSCRIPT measured with increasing concentration of pNPG (0.1-5 mM) and in the presence of various concentrations of each synthetic compound in buffer A (0.1 M NaPi, pH 7.0), at 25 °C for 10 min. In order to measure the inhibition of rat α-Gls, a reaction mixture, containing this enzyme (0.15 U/mL) and varying concentrations of each synthetic compound was pre-

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incubated for 3 min at 37 °C in Buffer B (0.01 M NaPi, pH 7.0) , before addition of pNPG (0.5-10 mM). The stock solutions of the synthetic compounds were prepared in dimethyl sulfoxide (DMSO). Mode of inhibition was determined by Lineweaver-Burk plot analysis

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and the concentration of each synthetic compound required 50% inhibition of α-Gls activity (IC50 value) was calculated, using Dixon plot [20]. Also, the experimental inhibition constant

2.3. Pancreatic α-Amy assay

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(Ki) was determined by Cheng- Prusoff equations [19].

The α-Amy inhibition was determined based on conventional methods [21, 22]. Each synthetic compound (250 µL, 0.4 M) was incubated with porcine α-Amy (250 µL, 0.5

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mg/mL) at 25 oC for 10 minutes in buffer C (20 mM NaPi, pH 6.9 containing 6 mM sodium chloride). After pre-incubation, 250 µL of a starch solution (0.5%) in buffer C was added to each tube. The reaction mixtures were then incubated at 25 oC for 10 minutes and then

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stopped with 1 mL of dinitrosalicylic acid (DNS). After that, the test tubes were incubated in

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a boiling water bath for 5 minutes. After cooling down to room temperature, the reaction mixture was then diluted by adding 10 mL distilled water and the absorbance measured at 540 nm. The readings were compared with the controls, containing buffer instead of sample extract and the results were expressed as percentage of α-Amy inhibition, according to the following equation: (1)

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ACCEPTED MANUSCRIPT 3. Results and Discussion 3.1. Chemistry of the α-Gls inhibitors In our previous studies, we have introduced the pyrimidine fused heterocycle (PFH) compounds as important class of α-Gls inhibitor in which the PFH ring plays a substantial

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role [15,16]. In structure of these inhibitors, three different substructures R1, R2 and R3 can be changed to obtain new compounds with an improved inhibitory action (Fig. 2).

HO OH

OH

HO OH

NH N H

N

R3

N H

HN OS

N

NH2

NH2

OH

HO O

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O

HN

NH

N H

O O

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N

O

N H

N

N H

NH

O

O

NH2

NH2

NH2

L4

L5

L3

O

N

OH

O

O

N

O

O

O

NH2

NH2

NH2

L6

L7

L8

N H

O

O O

HO

OH O

O

OH

O OH

HN

NH N H

OH

HO HO

O

HO

O

HO

HO HO HO

N H

OH

OH

HN O O

O

O

OH

NH

O

N

OH

HO O

NH N H

OH

HO O

O

N

HO

HO

N H

S

HO

OH

HO O

OH

OH

R2

N H

L2

HO

O

N H

O

HN

O

NH

O

L1

R1

OH

HO O

O

OH

HO

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O

OH

HO

OH

HO

HO O

O

HN

R2

OH

HO

HO O

HO

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R1

R3

HO

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HO

O

NH N H

N

N H

O

O

NH2 L9

►Figure 2◄ Therefore, it is possible to modulate their α-Gls inhibitory action, using selection of appropriate R1, R2, and R3 moieties. Although the significant role of substructures, R1

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ACCEPTED MANUSCRIPT (polyhydroxyl chain) and R3 (4-(4-aminophenoxy) phenyl) in α-Gls inhibitory action have been demonstrated already by us; in the current study the impact of substructure R2 (PFH ring) was evaluated. Therefore, new derivatives of the PFH compound were synthesized and their abilities to inhibit α-Gls/α-Amy enzymes were studied. As shown in Fig.2, compounds

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L1-L5 possess two constant structural moieties, R1 and R3, and one variable PFH ring (R2). For synthesis of these compounds (L1-L5), the following multicomponent approach was used. We used a protocol in which one equivalent of (+)-D-glucose and one equivalent of

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4,4'-oxydianiline were reacted with two equivalents of an appropriate diketone to produce corresponding compound (Scheme 1). After evaluating the impact of R2 in L1-L5, we realized

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that this substructure strongly influences the inhibitory action of the synthetic compounds. Due to the significant role of poly hydroxyl substructure in the inhibitory action of these compounds, we decided to change this substructure, while R2 and R3 remained constant. Also, variation of R1 with changing the type of sugar which participates in the multi component

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reaction (MCR) is possible. Therefore, we used different sugars (galactose, arabinose, lactose

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and maltose) and synthesized L6-L9 compounds (Scheme 1).

