N-Bridged 1-deoxynojirimycin dimers as selective insect trehalase inhibitors

Laura Cipolla a, Antonella Sgambato a, Matilde Forcella a, Paola Fusi a, Paolo Parenti b, Francesca Cardona c,
Davide Bini a,⇑
a Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy
b Department of Earth and Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy
c Department of Chemistry ‘Ugo Schiff’, University of Florence, Via della Lastruccia 3-13, I-50019 Sesto Fiorentino, Florence, Italy


A small set of N-bridged 1-deoxynojirimycin dimers has been synthesized and evaluated as potential inhibitors of insect trehalase from midge larvae of Chironomus riparius, porcine trehalase as the mamma- lian counterpart and a-amylase from human saliva. All the tested compounds (2–4) proved to be active (micromolar range activity) against insect trehalase, showing selectivity toward the insect glycosidase. No activity was observed against a-amylase.

1. Introduction

Trehalase (a-glucoside-1-glucohydrolase, EC is a specific glycosidase that catalyzes the hydrolysis of trehalose1 (1, a-D-glucopyranosyl-a-D-glucopyranoside, Fig. 1) to the two constituent glucose units. This disaccharide is found in many organisms as diverse as bacteria, yeast, fungi, nematodes, plants, insects and some other invertebrates, but is absent in mammals. In lower organisms, trehalose may serve as a source of energy, a carbohydrate store, or an agent for protecting proteins and cellular membranes from inactivation or denaturation caused by a variety of environmental stress conditions. In insects, trehalose hydrolysis by trehalase is fundamental in various physiological processes including chitin synthesis during molting,2 and thermotolerance in larvae.3 Moreover, trehalase activity is the basis for flight metab- olism,1,4 trehalose being the principal hemolymph sugar in insects5 that acts as an indispensable substrate for energy production and macromolecular biosynthesis.6 Given these premises, insect trehalases are attractive targets for the search of inhibitors as potential novel and selective insecticides.7 Some natural pseudodi- saccharides, such as validoxylamine A,8 trehazolin,9 casuarine-6-O- a-D-glucoside10,11 and its analogues12 have been shown to be potent inhibitors of trehalase, together with synthetic trehalose analogues.

In the search for new inhibitors that might be specific toward insect trehalase based on 1-deoxynojirimycin and its N-acyl derivatives13 we herein propose the synthesis of N-bridged 1-deoxynojirimycin dimers (Fig. 1, 2–4) and their biological evalu- ation toward both insect and porcine trehalase, compared to the human a-amylase enzyme (EC

The synthesis of compounds 2–4 was performed straightforward from protected 1-deoxynojirimycin,17 as outlined in Scheme 1. The reaction of compound 5 with oxalyl or succinyl chloride success- fully afforded compounds 6 and 8 in 66% and 49% yields, respec- tively; in contrast, the reaction of 5 with malonyl chloride in the same reaction conditions gave compound 7 only in traces. The unexpected outcome of the reaction with malonyl chloride is prob- ably due to by-products deriving from reaction of methylenic acidic protons of malonyl chloride with the basic pyridine used in the procedure. Dimer 7 was obtained in 70% yield by reaction of 5 with malonic acid in the presence of DCC. Direct hydrogenol- ysis of 6–8 quantitatively afforded the compounds 2–4.

Compounds 2–4 were tested for their inhibitory activity against insect trehalase of midge larvae of C. riparius,18 porcine trehalase (purchased from Sigma–Aldrich) as the mammalian counterpart and a-amylase from human saliva (purchased from Sigma– Aldrich), as a relevant glycolytic enzyme. Midge larvae are widespread in freshwater ecosystems, as sentinel organisms are widely used in ecotoxicological studies and environmental biomonitoring program and represent a good model for biochemi- cal studies. To examine the potential of each 1-deoxynojirimycin dimer as trehalase inhibitor, preliminary screening assays at a fixed concentration (1 mM) of potential inhibitors were carried out, and dose–response curves were established for most active compounds in order to determine the IC50 values. Experiments were performed at a fixed substrate concentration, in the presence of increasing inhibitor concentrations. The inhibitory activity is shown in Figure 2 as IC50 value.

Figure 1. Structures of trehalose (1), validoxylamine A, trehazolin, casuarine-6-O-a-D-glucoside, 1-deoxynojirimycin, and the synthesized dimers 2–4.

Scheme 1. Synthesis of nojirimycin dimers 2–4. Reagents and conditions: (a) Oxalyl or succinyl chloride, pyridine, DCM, 0 °C?rt, 3 h; (b) Malonic acid, DCC, DMAP, p-TsOH, DCM, rt, 30 min; (c) Pd(OH)2/C, H2, EtOAc/EtOH = 1:1.