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ACCEPTED MANUSCRIPT ►Scheme 1◄ The synthetic compounds were also characterized, using different spectroscopic techniques such as 1H-NMR,

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C-NMR and mass spectroscopy. The data revealed that these molecules

were produced successfully.

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The variable substructure, R2 in L1 (and L6-L9), L2, L3, L4 and L5 are pyrido[2,3-d:6,5-d'] dipyrimidine, thioxo-pyrido[2,3-d:6,5-d'] dipyrimidine, acridine, pyrimido [4,5-b]quinoline and chromeno [3',4':5,6]pyrido [2,3-d] pyrimidine, respectively [23,24]. The relatively small

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size and lipophilic properties of the synthetic compounds are to satisfy the Lipinski's rule of five; therefore, these compounds are likely to be orally active agents in human. The

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heterocyclic ring of the synthetic compounds is already well-known for its broad spectrum of biological activities, including anticancer, antimicrobial, antiviral, antifungal, anticoagulation and anti-inflammatory properties [19, 25]. Therefore these synthetic compounds may have further biological and therapeutic values. As a result of R2 variation, the synthetic

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compounds demonstrate significant alteration in their three-dimensional structures (Fig. 3). ►Figure 3◄

In this regard, the changes in spatial position of R1 and R3 clearly indicate the

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particular role of R2 in the unique conformation of each compound. Therefore, we believe

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that the conformational feature might be also an important parameter to consider for the inhibitory action of the synthetic compounds. As indicated in Fig.3, the synthetic compounds with the same scaffold also demonstrate nearly similar conformations which can explain their comparable biological activities. For instance L1 and L2 with similar conformations display almost similar inhibitory activity. Due to the occurrence of dimedone moiety in its structure; L3 is more lipophilic than L1 and L2 which possess respectively barbituric acid and thiobarbituric acid in their structures. For the synthesis of L4 and L5 two different diketones were used. Also, in the structure of these compounds there is a stereogenic center, allowing

12

ACCEPTED MANUSCRIPT them to appear in the stereoselective manner. Since we did not obtain their single crystals, the exact structure of these ligands (L4 and L5) remained unsolved. Compound L4 with a dimedone and a barbituric acid moiety has different conformation from either L1 or L3. Interestingly, in compound L5 which possesses a 4-hydroxycoumarin fragment, conformation

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is entirely different from other synthetic compounds. In compounds L6-L9 which differ only in R1, the conformational variation resulted from the nature of poly hydroxy chain. 3.2. Inhibition of α-Gls by different synthetic compounds

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The synthetic compounds were compared for their enzyme inhibitory action, according to the method described in the experimental section. Yeast α-Gls which is readily

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available in a pure form was used for the initial screening of the potential inhibitors. Also, the inhibitory activities of the synthetic compounds were studied against rat α-Gls which resembles the human enzyme counterpart. Since both enzymes belong to the family-II of αGls [27]; the results of rat enzyme inhibition are likely to be case for the human enzyme

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counterpart. Both inhibition mode and IC50 value were determined, respectively by Lineweaver-Burk (Figs. 4 and 5) and Dixon plots (Figs. 6 and 7).

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►Figure 4◄ ►Figure 5◄ ►Figure 6◄ ►Figure 7◄

Also, in this study, additional to IC50 value, the inhibition parameter, Ki was obtained

by Cheng-Prusoff equations to compare the affinity of each inhibitor for a particular enzyme. The inhibition constant, Ki is equated with a specific inhibitor concentration that leads to half-maximal saturation of the available enzyme binding sites [28]. According to ChengPrusoff equations [28], and the results presented in Tables 1 and 2, a direct correlation

13

ACCEPTED MANUSCRIPT between Ki and IC50 values further notifies the existence of competitive inhibition mode of the synthetic compounds. The Lineweaver-Burk plots revealed that these compounds except L4 and L7 act as competitive inhibitors against yeast α-Gls. The synthetic compounds L4 and L7 demonstrate

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a mixed-competitive inhibition mode (Table 1). Moreover, all of the synthetic compounds exhibit competitive inhibitory action against rat enzyme (Table 2). ►Table 1◄

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►Table 2◄

While, L2 and L5 were the most promising inhibitors against yeast α-Gls, only L5

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demonstrated significant inhibitory action on the rat enzyme counterpart (Tables 1 and 2). As shown in Table 1, the inhibitors, L2 and L5 respectively possess the IC50 values of 11.96 and 8.88 µM, against yeast α-Gls. Also, L5 inhibits the rat enzyme with an IC50 value of 43.21 µM. Surprisingly, the replacement of thioxo in L2 with oxo in L1, results in its significantly

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poor inhibitory action with an IC50 value of 209.8 µM, against the yeast enzyme (Table 1). This finding indicates the significant impact of a chemical modification on the α-Gls inhibitory action of this class of synthetic compounds. However, due to the structural

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difference in their corresponding binding sites, these compounds possess different inhibitory

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activity against rat α-Gls. In this study, L1 with an IC50 value of 88.13 µM demonstrates the second most promising inhibitor after L5, against rat α-Gls. The variable substructure, R3 in L1

and

L5

are

pyrido[2,3-d:6,5-d']dipyrimidine

and

chromeno[3',4':5,6]pyrido[2,3-

d]pyrimidine, respectively. Also, the results indicated that L3 did not exhibit any inhibitory action against rat α-Gls, and indicated a weak inhibitory activity against yeast enzyme counterpart (IC50=122.3 µM). The synthetic compound L3 is an acridine analogue which due to the existence of four methyl and methylene groups in its structure, demonstrates lower hydrophobic properties compared to the other compounds. Also it has a reduced number of

14

ACCEPTED MANUSCRIPT hydrogen binding sites in comparison to the other synthetic compounds, affecting its solubility in water and its capability for fine interaction with the target enzyme. These reasons may justify the absence of inhibitory activity of L3 in relation to the other synthetic compounds. Although, compound L4 with a pyrimido[4,5-b]quinoline structure shows a very

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poor inhibitory activity (IC50=141.5 µM) against rat α-Gls, it is not comparable with either L1 or L5. This compound also possesses poor inhibitory activity (177.1 µM) against yeast α-Gls. In chemical structure of L4, two methyl and methylene groups may contribute in the

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improvement of the compound’s inhibitory action. The structure-activity relationship of the synthetic compounds revealed that the presence of pyrimidine unit is more efficient than the

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existence of dimedone in substructure R2. The improvement of inhibitory activity due to the presence of coumarin unit in compound L5 is an important point. Coumarin has been found as a valuable structure in the synthesis of many drugs due to its high potential biological activities [29-33]. Its seems that the presence of coumarin moiety in substructure R2 of

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compound L5 creates suitable binding sites to accommodate relatively high inhibitory activity in this compound. Our results indicated that L5 was the most promising tested compound for the inhibition of both yeast and rat enzymes. Also, this study prompted us to synthesize new

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polyhydroxy-PFH based on L1 with the aim to examine the impact of nature of sugar moiety

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on the enzyme inhibitory action of the synthetic compounds. The synthetic compounds L1 and L6 which respectively contain glucose and galactose in their structures indicated different enzyme inhibitory properties. In comparison with L1, compound L6 did not show any activity, indicating the significance of stereochemistry of the sugar moiety in the inhibitory action of the synthetic compounds. The results suggest that L7 with a polyhydroxy chain which derived from arabinose demonstrates most promising inhibitory action with the IC50 values of 45.20 µM and 379.1 µM against yeast α-Gls and rat enzyme counterpart, respectively. These results also revealed 15

ACCEPTED MANUSCRIPT that the length of polyhydroxy chain is effective in their inhibitory action. For instance, L1 with a polyhydroxy chain which derived from glucose indicated lower inhibitory action than L7 that its polyhydroxy chain derived from arabinose. In addition, slight enzyme inhibitory activities were observed for L8 and L9 which their polyhydroxy chain are derived from

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lactose and maltose, respectively. Overall, our results may suggest an important function for the nature of the sugar moiety on the inhibitory action of this class of synthetic compounds against α-Gls.

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Acarbose is a widely used anti-diabetic drug which demonstrates inhibitory action

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with the IC50 values of 365.4 and 22.3 µM against yeast and rat enzymes, respectively (Tables 1 and 2). Comparing the results of inhibitory action for the rat enzyme system; it is clear that inhibitory properties of the synthetic compounds are not in the very satisfactory ranges and further chemical modifications are needed to obtain inhibitor compounds with improved activities. On the other hand, similar to Acarbose, the competitive inhibitory action

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of the synthetic compounds can be considered as a disadvantage, because with the higher feed, the higher concentration of these compounds would be needed to show the same

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inhibitory action.

3.3. Pancreatic α-Amy inhibition

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The selectivity of α-Gls inhibitors is of great importance, because non-specific inhibition of other glycosidase particularly pancreatic α-Amy may lead to the accumulation of non-digested carbohydrates in Gut, which in turn results in abdominal cramping, diarrhea and flatulence [11]. In this study, we examined the effects of synthetic compounds against porcine pancreatic α-Amy activity and the results are depicted in Fig.8. ►Figure 8◄

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ACCEPTED MANUSCRIPT As shown, the activity of α-Amy was reduced only to lower than 10% of its original value in the presence of these synthetic compounds. However in comparison with Acarbose (70% inhibition), these synthetic compounds show significantly reduced inhibitory activity against α-Amy. Therefore, the obtained results are important in term of the reduced

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susceptibility of the synthetic compounds for the possible development of intestinal disturbance side effects.