All the synthesized dimers were inactive against a-amylase,while they were similarly active against C. riparius trehalase, with the activity in the micromolar range. Compound 2 resulted to be the most active derivative of the series; all compounds 2–4 showed a slight selectivity toward the insect glycosidase, resulting more selective between mammalian and insect trehalase if compared to the parent compound 1-deoxynojirimycin.

We can conclude that despite the fact the both trehalase specif- ically hydrolyze trehalose, they might have significant differences in the catalytic pocket that can be exploited for the design and development of specific insect trehalase inhibitors, with potential follow up in the development of insecticides. However, further in- sights are needed into enzyme recognition features.

2.2. General procedure for the hydrogenolysis reaction

A 0.02 m solution of the appropriate dimer dissolved in EtOAc/ EtOH (1:1) was treated with Pd(OH)2/C (100% in weight). The reac- tion was stirred for 5 d under a H2 atmosphere. Palladium was then removed by filtration through a Celite pad followed by washing with EtOH and water. Evaporation of the solvents afforded the cor- responding deprotected compounds in quantitative yields.

2.3. 1,2-Bis((2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)piperidin-1-yl)ethane-1,2-dione (6)

To a solution of compound 5 (108 mg, 0.21 mmol) in dry DCM (1.1 mL), pyridine (33 lL, 0.41 mmol) and oxalyl chloride (9 lL, 0.10 mmol) were added at 0 °C. The temperature was slowly in- creased to rt (3 h); the mixture was then concentrated and the res- idue was purified directly on a silica gel column (petroleum ether/ EtOAc, 65:35) affording 6 (74 mg, 66% yield). 1H NMR (CDCl3): d = 7.37–7.01 (m, 40H, ArH), 4.77–4.19 (m, 18H, OCH2Ph, H-5),3.97–3.54 (m, 12H, H-1a, H-2, H-3, H-4, H-6), 3.14–3.02 (m, 2H, H- 1b) ppm. 13C NMR (CDCl3): d = 165.0, 164.7 (C@O), 138.5, 138.5,138.3, 138.2, 138.2, 138.1, 138.1, 137.9 (C Ar), 128.7–127.6 (CH Ar), 82.4 (C-3), 79.1, 76.1 (C-2, C-4), 73.7–70.1 (OCH2Ph), 68.9 (C-6), 58.6 (C-5), 43.8 (C-1) ppm. MS (TOF, m/z): [M+H]+ calcd for C70H73N2O10, 1101.5; found 1101.5. C70H72N2O10 (1101.33): calcd C, 76.34, H 6.59, N 2.54; found C 76.49, H 6.58, N 2.55.

Figure 2. Histogram of the inhibitory activity of compounds 2–4, compared to 1-deoxynojirimycin.

2.4. 1,3-Bis((2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2- ((benzyloxy)methyl)piperidin-1-yl)propane-1,3-dione (7)

Compound 5 (67 mg, 0,12 mmol), malonic acid (6,4 mg, 0,06 mmol), DMAP (3 mg, 0.02 mmol) and p-toluenesulfonic acid (4.7 mg, 0.02 mmol) were dissolved in DCM (1.14 mL). DCC (32 mg, 0.15 mmol) was added and the solution was stirred for 30 min at room temperature. Then the DCC–urea was filtered off and washed with a small volume of DCM. The solvent was evapo- rated, and the residue was purified by flash column chromatogra- phy (petroleum ether/EtOAc, 62.5:37.5) giving pure 7 (48 mg, 70% yield). 1H NMR (CDCl3) d = 7.34–7.15 (m, 40H, ArH), 4.77–4.59 (m, 6H, H-5, OCH2Ph), 4.59–4.44 (m, 8H, OCH2Ph), 4.41–4.25 (m, 4H, OCH2Ph), 4.03–3.93 (m, 4H, H-2, H-1a), 3.80–3.56 (m, 10H, H-6,
H-3, CH2C=O, H-4), 3.56–3.46 (m, 2H, H-1b) ppm. 13C NMR (CDCl3) d = 166.9 (C@O), 142.7–137.8 (C Ar), 128.4–127.5 (CH Ar), 80.9
(C-3), 78.1, 73.9 (C-2, C-4), 72.9–70.8 (OCH2Ph), 68.0 (C-6), 54.2 (C-5), 44.4 (C-1), 41.9 (CH2C@O) ppm. MS (TOF, m/z): [M+H]+ calcd for C71H75N2O10, 1115.5; found 1115.5. C71H74N2O10 (1115.35): calcd C 76.46, H 6.69, N 2.51; found C 76.51, H 6.67, N 2.52.