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4. Conclusion

In conclusion, nine poly-hydroxyl functionalized acridine derivatives (L1-L9) with

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two variable (R1&R3) and one constant (R2) substructures in their molecular scaffold were synthesized, according to our previously reported α-Gls inhibitors [18,19]. The results of this study notify the significant role of the variable substructure, R2 (PFH ring) on the inhibitory properties of the synthetic compounds. Also, L5 with a chromeno[3',4':5,6]pyrido[2,3-

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d]pyrimidine moiety and exceptional conformational feature was the most promising inhibitor among the synthetic compounds against both yeast and rat α-Gls enzymes. The comparison of α-Gls inhibitory action of the synthetic compounds L1 and L6-L9 led us to

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suggest an important role for the nature of the sugar moiety. Moreover, the considerably lower α-Amy inhibitory action of the synthetic compounds compared to Acarbose is highly

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important in the term of their likely reduced susceptibly for development of gastrointestinal discomforts. Overall, L5 with reasonable inhibitory action against α-Gls and poor ability for inhibition of α-Amy is potentially an important anti-diabetic agent for the more convenient controlling of postprandial hyperglycemia.

17

ACCEPTED MANUSCRIPT Acknowledgments We wish to acknowledge supports from the Iran National Science Foundation (grant no. 92001695). Also, the financial support of the research council of Shiraz University is

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gratefully acknowledged. References

[1] Palanuvej, C.; Hokputsa, S.; Tunsaringkarn, T.; Ruangrungsi, N. Sci. Pharm., 2009, 77,

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837-849.

[2] Agrawal, R.P.; Sharma, P.; Pal, M.; Kochar, A.; Kochar, D.K. Diabetes Res. Clin.

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Pract., 2006, 73, 211-214.

[3] Goh, S. Y.; Cooper, M. E. J. Clin. Endocrinol. Metab., 2008, 93, 1143-1152. [4] Engerman, R. L.; Kern T. S. Diabetes, 1984, 33, 97-100.

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[5] (a) Del-Corso, A.; Balestri, F.; Bugno, E. D.; Moschini, R.; Cappiello, M.; Sartini, S.; LaMotta, C.; Da-Settimo, F.; Mura, U. PLoS One, 2013, 8, 74076;

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(b) Vlassara, H. Diabetes Metab. Res. Rev., 2001, 17, 436-43. [6] (a) Engelen, L.; Stehouwer, C.D.; Schalkwijk, C.G. Diabetes Obes. Metab., 2013, 15,

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677-89;

(b) Srivastava, S. K.; Ramana, K.V.; Bhatnagar, A. Endocr. Rev., 2005, 26, 380-92. [7] Jaiswal, N.; Srivastava, S.P.; Bhatia1, V.; Mishra, A.; Sonkar, A. K.; Narender, T.; Srivastava, A. K.; Tamrakar, A. K. J. Diabetes Metab., 2012, S6:004, doi:10.4172/21556156.S6-004 [8] Kim, Y.M.; Y. Jeong, K.; Wang, M. H.; Lee, W.Y.; Rhee H. I. Nutrition, 2005, 21, 756761. 18

ACCEPTED MANUSCRIPT [9] Sama, K.; Murugesan, K.; Sivaraj, R. Asian J. Plant Sci. Res., 2012, 2, 550-553. [10] Casirola, D. M.; Ferraris, R.P. Metabolism, 2006, 55, 832-841. [11] Levetan, C. Curr. Med. Res. Opin., 2007, 23, 945-952.

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[12] Ramdanis, R.; Soemiati, A.; Abdul, M. Int. J. Med. Arom. Plants, 2012, 2, 447-452. [13] Yamazaki, K.; Inoue, T.; Yasuda, N.; Sato, Y.; Nagakura, T.; Takenaka, O.; Clark, R.;

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Saeki, T.; Tanaka, I. J. Pharmacol. Sci., 2007, 104, 29-38.

[14] Bachhawat, J. A.; Shihabudeen, M.S.; Thirumurugan, K. Int. J. Pharm. Pharmaceut.

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Sci., 2011, 3, 267-274.

[15] Shihabudeen, H. M. S.; Priscilla, D. H.; Thirumurugan, K. Nutr. Metabol., 2011, 8, 4657.

[16] Standl, E.; Schnell, O. Diab. Vasc. Dis. Res., 2012, 9, 163-169.

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[17] Lossow, W.J.; Migirini, R.H.; Brot, N.; Chaikoff, I. L. J. Lipid Res., 1964, 5, 198-202. [18] Yousefi, R.; Alavian mehr, M. M.; Mokhtari, F.; Panahi, F.; Mehraban, M.H.; Khalafi-

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Nezhad, A. J. Enzyme Inhib. Med. Chem., 2013, 28, 1228-1235. [19] Panahi, F.; Yousefi, R.; Mehraban, M.H.; Khalafi-Nezhad, A. Carbohydrate Res., 2013,

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380, 81-91.

[20] Cortés, A.; Cascante, M.; Cárdenas, M. L.; Cornish-Bowden, A. Biochem. J., 2001, 357, 263-268.

[21] Kwon, Y. I. I.; Vattem, D. A.; Shetty, K. Asia Pac. J. Clin. Nutr., 2006, 15, 107-118. [22] Zhang, L.; Hogan, S.; Li, J.; Sun, S.; Canning, C.; Zheng, S. J. Food Chem., 2011, 126, 466-471.

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ACCEPTED MANUSCRIPT [23] Dabiri, M.; Arvin-Nezhad, H.; Khavasi, H.R.; Bazgir, A. Tetrahedron, 2007, 63, 17701774. [24] Khalafi-Nezhad, A.; Panahi, F.; Golesorkhi, B. Helv. Chim. Acta, 2013, 96, 1155-1162.

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[25] Dinakaran, V.S.; Bomma, B.; Srinivasan, K. K. Der. Pharma. Chemica, 2012, 4, 255265.

[26] Frisch, M. J.; Trucks, G.W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M.A.; Cheeseman,

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J. R.; Zakrzewski, V. G.; Montgomery Jr., J.A.; Stratmann, R.E.; Burant, J.C.; Dapprich, S. Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone,

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V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D.

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J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle E. S.; Pople, J. A. GAUSSIAN98 (A. 9 Revision), Gaussian, Inc., Pittsburgh, PA,

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2003.

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[27] Kimura, A.; Lee, J. H.; Lee, I.S.; Lee, H.S.; Park, K.H.; Chiba, S.; Kim, D. Carbohydrate Res., 2004, 339, 1035-40. [28] Cheng, Y.; Prusoff, W.H. Biochem. Pharmacol., 1973, 22, 3099-108. [29] Bariana, D.S. J. Med. Chem., 1970, 13, 544-546. [30] Sashidhara, K.V.; Kumar, M.; Khedgikar, V.; Kushwaha, P.; Modukuri, R.K.; Kumar, A.; Gautam, J.; Singh, D.; Sridhar, B.; Trivedi. R. J. Med. Chem., 2013, 56, 109-122.

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ACCEPTED MANUSCRIPT [31] Rempel, V.; Volz, N.; Gläser, F.; Nieger, M.; Bräse, S.; Müller, C. E. J. Med. Chem., 2013, 56, 4798-4810. [32] Zhao, H.; Neamati, N.; Hong, H.; Mazumder, A.; Wang, S.; Sunder, S.; Milne, G. W.

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A.; Pommier, Y.; Burke, T. R. Jr. J. Med. Chem., 1997, 40, 242-249. [33] Hwu, J. R.; Lin, S.Y.; Tsay, S.C.; Clercq, E. D.; Leyssen, P.; Neyts. J. J. Med. Chem.,

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2011, 54, 2114-2126.

Legends

Scheme 1: The synthetic approach for preparation of compounds L1-L9. Figure 1. Schematic representation of major available inhibition sites for reducing the post

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prandial hyperglycemia and its complications in diabetic patients. Also, the general structure of the synthetic compounds is shown. As indicated, they demonstrate strong and weak

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inhibitory actions against α-Gls and α-Amy, respectively. Figure 2. The chemical structure of synthesized compounds. The variable substructure in the

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synthetic compounds is R2.

Figure 3. The optimized molecular structures for the most stable conformations of the synthesized compounds, using B3lYP-3-21g method [26]. Figure 4. The Lineweaver-Burk plots derived from the inhibition of yeast α-Gls by the synthetic compounds. The α-Gls activity was measured as a function of pNPG concentration (0.1-5.0 mM) in the absence and presence of the synthetic inhibitors. For details, see “Material and Methods”.

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ACCEPTED MANUSCRIPT Figure 5. The Lineweaver-Burk plots derived from the inhibition of rat α-Gls by the synthetic compounds. The α-Gls activity was measured as a function of pNPG concentration (0.5-10 mM) in the absence and presence of inhibitors. For details, see “Material and Methods”.

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Figure 6. The Dixon plots derived from the inhibition of yeast α-Gls by the synthetic compounds, in which the intercepts on the abscissa shows the -IC50 values.

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Figure 7. The Dixon plots derived from the inhibition of rat α-Gls by the synthetic compounds, in which the intercepts on the abscissa shows the -IC50 values.

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details, see “Material and Methods”.

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Figure 8. The inhibitory assessment of the synthetic compounds on pancreatic α-Amy. For

22

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Table 1. The IC50/Ki values and inhibition mode of synthetic compounds against yeast α-Gls.