2.5. 1,4-Bis((2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)piperidin-1-yl)butane-1,4-dione (8)

To a solution of compound 5 (177 mg, 0.34 mmol) in dry DCM (1.9 mL), pyridine (55 lL, 0.67 mmol) and succinyl chloride (19 lL, 0.17 mmol) were added at 0 °C. The temperature was slowly increased to rt (3 h); the mixture was then concentrated and the residue was purified directly on a silica gel column (petroleum ether/EtOAc, 50:50) affording 8 (93 mg, 49% yield). 1H NMR (CDCl3): d = 7.38–7.16 (m, 40H, ArH), 4.82–4.23 (m, 18H, OCH2Ph,H-5), 4.05–3.41 (m, 12H, H-1a, H-2, H-3, H-4, H-6), 2.97–2.58 (m, 6H, H-1b, CH2C@O) ppm. 13C NMR (CDCl3): d = 171.8 (C@O),138.4–137.8 (C Ar), 128.5–127.6 (CH Ar), 82.4 (C-3), 78.9, 74.3 (C-2, C-4), 73.3–71.1 (OCH2Ph), 68.3 (C-6), 54.5 (C-5), 44.0 (C-1),32.0 (CH2C@O) ppm. MS (TOF, m/z): [M+H]+ calcd for C72H77N2O10,1129.6; found 1129.6. C72H76N2O10 (1129.38): calcd C 76.57,H 6.78, N 2.48; found C 76.49, H 6.80, N 2.47.

2.6. 1,2-Bis((2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidin-1-yl)ethane-1,2-dione (2)

1H NMR (D2O): d = 3.75–3.65 (m, 4H, H-6), 3.60–3.53 (m, 2H,H-2), 3.42–3.26 (m, 6H, H-1a, H-3, H-4), 3.02–2.98 (m, 2H, H-5),2.82–2.72 (m, 2H, H-1b) ppm. 13C NMR (D2O): d = 76.2 (C-3),67.7, 66.9 (C-2, C-4), 60.0 (C-5), 57.6 (C-6), 45.8 (C-1) ppm. MS (TOF, m/z): [M+H]+ calcd for C14H25N2O10, 381.1; found 381.4. C14H24N2O10 (380.35): calcd C 44.21, H 6.36, N 7.37; found C 44.27, H 6.34, N 7.38.

2.7. 1,3-Bis((2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidin-1-yl)propane-1,3-dione (3)

1H NMR (D2O) d = 4.36 (d, 1H, J = 14.4 Hz, CH2C@O), 3.96–3.52 (m, 16H, H-1, H-2, H-3, H-4, H-5, H-6), 3.20 (d, 1H, J = 14.8, CH2-
C@O) ppm. 13C NMR (D2O) d = 74.1 (C-3), 69.3, 68.4 (C-2, C-4), 63.0 (C-5), 59.8 (C-6), 47.7 (C-1), 40.6 (CH2C@O) ppm. MS (TOF,m/z): [M+H]+ calcd for C15H27N2O10, 395.2; found 395.3. C15H26N2O10 (394.37): calcd C 45.68, H 6.65, N 7.10; found C 45.74, H 6.63, N 7.11.

2.8. 1,4-Bis((2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidin-1-yl)butane-1,4-dione (4)

1H NMR (D2O): d = 3.82–3.71 (m, 4H, H-6), 3.68–3.61 (m, 2H, H-2), 3.48–3.34 (m, 6H, H-1a, H-3, H-4), 3.09–3.05 (m, 2H, H-5), 2.86–
2.80 (m, 2H, H-1b), 2.61–2.50 (m, 4H, CH2C@O) ppm. 13C NMR (D2O): d = 76.0 (C-3), 67.5, 66.7 (C-2, C-4), 59.7 (C-5), 57.4 (C-6),
45.6 (C-1), 28.6 (CH2C@O) ppm. MS (TOF, m/z): [M+H]+ calcd for C16H29N2O10, 409.2; found 409.4. C16H28N2O10 (408.40): calcd C
47.05, H 6.91, N 6.86; found C 47.14, H 6.89, N 6.87.

2.9. Biological assays

Trehalase activity was measured through a coupled assay with glucose-6-phosphate dehydrogenase and hexokinase according to Wegener at al.19. To examine the potential of each compound as a trehalase inhibitor, screening assays of potential inhibitors were carried out at a fixed concentration of 1 mM and dose–response curves were established to determine the IC50 values. Experiments were performed at fixed substrate concentration close to the Km value (0.5 mM for C. riparius trehalase and 2.5 mM for porcine trehalase), in the presence of increasing inhibitor concentrations. Initial rates as a function of inhibitor concentration were fitted to the following equation:where mi and m are the initial rate in the presence and in the absence of inhibitor, respectively, [I] is the inhibitor concentration, IC50 is the inhibitor concentration producing half-maximal inhibition, and n is the Hill coefficient.


We gratefully acknowledge University of Milano-Bicocca FAR 2009 and MIUR (PRIN2008/24M2HX) for financial support.


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