IC50 (µM)

*Kic (µM)

*Kiu(µM)

Mode of inhibition

L1

209.8 ± 1.29

173.39 ± 1.07

-

Competitive

L2

11.96 ± 0.24

9.88 ± 0.61

-

L3

122.3 ± 2.75

101.07 ± 1.71

-

L4

177.1 ± 3.19

159.67 ± 3.55

591.11 ± 4.98

L5

8.88 ± 0.11

7.33 ± 0.25

L6

-

-

L7

45.20 ± 2.98

L8

-

L9

-

Acarbose

365.4 ± 1.67

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Competitive

Mixed-competitive Competitive

-

-

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-

336.36 ± 2.8

Mixed-competitive

-

-

-

-

-

-

304.5 ± 3.07

-

Competitive

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Competitive

37.73 ± 2.81

* c and u respectively stand for competitive and un-competitive.

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Ligand

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Table 2. The IC50/Ki values and inhibition mode of synthetic compounds against rat α-Gls.

IC50 (µM)

Ki (µM)

Mode of inhibition

L1

88.13 ± 2.01

70.51 ± 1.61

Competitive

L2

119.1 ± 1.85

95.28 ± 1.48

L3

-

-

L4

141.5 ± 0.85

93.71 ± 0.68

L5

43.21 ± 3.52

34.57 ± 2.82

Competitive

L6

-

-

-

L7

379.1 ± 0.47

361.04 ± 0.67

Competitive

L8

-

-

-

L9

-

-

-

Acarbose

22.3 ± 0.97

17.42 ± 0.82

Competitive

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Competitive -

Competitive

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ACCEPTED MANUSCRIPT Highlights ►Novel poly-hydroxyl functionalized acridine derivatives (L1-L5) with ability for αGls inhibition was synthesized. ►Compared to Acarbose they weakly inhibit the activity of pancreatic α-Amy. ► The PFH variable group plays an important role in

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their enzyme inhibitory action. ►L5 with a chromeno[3',4':5,6]pyrido[2,3-

d]pyrimidine moiety demonstrates the highest inhibitory activity.► These synthetic

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compounds potentially offer an opportunity to develop new anti-diabetic drugs .

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Synthesis of novel poly-hydroxyl functionalized acridine derivatives as inhibitors of α-Glucosidase and α-Amylase

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Zahra Toobaei1, Reza Yousefi,*1,2 Farhad Panahi,3 Sara Shahidpour,1 Maryam Nourisefat,3 Mohammad Mahdi Doroodmand3, Ali Khalafi-Nezhad*3 1

Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences,

Shiraz University, Shiraz, Iran Institute of Biotechnology, Shiraz University, Shiraz, Iran

3

Department of Chemistry, College of Sciences, Shiraz University, Shiraz, Iran

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2

Outline 1. Experimental

2. Spectral data for synthesized compounds L1, L6-L9

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L9

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3. Copy of 1H NMR, 13C NMR and IR of synthesized compounds L1, L6-

1

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1. Experimental 1.1. General

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1.2. General Procedure for the synthesis of compounds A mixture of suger (1mmol), barbituric acid (2 mmol) and 4-(4-aminophenoxy) benzenamine (1 mmol) in presence of PSA (1g) as catalyst in round bottom flask was

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stirred at 50 °C for appropriate time (table 1). After completion of the reaction, as indicated by TLC, the reaction mixture was filterd and washed with ethanol to obtain the

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pure product. All the isolated products gave satisfactory spectral and physical data. The isolated catalyst could be recycled, see Supplementary data for details.

2. Experimental characterization data for compounds: 10-(4-(4-aminophenoxy)phenyl)-5-((1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl)-5,10-

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1.

dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L1)

OH O

HN

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H O N

OH

OH O

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N

O

HN

NH

O

NH2

Brown crystal, Yield: 82% (0.48 g); m.p. 139-141 ºC. IR

HO HO

L1

(KBr, cm-1): 3834, 3741 (Amide N-H Stretch), 3409

(Amine N-H Stretch), 3217 (Alcohol O-H Stretch), 2931 (Aromatic C-H Stretch, Alkyl C-H Stretch), 2638 (Alkyl C-H Stretch), 2360, 2083, 1697 (Amide C=O Stretch), 1589 (Aromatic C=C Bending), 1496 (Aromatic C-C Stretch), 1365, 1249, 1203 (C-O Stretch), 1080, 1041, 825

(Aromatic C-H Bending), 779, 540. 1H-NMR (250 MHz, DMSO-d6/TMS) δ (ppm): 3.273.43 (m, 6H), 3.56-3.64 (m, 4H), 3.86 (s, 1H, CH), 4.16 (s, 1H, CH), 4.77 (m, 2H), 6.796.91 (m, 8H, Ar), 11.07-11.21 (m, 4H, NH).

13

C-NMR (62.5 MHz, DMSO-d6/TMS) δ

(ppm): 18.4, 43.4, 55.9, 63.1, 65.3, 70.5, 72.0, 76.2, 118.1 (C, Ar), 119.0 (C, Ar), 138.2 2

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(C, =C), 150.6 (C, C=O), 212.6 (C, C=O). MS: 582 (17.5%, M+). Anal. Calcd for C26H26N6O10 (582.53): C, 53.61; H, 4.50; N, 14.43. Found: C, 53.45; H, 4.39; N, 14.32.

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2. 10-(4-(4-aminophenoxy)phenyl)-5-((1S,2R,3S,4R)-1,2,3,4,5-pentahydroxypentyl)-5,10dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L6)

Brown crystal, Yield: 76% (0.44g); m.p. 238-240 ºC. IR (KBr, cm-1): 3834, 3749 (Amide

L6

(Alcohol O-H Stretch), 2923 (Aromatic C-H Stretch, Alkyl C-H Stretch), 2360, 1759, 1689, 1620

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HO H HO O N O OH HN OH N O HN NH O O

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N-H Stretch), 3456 (Amine N-H Stretch), 3263 OH

(Amide C=O Stretch), 1504, 1458, 1419 (Aromatic C-C Stretch), 1357, 1249 (C-O Stretch), 1080, 1010, 910, 825 (Aromatic C-H Bending), 786, 509, 439. 1H-NMR (250 MHz, DMSO-d6/TMS) δ (ppm):

NH2

3.21 (s, 1H, CH), 3.24 (s, 1H, CH), 3.28-3.33 (m,

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2H), 3.39-3.52 (m, 5H), 3.58 (s, 1H, CH), 3.62 (s, 1H, CH), 3.90 (t, 1H, J = 9.5 Hz, CH), 4.90 (m, 2H), 6.76-6.84 (m, 8H, Ar), 11.04 (s, 1H, NH), 11.10 (s, 1H, NH), 11.16-11.20 (m, 2H, NH).

13

C-NMR (62.5 MHz, DMSO-d6/TMS) δ (ppm): 18.4, 43.5, 48.3, 55.9,

63.1, 70.5, 72.0, 76.2, 78.1, 80.9 (C, =C), 118.1 (C, Ar), 119.0 (C, Ar), 138.2 (C, =C),

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150.0 (C, C=O), 212.6 (C, C=O). MS: 582 (8%, M+). Anal. Calcd for C26H26N6O10

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(582.53): C, 53.61; H, 4.50; N, 14.43. Found: C, 53.53; H, 4.46; N, 14.32.

4. 10-(4-(4-aminophenoxy)phenyl)-5-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-5,10dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L7) OH

H HO O N O HN

OH O

N O

HN

NH O

NH2

OH

Brown crystal Yield: 70% (0.38g) ; m.p. 168-172 ºC. IR L7

(KBr, cm-1): 3834, 3818, 3749 (Amide N-H Stretch), 3394 (Amine N-H Stretch), 3217, 3217 (Alcohol O-H Stretch), 2962, 2638 (Aromatic C-H Stretch, Alkyl C-H Stretch), 2360, 1697, 1620 (Amide C=O Stretch), 1496, 3

ACCEPTED MANUSCRIPT

1458 (Aromatic C-C Stretch), 1365, 1242, 1203, 1118 (C-O Stretch), 1072, 1033, 833 (Aromatic C-H Bending), 779, 540. 1H-NMR (250 MHz, DMSO-d6/TMS) δ (ppm): 3.153.44 (m, 7H), 3.59 (t, 1H, J = 5.0 Hz, CH), 3.82-3.91 (m, 2H), 4.67 (m, 2H), 6.73-6.81 (m, 8H, Ar), 11.08 (s, 2H, NH), 11.18 (s, 2H, NH).

13

C-NMR (62.5 MHz, DMSO-

RI PT

d6/TMS) δ (ppm): 22.0, 24.1, 61.7, 73.2, 77.4, 119.0 (C, =C), 119.1 (C, Ar), 119.2 (C, Ar), 120.2 (C, Ar), 120.5 (C, Ar), 125.4 (C, Ar), 128.1 (C, Ar), 136.7 (C, =C), 150.9 (C, Ar), 151.2 (C, C=O), 151.5 (C, C=O), 167.6 (C, C=O), 210.8 (C, C=O). MS: 552 (14%, M+). Anal. Calcd for C25H24N6O9 (552.50): C, 54.35; H, 4.38; N, 15.21. Found: C, 54.28;

10-(4-(4-aminophenoxy)phenyl)-5-((1S,2S,3R,4R)-1,2,4,5-tetrahydroxy

3-

M AN U

4.

SC

H, 4.32; N, 15.14.

(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)pentyl)-5,10-dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)tetraone (L8)

Pale Yellow crystal Yield: 63% (0.47g);

H N

O

OH

TE D

HO HO O HO

O

O

HN N

m.p. 150-151 ºC. IR (KBr, cm-1): 3895, 3834 (Amide N-H Stretch), 3379 (Amine N-

OH

O

EP

OH

OH

O

H Stretch, Alcohol O-H Stretch), 2900 (Aromatic C-H Stretch, Alkyl C-H Stretch), 2360, 2067, 1697, 1620 (Amide C=O

AC C

O

OH

NH

HN

L8

Stretch),

NH2

1496,

1458

(Aromatic

C-C

Stretch), 1357, 1249, 1203 (C-O Stretch),

1072, 1033, 879, 833 (Aromatic C-H Bending), 779, 632, 540. 1H-NMR (250 MHz, DMSO-d6/TMS) δ (ppm): 3.24-3.27 (m, 6H), 3.40 (s, 1H, CH), 3.43 (s, 1H, CH), 3.453.49 (m, 6H), 3.59 (s, 4H), 3.71-3.72 (m, 2H), 4.44 (m, 2H), 6.73 (m, 8H, Ar), 11.0911.27 (m, 4H, NH).

13

C-NMR (62.5 MHz, DMSO-d6/TMS) δ (ppm): 18.4, 48.11, 56.0, 4

ACCEPTED MANUSCRIPT

60.3, 60.4, 68.0, 69.3, 69.7, 70.4, 71.2, 71.9, 73.1, 75.1, 75.3, 80.5, 81.1, 91.9 (C, =C), 103.6, 103.7, 117.8 (C, Ar), 119.0 (C, Ar), 138.6 (C, =C), 150.5 (C, Ar), 150.9 (C, C=O), 151.5 (C, C=O), 167.8 (C, C=O). MS: 744 (6%, M+). Anal. Calcd for C32H36N6O15

RI PT

(744.67): C, 51.61; H, 4.87; N, 11.29. Found: C, 51.52; H, 4.80; N, 11.21.

5. 10-(4-(4-aminophenoxy)phenyl)-5-((1S,2S,3R,4R)-1,2,4,5-tetrahydroxy-3

SC

(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-

yl)oxy)pentyl)-5,10-dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-

OH HO O OH HN O HN

OH

O

OH

O OH O

OH L9

NH OH

N HN

O

Brown crystal, Yield: 65% (0.48g); m.p. 178-180 ºC. IR (KBr, cm-1): 3834, 3818 (Amide N-H

Stretch), 3409 (Amine N-H Stretch, Alcohol O-H Stretch), 2931 (Aromatic C-H Stretch, Alkyl C-H

TE D

O

M AN U

tetraone (L9)

Stretch), 2376, 1697, 1620 (Amide C=O Stretch),

NH2

1504 (Aromatic C-C Stretch), 1365, 1242, 1149

EP

(C-O Stretch), 1033 (Aromatic C-H Bending), 779, 532, 501. 1H-NMR (250 MHz, DMSO-d6/TMS) δ (ppm): 3.32-3.43 (m, 14H), 3.58-3.63 (m, 6H), 3.71-3.72 (m, 2H),

AC C

4.67 (m, 2H), 6.75 (s, 8H, Ar), 11.08 (s, 4H, NH).

13

C-NMR (62.5 MHz, DMSO-

d6/TMS) δ (ppm): 18.4, 55.9, 60.6, 60.7, 69.7, 73.2, 118.9 (C, Ar), 119.0 (C, Ar), 136.8 (C, =C), 151.2 (C, Ar), 151.5 (C, C=O), 167.6 (C, C=O), 167.7 (C, C=O). MS: 744 (9%, M+). Anal. Calcd for C32H36N6O15 (744.67): C, 51.61; H, 4.87; N, 11.29. Found: C, 51.53; H, 4.82; N, 11.22.

5

ACCEPTED MANUSCRIPT

RI PT

3. Copy of 1H NMR, 13C NMR and IR of synthesized compounds

AC C

EP

TE D

M AN U

SC

10-(4-(4-aminophenoxy)phenyl)-5-(1,2,3,4,5-pentahydroxypentyl)-5,10dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L1)

6

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

7

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

10-(4-(4-aminophenoxy)phenyl)-5-((1S,2R,3S,4R)-1,2,3,4,5-pentahydroxypentyl)-5,10dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L6)

8

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

10-(4-(4-aminophenoxy)phenyl)-5-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-5,10dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)-tetraone (L7)

9

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

10

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

10-(4-(4-aminophenoxy)phenyl)-5-((1S,2S,3R,4R)-1,2,4,5-tetrahydroxy-3(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)pentyl)-5,10-dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)tetraone (L8)

11

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

12

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

10-(4-(4-aminophenoxy)phenyl)-5-((1S,2S,3R,4R)-1,2,4,5-tetrahydroxy-3(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)pentyl)-5,10-dihydropyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,9H)tetraone (L9)

13

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

14

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

15